Anxiolytics and Antidepressants

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INTERNATIONAL ENCYCLOPEDIA OF
PHARMACOLOGY A N D THERAPEUTICS
Section 136
PSYCHOPHARMACOLOGY
OF ANXIOLYTICS
AND ANTIDEPRESSANTS
SPECIALIST SUBJECT EDITOR
SANDRA E. FILE
Psychopharmacology Research Unit
UMDS Division of Pharmacology
University of London
Guy's Hospital
England
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PREFACE
The chapters in this volume provide the pieces of our jigsaw puzzle that are available
in 1990. Chapter 1 is concerned with the clinical pharmacology of anxiety and depres
sion. Chapter 2 reviews the clinical and animal literature on the roles of the hormones
of the hypothalamic-pituitary-adrenal (ΗΡΑ) axis in anxiety and depression. Chapter 3
is concerned with the response of central catecholamines to chronic stress. Chapters 3
and 4 discuss experimental test conditions in which both anxiolytic and antidepressant
drugs are active. Chapter S reviews the technique of drug discrimination, which is suit
able for pharmacological, rather than clinical, classification of drugs. Chapters 6 through
9 discuss tests that specifically detect anxiolytic drugs, and Chapter 10 reviews a model
that is specific for antidepressants. Chapter 11 covers the range of pharmacological agents
that can modify distress vocalizations. The next decade of research will determine how
these pieces should be fitted together, but the interconnections between chapters that are
discussed below provide some guidelines as to the pictures we are trying to complete.
Most of the chapters in this volume are concerned with the effects of drugs in animal
tests. There are two main reasons for using animal tests to study the effects of anxiolytic
and antidepressant drugs. One is to screen new compounds for their potential clinical
use and the other is to study the neurobiology of anxiety and depression. In both cases,
it is essential to make constant reference to the clinical literature to establish clear cases
of positive and negative drug treatments for the disease in question. This is not as easy
as it sounds. Often, the clinical data are partial and equivocal and, of course, there is a
considerable time lag between the initial screening of compounds in animal tests and the
publication of results from clinical trials. A decade ago, the clinical situation seemed
straightforward: anxiety and depression were distinct disorders, each treated by a distinct
group of drugs. So clear was the situation that the two groups of drugs became known as
anxiolytics and antidepressants. All this is changing. As Nutt and Glue point out in
Chapter 1, the dichotomous view of anxiety and depression is no longer tenable and
while either can exist alone, there are certainly many cases where both symptoms coexist.
Not only has the clinical diagnosis of these states undergone considerable change, but
the drug treatments are also changing. Thus, there is growing evidence for the anxiolytic
action of antidepressant drugs and for an antidepressant action of anxiolytic drugs. Faced
with this state of flux, it is a hard task for those developing animal tests, since it is essen
tial to know the difierences between anxiety and depression in their etiologies and/or
treatments.
The first animal test used to screen anxiolytic drugs (the conditioned emotional
response) is reviewed in Chapter 8 by Davis, and the current use of animal tests for
screening is reviewed in Chapter 6 by Howard and Pollard. In Chapter 7, Lister discusses
the development and evaluation of animal tests with respect to their use as models of
psychiatric disorders. The use of an animal test (potentiated startle) to study the neuro-
biological basis of conditioned fear is described in Chapter 8 by Davis; the anatomical
basis is reviewed, and the importance of the central nucleus of the amygdala is stressed.
The relationship between conditioned fear and clinical anxiety states is not known, but
Davis proposes a theoretical framework for relating the two. An important note of warn
ing is sounded by Andrews and Stephens in Chapter 5, on the use of the technique of
drug discrimination. Contrary to many claims, this technique may not discriminate
compounds on the basis of their cUnical action (e.g., anxiolytics or antidepressants) and
VI Preface
therefore may be of limited use either in screening new compounds or in investigating

the neurobiology of anxiety or depression. Rather, the internal cues provided by a drug
reflect all of its pharmacological actions and are therefore best considered as relating to
the pharmacological category of a drug. The use of drug discrimination studies as part
of the pharmacological classiñcation of drugs is discussed in Chapter 5.
One important difference between the two disorders is that anxiety, unlike depression,
is both a normal emotion and a psychiatric disorder. Because anxiety is a response to an
acute situation, it is easier to "model" in animals, as is reflected in the number of chap-
ters in this volume that are devoted to tests for anxiolytic drugs. However, it must be
remembered that the responses of animals to acute stress or anxiogenic test situations
are likely to be adaptive and to reflect "state" anxiety. The hormones of the hypotha-
lamic-pituitary-adrenal axis play important roles in the immediate response to an anx-
iogenic situation, but are less important in the development of a pathologic^ response
to chronic stress (see Chapter 2 by File). The effect of corticotropin releasing hormone
on gamma-amino butyric acid (GABA) release provides a possible mechanism for medi-
ating very rapid adaptive changes and of linking the ΗΡΑ axis with the GABA-benzo-
diazepine receptor complex. Several test situations show excellent speciñcity for benzo-
diazepines and anxiolytic drugs and do not detect any action of antidepressant drugs.
Most of these measure an animal's response to an acute test condition: potentiated startle
(see Chapter 8 by Davis), extinction and negative contrast (see Chapter 9 by Raherty),
and ethologically based tests (see Chapter 7 by Lister and Chapter 11 by Newman). How-
ever, in tests of punished responding (see Chapter 6 by Howard and Pollard), it is possible
to obtain responses to a chronic anxiogenic stimulus. If the existing animal tests can be
extended beyond state anxiety, they are most likely to be applicable to generalized anx-
iety disorder; at present, there is a lack of animal tests that might relate to posttraumatic
stress disorder, phobias, obsessive-compulsive, or panic disorders. Lister's chapter pro-
vides useful pointers for developing these using ethologically based models. The similar-
ity of panic disorder to kindling has been raised by Nutt and Glue (Chapter 1) and by
File (Chapter 2); this could perhaps form a useful guide to the development of an animal
model. Intriguingly, in negative contrast, we seem to have an animaJ correlate of disap-
pointment (see Chapter 9 by Flaherty). The test has not yet received extensive pharma-
cological study, but benzodiazepines and ethanol (but not antidepressants) reduce neg-
ative contrast. While acute disappointment is not usually treated clinically, self-
medication with ethanol is a frequent response to disappointment.
The response to negative contrast is an acute one and, with continued exposure, there
is habituation, rather than the development of a depressive reaction. This may again
reflect the adaptive nature of the rat's response, but also raises an important question of
etiology. Under what circumstances is there habituation of disappointment or of an anx-
iogenic response, under what circumstances will the responses persist, and what condi-
tions will lead to an increased response (kindling) or the development of depression?
Why does the response to negative contrast habituate, whereas that to punished respond-
ing in the Cook-Davidson test (see Chapter 6) persist for up to 2 years? The importance
of chronic stress to the development of depression is discussed in Chapter 3. The olfac-
tory bulbectomized rat shows behavioral changes in response to antidepressant treat-
ment, but little response to anxiolytics. Although van Riezen and Leonard (Chapter 10)
emphasize that olfactory bulbectomy is an inexact model of depression, it does have
many positive features and the long-lasting behavioral, biochemical, and immunological
abnormalities respond to chronic antidepressant treatment. Two animal test situations
seem promising for revealing the inteφlay between anxiety and depression, particularly
with respect to etiology. Both anxiolytic and antidepressant drugs affect behavior in
"learned helplessness" and "behavioral despair" tests, but they are effective at clearly
different stages of the tests (see Chapter 3 by Anisman and Zacharko). Similarly, both
classes of drug can change nonhuman primate behavior, but as Vellucci discusses in
Chapter 4, it is the behavior of the dominant animal that is most susceptible to anxioly-
Preface vii
tics and that of the subordinates, to antidepressants. Thus, detailed ethological studies of
factors that lead to the development of dominant or subordinate behavior would provide
valuable data on the etiology of anxiety and depression. As Vellucci reviews, factors such
as social stress and overcrowding have powerful effects on subordinate animals, whereas
dominant animals are more disrupted by exposure to novelty. Both anxiolytic and anti
depressant drugs can modify separation-induced vocalizations in a wide range of species,
but the opiate system plays a more important role (see Chapter 11 by Newman).
With the diversity of animal tests now available and with future refinements, partic
ularly with respect to etiology, it may be possible to relate particular animal tests to par
ticular types of clinical disorder. What is already emerging is that, whereas some drugs,
for example, the benzodiazepines, are effective in a very wide range of animal tests, oth
ers such as 5HT3 antagonists may be effective only in certain tests. Thus, it may be pos
sible in the future to determine which neurotransmitters and receptor subtypes mediate
behaviors in particular tests; this, of course, will be helped by the development of drugs
specific for particular receptor subtypes. In responding to chronic stress and in the devel
opment of depression, the evidence for the role of norepinephrine is still stronger than
that for other neurotransmitters, whereas evidence continues for a stronger role for
5-HT in anxiety. There may also be an important role for norepinephrine in panic dis
order, which has been linked to depression clinically and in drug treatment. However, it
is naive to assume that only one transmitter-receptor system will control behavior in a
particular task, and as is clear from all the chapters, it is likely that a constellation of
mechanisms will emerge for each and that it will be necessary to determine the interac
tions between the various pathways. There is a long way to go, but I hope that the chap
ters in this volume will provide the reader and the experimenter with a sound back
ground for making the voyage.
Sandra E. File
April 1990
LIST OF CONTRIBUTORS
J. S. Andrews Richard G. Lister

Scientific Development Group Laboratory of Clinical Studies
Organon International BY NIAAA
Oss
The Netherlands Bethesda, Md, USA
Hymie Anisman
Psychology Department John D. Newman
Unit for Behavioral Medicine and Laboratory of Comparative Ethology
Pharmacology NICHD
Carleton University
Ottawa, Ontario PoolesviUe, Md, USA
Canada
David J. Nutt
Michael Davis
Yale University School of Medicine and Reckitt and Colman Psychopharmacology
Ribicoff Research Facilities of the Unit
Connecticut Mental Health Center Department of Pharmacology
New Haven, Conn., USA School of Medical Sciences
Bristol
Sandra E. File United Kingdom
Psychopharmacology Research Unit Gerald T. Pollard
UMDS Division of Pharmacology Division of Pharmacology
University of London Burroughs Wellcome Co.
Guy's Hospital Research Triangle Park, NC, USA
London D. N. Stephens
United Kingdom Department of Neuropsychopharmacology
Research Laboratories of Schering AG
Charles F. Flaherty Beriin
Department of Psychology
Rutgers University Federal Republic of Germany
New Brunswick, NJ, USA
H. van Riezen
Paul Glue
Reckitt and Coleman Psychopharmacology Department of Pharmacology
Unit CIBA-GEIGY Ltd.
Department of Pharmacology Basel
School of Medical Sciences Switzerland
Bristol Sandra V. Vellucci
United Kingdom Department of Anatomy
University of Cambridge
James L. Howard Cambridge
Division of Pharmacology United Kingdom
Burroughs Wellcome Co.
Research Triangle Park, NC, USA Robert M. Zacharko
Psychology Department
B. E. Leonard Unit for Behavioral Medicine and
Department of Pharmacology Pharmacology
University College Carlton University
Galway Ottawa, Ontario
Republic of Ireland Canada
XV
File, S. Ε., editor.
(1991) PsyOmpharmacohty ofAnxiolytics and Antidepressants.
Vavmm Press, Inc. (New York), pp. 1-28
CHAPTER 1
CLINICAL PHARMACOLOGY OF ANXIOLYTICS

AND ANTIDEPRESSANTS:
A PSYCHOPHARMACOLOGICAL PERSPECTIVE
DAVID J. NUTT AND PAUL GLUE
Reckitt and Colman Psychopharmacology Unit, Department of Pharmacology, School of Medical Sciences,
Bristol, United Kingdom
1. INTRODUCTION
Both anxiety and depression are common clinical disorders as demonstrated by a
number of epidemiological studies. For instance, a recent major study from the USA
reported lifetime rates of major depression at 5.8%, panic disorder at 14.6%, and rates
for both diagnoses at 3.6% (Regier et al., 1989). Both can be effectively treated by phar
macological means, and it is becoming clear there is therapeutic overlap, since antide
pressants are being shown to be effective in some anxiety disorders. Before concentrating
on the psychopharmacology of anxiety and depression, it is germane to clarify some diag
nostic and conceptual issues that commonly cause confusion.
2. DIAGNOSTIC ISSUES
2.1. ANXIETY
Anxiety, unlike depression, is both a normal emotion and a psychiatric disorder. The
best way of conceptualizing this may be to consider a graded progression of anxiety, from
normal to pathological degrees, or of functionally useful arousal through to disruptive
and maladaptive (pathological) anxiety. Whether the neuropharmacological basis of anx
iety is the same at all points on this spectrum is still unclear, and the evidence that panic
attacks, social phobia, and simple anxiety respond to different treatments suggests a bio
logical diversity that will be discussed below.
Another important conceptual issue is that of state-vs-trait anxiety (Speilberger et al.,
1980). State anxiety is that seen at any given time and when elevated is usually associated
with a transient stressor and remits once the stimulus is removed. Trait anxiety is a per
sistent, enduring feature of an individual, which is a facet of their personality and which
may have an inheritable component. Anxious patients usually rate themselves highly on
scales that assess both forms of anxiety, although for those with simple phobias, state
anxiety may be quite low until they confront their feared stimulus or situation.
Anxiety occurs in almost all psychiatric and in most physical illnesses. It also is a
prominent and often debilitating component of drug withdrawal especially from the opi
ates, benzodiazepines, and alcohol. These latter observations have contributed to the the
ories of the pharmacology of anxiety in particular those that emphasize the noradrenergic
aspects.
2.1.1. Diagnostic Classification

A number of different types of anxiety disorders are recognized. In the USA, the 3rd
Edition of the Diagnostic and Statistical Manual (DSM-III-R) classiñcation system is
most commonly used (American Psychiatric Association, 1982; revised in 1987),
whereas in the UK the 9th Edition of the International Classification of Diseases (ICD-9)
2 D. J. NUTT AND P. GLUE
system is presently employed, soon to be replaced by the ICD-10, which is presently

being used in field trials. Details of these are given in Table 1.1. As an overview of dif
ferences between these classification systems, the ICD-9 was not intended to be used as
a research tool, and so did not provide any explicit or operational criteria for different
illness categories. As well as providing operational criteria, the DSM-III-R also used a
multiaxial classification system, meaning that emphasis was put on identifying other
aspects that might be important in diagnosis apart from the main presenting problem
(e.g., personality factors, physical illness, etc.). The ICD-10 differs from the ICD-9 in a
number of ways. For instance, it groups together a number of disorders with common
themes (e.g., mood disturbance) that were previously separated, and has features more
in common with the DSM-III-R (e.g., multiaxial classification, use of diagnostic guide
lines). Further information on this area is available in the DSM-III-R or in a recent ICD-
10 supplement in the British Journal of Psychiatry (Sartorius et al., 1988).
One notable recent change in the diagnosis of anxiety disorders is the distinction
between generalized anxiety disorder and panic disorder, as in the DSM-III-R. This is
based on pharmacological observations that antidepressant drugs such as Imipramine (a
tricyclic antidepressant) and phenelzine (a monoamine-oxidase inhibitor, or MAOI) will
stop panic attacks, yet have only a limited effect on anxiety levels between attacks. The
syndrome of anxiety that is accompanied by panic attacks has become known as panic
disorder, and patients with chronic high levels of anxiety that do not have panic attacks
are referred to as having generalized anxiety disorder (GAD). This latter syndrome seems
to respond best to tranquillizers such as the benzodiazepines (see below). It should be
noted that there are reservations about the validity of this classification among some UK
psychiatrists (Ashcroft et al., 1987; Gelder, 1986; Marks, 1987; Tyrer, 1984). In view of
the different treatments suitable for the range of anxiety disorders, it becomes important
that the correct diagnosis is made prior to starting treatment. In practice, many patients
suffer from more than one type of anxiety disorder and so may need a combination of
therapeutic approaches.
2.1.2. Symptom Variables

As well as diagnostic subtypes with the anxiety disorders, symptoms that are reported
by patients also differ. Complaints can include lightheadedness or dizziness, which may
not appear to be related to anxiety at all. The supposition that there is a single underlying
abnormality that leads to this variety of symptoms is still only theoretical, although the
TABLE 1.1. Diagnostic Classifications of Anxiety: Comparison o / D S M - I I I - R , I C D - 9 and I C D - 1 0
DSM-III-R diagnosis ICD-9 diagnosis ICD-10 diagnosis
Phobic Disorders Phobic States Phobic Disorders

Agoraphobia Agoraphobia Agoraphobia ( ± panic
Panic disorder with attacks)
agoraphobia
Social phobia Social phobias
Simple phobia Simple phobia Specified (isolated) phobias
Anxiety States Anxiety States Other Anxiety Disorders
Panic disorder Anxiety neurosis Panic disorder
Generalized anxiety disorder Generalized anxiety disorder
Mixed anxiety and
depressive disorder
Obsessive compulsive Obsessive compulsive Obsessive compulsive
disorder disorder disorder
Acute stress reaction Acute stress reaction
Adjustment disorder with Adjustment disorder Adjustment disorder
anxious mood
Post-traumatic stress disorder Post-traumatic stress
disorder
Clinical pharmacology of anxiolytics and antidepressants
TABLE 1.2. Symptom Subcomponents of Anxiety and Depression
Symptom Anxiety Depression
Mood Apprehension, worry, irritability Sadness, flatness, loss of feeling, anhedonia

In severe cases, mood-congruent
hallucinations and delusions
Cognitions Thoughts of impending disaster or Thoughts of guilt, worthlessness, uselessness,

illness, social embarrassment hypochondriasis, suicide
Physiology Tachycardia, sweating, Anorexia, constipation, sexual impotence,

palpitations, flushing, nausea, early morning waking
diarrhea, tremor, onset
insomnia
Behavior Hand-wringing, pacing, scratching, Agitation or retardation, suicide attempts,

hypervigilance tearfulness, reduction in normal activity,
slowed speech
separation of panic and generalized anxiety disorders may prove to be the first of many.
For instance, recent work has suggested that the benzodiazepines are particularly good
at treating the insomnia of anxiety whereas Imipramine may be better for the tension
(Kahn et al., 1986). One way of conceptualizing the symptom subcomponents of anxiety
is shown in Table 1.2. Avoidance behavior has not been included in this table because it
is not clear whether it is best considered as a simple motor behavior or as a cognitive
state. Clearly it has components of both.
Most studies to date looking at treatment or provocation of anxiety tend to emphasize
mood, physiological, and behavioral aspects of anxiety, rather than cognitive aspects.
The cognitions of anxiety are often difficuh to assess for therapists as well as patients
(Beck et al., 1985a). Hopefully, this will be rectified in the near future, since there is
accumulating data that cognitive inteφretations of stimuli may be very different in anx
ious subjects and may be of etiological significance (Clark, 1986).
2.2. DEPRESSION
Depression is a pathological mood state and, unUke anxiety, is not seen in normal
individuals. Commonly, there is confusion of depression with sadness, which is a normal
response to unhappy events. Perhaps the closest that most people come to experiencing
depression is the sadness and distress of grief following a bereavement. Soon after a
death, bereaved people may describe many symptoms of depression except those of
pathological guilt, suicidal thoughts, and psychomotor retardation. Those symptoms
they do describe tend to remit over time, which distinguishes grief from depression (see
Clayton, 1982). Prolonged grief is pathological and probably indicates a depressive
illness.
2.2.1. Diagnostic Classification

Classification of depressive disorders is more complicated than classification of anxiety
disorders. Clinically, patients presenting with depression may have illnesses that range
from mild to very severe, and symptoms at each extreme are different. For instance,
patients with mild depression do not have symptoms such as severe weight loss or delu
sions of guilt, but do have a pattern of sleep disturbance where they have difficulty falling
asleep, and have mood reactivity (i.e., their mood can lift temporarily in some circum
stances), whereas those with severe depression almost invariably have weight loss and
guilt, a pattern of early morning waking, and no mood reactivity. Patients with mild
depression often feel worse at night, whereas those with severe depression are at their
4 D . J . NUTT AND P. GLUE
worst in the morning and improve during the day. Their response to treatment may
differ, in that severe depression with hallucinations and delusions of guilt may only
respond to electroconvulsive therapy, whereas more mild depression responds to drug
treatment alone.
Attempts to quantify these differences have led to a number of proposals being made
for subClassification of depression. It has been suggested that depression is a unitary con
dition, with a continuum running from mild to severe types (e.g., Kendell, 1976). Other
proposals have argued a number of subtypes, usually dichotomous; e.g., according to
preceding illness (primary vs secondary to other psychiatric disorder), illness history (uni
polar vs bipolar), measures of severity (neurotic or mild vs psychotic or severe), or
according to supposed etiology (reactive vs endogenous) (Andreasen, 1982). These
dichotomies are not synonymous, as severe or endogenous depression may occur in reac
tion to a stressful life event (Paykel, 1982). The unipolar-bipolar distinction has been
the most enduring and separates patients with recurrent manic and depressive episodes
of illness (bipolar) from those with recurrent depressions only (unipolar) (see review by
Perris, 1982). Another approach has been to apply mathematical techniques, such as
cluster analysis, to patient data to generate clusters or groups of patients (Paykel, 1971;
Andreasen, 1982). Although these studies have consistently yielded a cluster that corre
sponds with an endogenous/psychotic depressive subtype, no such consistency has been
found for patients with mild depression. This may be due in part to differences between
computer models used to generate clusters.
Table 1.3 compares the current diagnostic classification systems, the DSM-III-R
(American Psychiatric Association, 1987), and the ICD-9 and -10. It is beyond the scope
of this article to look at this area exhaustively, except to point out that depressive dis
orders in the ICD-9 may be placed within several diagnostic groups (with no good jus
tification for doing this), whereas the DSM-III-R has these as several categories within
one group and has dropped the neurotic/psychotic division as a means of describing
illness severity. Melancholia in DSM-III-R refers to a severe type of major depression,
with delusions of guilt and agitation. ICD-10 is still undergoing field trial testing, but
appears to resemble DSM-III-R more than ICD-9. Of interest is that it contains some
nonexclusive categories (e.g., bipolar affective disorder, and manic and depressive epi
sodes as individual categories).
2.2.2. Symptom Variables

A list of some depressive symptoms is given in Table 1.2, with subclassification into
abnormalities in mood, cognitions, physiology, and behavior. Some symptoms may
encompass more than one category (e.g., although there are clear sleep electroencepha
logram [EEG] changes in depression [disturbed physiology]), insomnia may also be con
sidered a disturbance of behavior. Although some patients may emphasize physical
aspects of their illness, generally complaints of depressed mood and associated distur
bances are common. As a useful means of rating severity of depression, objective and
subjective symptom rating scales may be used (see Hamilton, 1982).
2.3. COEXISTENCE OF DEPRESSION AND ANXIETY
It is clear that there is a considerable overlap between depressive and anxiety disorders,
even though they may also exist in pure forms. The exact relationship is compUcated and
has been studied in a number of ways. Prevalence studies in patients with panic disorder
have shown rates of secondary major depression of 31 to 40% and rates of primary major
depression of 22 to 26% (Breier et al., 1985; Qancy et al., 1978; Lesser et al., 1988). Panic
patients with coexisting depressive illness tend to have more severe symptoms of both
than those without depressive symptoms (Breier et al., 1985; Clancy et al., 1978) and
may have a worse prognosis (Buller et al., 1986; Qancy et al., 1978; Lesser et al., 1988;
see review by Grunhaus, 1988). Patients with both illnesses may have less favorable treat-
Clinical pharmacology of anxiolytics and antidepressants
TABLE 1.3. Diagnostic Classifications of Depression: Comparison of DSM-III-R, ICD-9, and ICD-10
DSM-III-R diagnosis ICD-9 diagnosis ICD-10 diagnosis
Affective disorders Affective psychoses Mood disorders

Major affective Manic-depressive psychosis, Manic episode (mild/severe)
disorders manic
Bipolar disorder Manic-depressive psychosis, Depressive episode (mild/severe)
depressed
Manic Manic-depressive psychosis, Bipolar affective disorder
circular type, currently
Depressed manic Recurrent depressive disorder
Mixed Manic-depressive psychosis, Other affective disorders
circular type, currently
Major depression depressed Affective disorders not
Single episode/ Manic-depressive psychosis, otherwise specified
recurrent circular type, currently
± melancholia mixed
± psychotic Manic-depressive psychosis,
features circular type, currently
Atypical affective unspecified
disorder
Atypical bipolar Manic depressive psychosis,
disorder other and unspecified
Atypical depression
Other nonorganic psychoses
Depressive type
Excitative type
Other specific affective Neurotic disorders
disorders Neurotic depression
Dysthymia Personality disorders
Cyclothymia Affective
Acute reaction to stress Acute stress reaction
Predominant disturbance of
emotions
Adjustment disorders with Adjustment reaction Adjustment reaction
depressed mood Brief depressive reaction Brief depressive reaction
Prolonged depressive reaction Prolonged depressive reaction
Depressive disorder not Depressive disorder, not
otherwise specified otherwise specified
Psychotic reactions not Schizophrenia Schizo-affective disorders
elsewhere classified Schizo-affective type
Schizo-affective
disorder
ment responses than patients with depression alone (Grunhaus et al., 1986). Differences
in the phenomenology of anxiety and depressive disorders have been looked at in several
studies, using factor analysis of a number of rating scales and personal history items.
Patients with mild anxiety and depressive illnesses can be differentiated using this tech
nique, although a number of items are common to each group (Derogatis et al., 1971;
Roth et al., 1972; Roth and Mountjoy, 1982). As a reflection of this, it is of interest to
compare items in two widely used rating scales for depression and anxiety, the Hamilton
Anxiety and Depression scales (Table 1.4). These show considerable overlap. If factor
analysis is not used, patients with mild anxiety and depression are only poorly distin
guished by rating scales alone (Mendels et al., 1972; Prusoff and Klerman, 1974).
Genetic studies of depression and anxiety have shown that relatives of patients with
major depression or panic disorder tended to "breed true" (Coryell et al., 1988), and this
is supported by the finding of high rates of anxiety disorders and normal rates of depres
sive disorders in relatives of patients with anxiety disorders (Noyes et al., 1978). In
patients with both illnesses, rates of major depression, alcoholism, and a range of anxiety
disorders in family members were increased compared to patients who only had depres
sion (Leckman et al., 1983). Qear evidence of a genetic component in major depression
has been demonstrated by twin studies (see reviews by Bertelsen et al., 1977; Gershon et
al., 1976) and family studies (see reviews by McGuffin and Katz, 1986, 1989). For less
D . J . NUTT AND P . GLUE
TABLE 1.4. Comparison of Items on the Hamilton Anxiety and Depression Rating Scales
Anxiety Shared Depression
Fears (specific) Depressed mood Guilt

Poor concentration/memory Agitation Suicidal thoughts/attempts
Autonomic symptoms Psychic anxiety Retardation
Behavior/appearance Somatic anxiety Loss of insight
Gastrointestinal symptoms Reduced interests/ability
Tension/general somatic symptoms to work
Genital symptoms (reduced libido, ?Hypochondriasis
etc.) ?Weight loss
severe ("neurotic") depression, family studies do not support a genetic vulnerability in

this disorder (Stenstedt, 1966). High rates of anxiety disorders have been found in first-
degree relatives of patients with panic disorder and agoraphobia (Crowe et al., 1983; Har
ris et al., 1983), and these are supported by similar findings of two twin studies (Slater
and Shields, 1969; Torgersen, 1983). They were not associated with increased risk of
generalized anxiety disorder (Crowe et al., 1983; Torgersen, 1983). However, it is still
unclear whether the risk of depression is increased. Interestingly, there is a marked
increase in alcoholism in the families of panic probands (Leckman et al., 1983), perhaps
due to underlying abnormalities in «j-adrenoceptor function (Nutt et al., 1988). Illness
outcome and response to treatment also indicate some differences between these ill
nesses. Outcome studies suggest that these diagnoses are stable over time (Roth and
Mountjoy, 1982). The differences in response to treatment will be discussed more fully
in the treatment section (see below). Treatments for mild depression and anxiety overlap,
although for more severe and purer illnesses, they are different. However, the response
of different patients to these shared treatments may differ, as shown by the anxiogenic
effect of tricyclic antidepressants in anxious patients and the absence of any such effect
in depressed patients (see below). More recently, it has been suggested that panic patients
may show altered sensitivity of benzodiazepine receptors as revealed by hypersensitivity
to antagonists (see Nutt et al., 1990).
Notwithstanding the extensive evidence that these illnesses are separate diagnostic
entities, it is a common clinical observation that the two conditions coexist. In a pro
portion of patients, anxiety is secondary to depression, and in another group, the depres
sion appears to be a secondary reaction to the anxiety disorder. It would appear that both
psychological and biochemical processes are involved in this depressive reaction, and
these are summarized in Fig. 1.1. More details on the biochemistry are given in the fol
lowing section.
Anxiety/Panic Phobic Avoidance
4 Self Image
• Social Reinforcement
Biochemical Changes
Negative
Cognitive Set
Depression '4- ' Demoralization
FIG. 1.1. Relationship between anxiety and depression.

Clinical pharmacology of anxiolytics and antidepressants 7
3. CAUSATIVE FACTORS
3.1. NEUROCHEMISTRY
Within this article, we do not have the scope to fully cover the neurochemical studies
in anxiety and depression, but will focus on theories of monoamine dysfunction. Those
studies examining the role of norepinephrine (NE) will be emphasized, as this neuro
transmitter is most implicated in these disorders. It is likely that disorders of other neu
rotransmitters are also important (e.g., serotonin in anxiety [Kahn et al., 1988] and
depression [Cowen, 1988]; 7-aminobutyric acid [GABA]/benzodiazepine receptors in
anxiety [Enna and Möhler, 1987, Nutt et al., 1990]), although these will not be discussed
further here. It is fair to say that hypotheses regarding these transmitters are less com
prehensive than for NE. Moreover, they do not readily explain the efficacy of antide
pressants in treating both anxiety and depressive disorders.
As originally hypothesized, depression was a syndrome of relative NE deficit (Schild
kraut, 1965). Evidence for this included low concentrations of urinary and cerebrospinal
fluid (CSF), 3-methoxy-4-hydroxyphenylethylene glycol (MHPG) in a number of depres
sives (Schildkraut, 1965), and the realization that antidepressants potentiated NE syn
aptic availability (Carlsson, 1966). Over the past decade, it has become apparent that this
simple view is not entirely correct, but it is generally agreed that a dysfunction of NE
transmission does exist. This has been conceptualized as NE inefficiency (Linnoila et al.,
1982; Rudorfer et al., 1984) or dysregulation (Siever and Davis, 1985). It is difficult to
directly test these hypotheses in man, especially those involving central synapses,
although the melatonin output of the pineal gland offers potential in this direction
(Arendt, 1989; Cowen et al., 1985; Nutt and Cowen, 1987; see Section 4.7).
A considerable body of evidence supports the view that anxiety disorders may be
related to excessive NE activity. For instance, episodes of increased anxiety, especially
with panic attacks, are associated with marked peripheral sympathetic activation (Char-
ney and Redmond, 1983; Lader and Mathews, 1970; Tyrer and Lader, 1976; Villacres
et al., 1987). This may reflect excessive NE release (Chamey et al., 1984; Nutt, 1989;
Wyatt et al., 1971) or reduced parasympathetic drive (George et al., 1989). Evidence for
increased central NE release in man is hard to come by, although correlations have been
found between CSF levels of NE and MHPG and anxiety (van Kämmen et al., 1990;
Post et al., 1989). There is extensive evidence from animal work, including primates,
that direct activation of the locus coeruleus can produce signs of arousal that strongly
resemble those seen in human anxiety states (Redmond, 1985; Redmond and Huang,
1979, 1982). On the basis of these data, the most persuasive explanation of the nature of
anxiety, especially panic disorder, is one of paroxysmal NE excess (Ko et al., 1983). This
may reflect dysfunction of inhibitory «j-adrenoceptors, as has been suggested by chal
lenge testing using Clonidine (Chamey and Heninger, 1986; Nutt, 1986a, 1989) and
yohimbine (Chamey et al., 1984).
3.2. STRESS
It is well recognized that life stress is implicated in most psychiatric disorders. Many
studies have demonstrated that stressful negative life events cluster before depression
(Brown et al., 1973; Paykel, 1982), and more recent data suggest the same is tme for
panic disorder (Roy-Byme et al., 1986; Lelliott et al., 1989). Stress is well recognized to
activate the sympathetic nervous system and, by inference, central adrenergic mecha
nisms, and this is supported by an extensive animal literature (see reviews by Stanford,
1990; Stone, 1983). In brief, certain forms of stress can alter brain adrenoceptors, includ
ing upregulation of β- and a2-adrenoceptors (Stanford, 1989). These changes are similar
to those seen in animal models of depression and may be reversed or prevented by the
use of antidepressant drugs (Stanford et al., 1987).
A link between benzodiazepine receptors, anxiety, and stress-related adrenergic
D . J. NUTT AND P. GLUE
t ß adrenoceptors 0
t a a adrenoceptors^
A
Stress
Benzodiazepines Antidepressants
Anxiety
" O ^ i e g FG 7142^1
learned helplessness
("depression") ^ ?
FIG. 1.2. Neurochemical aspects of stress and anxiety.
changes is given by studies with the benzodiazepine inverse agonist FG 7142. This drug
is anxiogenic (Dorow et al., 1983), potentiates learned helplessness (Drugan et al., 1985),
and produces adrenoceptor changes in rats (Stanford et al., 1986a, b), which are reversed
by antidepressant drugs (Stanford et al., 1987). This is illustrated in Fig. 1.2, which also
shows where benzodiazepines act.
As well as activating NE systems, stress is well recognized to stimulate hypothalamic-
pituitary-adrenocortical activity. It has been established that corticotropin releasing fac
tor (CRJF) release is increased at the hypothalamic level (Koob and Bloom, 1985), which
leads to elevations of adrenocorticotropic hormone (ACTH) and Cortisol (Vale et al.,
1981). Recent elegant studies in wild baboons have demonstrated a link between social
rank and plasma concentrations of corticosterone, with subordinate males having higher
levels. Challenge testing with CRF showed evidence for reduced central CRF receptor
sensitivity in the more stressed animals, since the subordinate males had lower responses
despite higher baselines (Sapolsky, 1989). This is good naturalistic evidence that social
and biological variables are interiinked.
Central administration of CRF produces a range of autonomic and behavioral changes
in rats that have led to the suggestion that it may mediate the effects of stress upon behav
ioral arousal (Brown et al., 1982; Koob and Bloom, 1985). There is now evidence of
interaction between adrenergic and CRF-containing neurons, including activation of the
locus coeruleus (Al-Damluji et al., 1987; Valentino et al., 1983). This means there is a
positive feedback loop, as illustrated in Fig. 1.3. Recently, it has also been established
that CRF, when given intraventricularly, produces kindling (Weiss et al., 1986). This
may explain some of the phenomenology of panic disorder, since kindling-like processes
are seen in this condition, in that panic symptoms tend to increase in frequency and
severity over time (Klein, 1981). It should also be noted that stress-induced CRF release
may well be the reason for abnormalities of Cortisol secretion in depression (Carroll et
al., 1981), and this may relate to elevated CSF levels of CRF (Nemeroff et al., 1984).
Furthermore, indices of central NE activity are higher in patients with Cortisol non-
suppression to dexamethasone (Roy et al., 1987, 1988). A link between stress hormones
and the benzodiazepine receptor is suggested by the work of Roy et al. (1989) who
reported significant positive correlations between the concentrations of DBI and CRF in
the CSF of depressed patients.
stress > panic
> kindling
FIG. 1.3. Relationship between stress and panic attacks.
4. THERAPEUTIC ISSUES
There are a number of established treatments for anxiety and depressive states, which
are summarized in Table 1.5. These have been obtained from many controlled studies
carried out over the last 30 years. Whereas mild degrees of either disorder may respond
equally as well to somatic as to psychological therapies, more severe illness invariably
requires somatic treatment of some sort (e.g. Gelder et al., 1983; Tyrer, 1982).
The following sections will address in more detail the issues of drug treatment of
depression and the anxiety disorders, especially the role of antidepressants as anxiolytic
and anxiogenic agents. We will then focus on, and attempt to explain, this apparent con
tradiction by developing an hypothesis that may encompass most of the known facts.
4.1. PHARMACOTHERAPY OF DEPRESSION
Drug treatment of depression has been well validated since the introduction of Imip
ramine 30 years ago (Tyrer, 1982). There is little new to review in terms of new, proven
therapeutic agents, although some promising new agents are presently being investigated.
One class of these is the a2-adrenoceptor antagonists (e.g., yohimbine and more selective
analogs such as idazoxan), which prevent autoreceptor inhibition and thus increase nor
adrenergic function (Cedarbaum and Aghajanian, 1976). The rationale for employing
these drugs is on the basis that there is reduced NE synaptic availability in depression
(Schildkraut, 1965) and that tricyclics and MAOIs tend to downregulate central
a2-adrenoceptor number and sensitivity (for review, see Green and Nutt, 1983, 1985).
Preliminary trials suggest that idazoxan is an effective antidepressant (Crossley, 1984;
Osman et al., 1989), although it is not yet available for treatment.
Another new approach stems from the observation that selective 5-hydroxytryptamine
(5-HT) uptake blockers work in depression and involve the use of 5-HT agonists. Prelim
inary work suggests that buspirone and gepirone are more effective than placebo in treat
ing depression (Robinson et al., 1988). However, since the present clinical use of these
drugs is to reduce anxiety, they are discussed below in the section on anxiolytics. One
final point regarding treatment of depression and 5-HT is the observation that lithium,
when added to unsuccessful tricyclic antidepressant therapy, may lead to recovery in
treatment-resistant depressed patients (de Montigny et al., 1983). Recent data suggest
this may be due to the enhancing effect of lithium on 5-HT function (Glue et al., 1986;
Price et al., 1989, 1990). Anticonvulsants such as carbamazepine have been shown to be
effective in treatment-resistant depressive illness, either alone or in combination with
lithium or other antidepressants (Elphick, 1989; Kramlinger and Post, 1989; Post and
Uhde, 1987).
Recent work has confirmed a very early finding on the efficacy of MAOIs in treating
atypical depressive illness (West and Dally, 1959). Patients with atypical depression pre
sent with hysterical, phobic or anxious symptoms, hypersomnia, hypeφhagia, sensitivity
TABLE 1.5. Established Treatments for Anxiety and Depression
Anxiety Depression
Acute Maintenance Acute Maintenance
Tricyclics Tricyclics E C T (severe) Tricyclics

MAOIs MAOIs Tricyclics Lithium
Benzodiazepines (Benzodiazepines) MAOIs MAOIs
Behavioral/cognitive Cognitive therapy (?ECT)
therapy (Lithium) (?Cognitive)
MAOIs = monoamine-oxidase inhibitors; ECT = electroconvulsive treatment.

10 D. J. NuTT AND p. GLUE
to interpersonal rejection, and reactivity of mood. Such patients responded better to

phenelzine compared with imipramine (Quitkin et al., 1989; Stewart et al., 1989).
4.2. BENZODIAZEPINES IN ANXIETY REDUCTION
A large body of clinical literature attests to the efficacy of the benzodiazepines in the
anxiety disorders (see reviews by Rickels and Schweizer, 1986; Sheehan, 1987; Tyrer,
1982). Rather than reiterate this substantial literature, which covers anxiety disorders
that would now be considered GAD, in this section we will look at their efficacy and
clinical use in panic disorder, which is more controversial.
A number of studies have examined whether benzodiazepines are effective in panic
disorder. Most interest in the last few years has centered on alprazolam, which has been
used recently in a large international trial in panic patients. This drug would appear to
be an effective alternative to the antidepressants (Ballenger et al., 1988; Chouinard et al.,
1982; Rizley et al., 1986; Sheehan et al., 1984). Clinical consensus suggests that the other
benzodiazepines such as diazepam and chlordiazepoxide are not as effective (Kahn et al.,
1986; McNair and Kahn, 1981), even though there are a few studies showing that diaz
epam and other benzodiazepines are of benefit in panic disorder (Dunner et al., 1986;
Noyes et al., 1984; Rickels et al., 1983). This is perhaps not so suφrising, since these
patients often have high levels of generalized or anticipatory anxiety as well. The com
mon clinical experience, that patients taking high doses of diazepam often complain of
panics, may reflect development of tolerance and withdrawal (Nutt, 1986b; Petursson
and Lader, 1984).
However, it is unlikely that the superior effect of alprazolam is due to some unique
pharmacological properties. Alprazolam is a particularly high affinity ligand for the ben
zodiazepine receptor. Ahhough all the benzodiazepines in clinical use are thought to be
full agonists with equivalent efficacy (Braestrup et al., 1983) and so vary only in receptor
affinity, it is possible that either a proportionally higher dose of drug is being adminis
tered or that high affinity ligands may have clinically distinct effects. The latter possibility
is more likely to be correct, since the high affinity benzodiazepines, clonazepam and
lorazepam, have recently been reported to have antipanic properties (Schweizer et al.,
1988; Spier et al., 1986).
There are considerable problems with the use of benzodiazepines in panic disorder.
Rebound anxiety is common after even gradual discontinuation of alprazolam (Fyer et
al., 1987; Mellman and Uhde, 1986; Pecknold et al., 1988), with the latter group finding
that 35% of their patients experienced a withdrawal syndrome after 8 weeks of treatment
and that most of their patients relapsed subsequently. This drawback must be tempered
against the faster onset of action of alprazolam compared with the tricyclics (Rizley et
al., 1986) and MAOIs, its immediate anxiolysis, its lack of anxiogenic effects, and its
initial beneficial effects on sleep.
Recently alprazolam has been claimed to have antidepressant effects equal to those of
imipramine or amitriptyline (Feighner et al., 1983; Rickels et al., 1985). However, these
studies did not select patients with severe depressive illnesses, since most trial subjects
were managed as outpatients, and it would appear that alprazolam was only effective in
mild-moderate depression. Further studies are required with alprazolam and its deriva
tive, adinazolam (Hester et al., 1980), to properly assess the effectiveness of these tria-
zolobenzodiazepines as antidepressants.
Benzodiazepines are ineffective in phobic states, e.g., in the treatment of specific or
simple phobias (Sheehan et al., 1980; Zitrin et al., 1983). This argues against the possi
bility that such anxiety is caused by the release of putative, endogenous, anxiogenic, ben
zodiazepine receptor ligands (Nutt, 1983; File et al., 1982). There is some evidence that
benzodiazepines may enhance adjunctive treatment. For patients with agoraphobia, ben
zodiazepines may increase the ease of exposure in behavior therapy (Hafner and Marks,
1976; Johnston and Gath, 1973). Theoretically, it is also possible that the amnesic (Lis-
ter, 1985; Lister et al., 1988) and performance reducing (Brosan et al., 1986) eflfects of
these drugs might antagonize behavioral learning processes. More work is needed to
establish whether adjunctive benzodiazepines are of any use in facilitating behavior ther
apy (Marks, 1987).
4.3. BENZODIAZEPINES A N D ANXIETY PROVOCATION
The role of the benzodiazepine receptor in anxiety is well established from clinical and
basic work. Benzodiazepine agonists such as diazepam act at specific high affinity recep
tors in the brain with sedative, anxiolytic, and anticonvulsant actions. Recent discoveries
have included a unique class of compounds called inverse agonists, or contragonists
(Nutt, 1983), which may be of benzodiazepine or /J-carboline structure. These have
opposite effects to those of the classical benzodiazepines in that they are proconvulsant
(Cowen et al., 1981; Nutt et al., 1982) and anxiogenic (File et al., 1982; Ninan et al.,
1982; Petersen et al., 1982) in animals. Clinically, it was reported that, in two normal
controls (Dorow et al., 1983), the /S-carboline FG 7142 caused severe anxiety, similar to
a panic attack, which was aborted by the IV administration of a benzodiazepine in one
subject in whom the anxiety became unbearable. Although this was a chance observation
in only two subjects, it clearly suggests that one form of anxiety may be directly induced
by altering benzodiazepine receptor function.
A third class of drugs is the benzodiazepine antagonists such as flumazenil (Ro 15-
1788). These block the effects of both benzodiazepine agonists and inverse agonists, but
have few intrinsic effects in man (Higgitt et al., 1986), although they may be somewhat
anxiogenic in rats (File et al., 1982).
Although most of the data produced so far has been from animal work, they raise
interesting clinical questions. One is whether there are endogenous equivalents to FG
7142 that might be released in panic attacks. This could readily be resolved by treating
patients during panic attacks with benzodiazepine receptor antagonists such as fluma
zenil. Another relates to the well-recognized clinical phenomenon of sensitization,
whereby panic attack severity often increases after the first attack (Klein, 1981).
Repeated administration of FG 7142 in rats and mice gradually leads to sensitization to
its effects (kindling) with development of full seizures (Little et al., 1984). Early studies
suggested that this might be accompanied by increased anxiety-related behavior (Corda
et al., 1986; Jeevanjee et al., 1985). Although this does not occur in all anxiety models
(Taylor et al., 1988), it is interesting to speculate on whether similar processes might
underlie both the animal and human observations.
4.4. ANTIDEPRESSANT DRUGS FOR ANXIETY
In 1962, two influential reports were published on the effectiveness of antidepressants

in the treatment of anxiety states. Sargant and Dally (1962) described an open trial of
several types of MAOIs in patients with atypical depression and anxiety disorders. Klein
and Fink (1962) reported that Imipramine would reduce panic but not other (e.g., antic
ipatory) anxiety. A number of additional studies using MAOIs in anxious patients were
carried out in the 1970s; however, it was not until the early 1980s that similar trials were
reported with Imipramine. These confirmed the original observations that both sorts of
antidepressant could effectively treat anxiety disorders, especially those in which panic
attacks were the major symptom. Also of interest was the finding that both of these drugs
could prevent lactate-induced panics, an experimental model of panic anxiety (Liebowitz
et al., 1984; Rifkin et al., 1981). In the following section, these drug studies will be
described, and mention will be made of other drugs that have been tried in anxiety dis
orders. Since this is an area of extensive present interest that challenges a dichotomous
view of anxiety and depression, it will be covered in some detail.
12 D . J . NUTT AND P . GLUE
4.4.1. Tricyclic Antidepressants as Anxiolytic Agents

It is now well established that antidepressants have antipanic activity. Most of the stud
ies published to date have used Imipramine or clomipramine. There have been 11 studies
comparing Imipramine with placebo (Table 1.6), and apart from the studies by Marks et
al. (1983) and Evans et al. (1986), all have found Imipramine to be more effective than
placebo in treating aspects of anxiety, usually panic attacks (see below). In the negative
studies, reanalysis of the data of Marks et al. (1983) demonstrated some superiority of
imipramine over placebo on some measures (Raskin, 1983). The report of Evans et al.
(1986) is flawed by the short duration of the trial and the small number of subjects used.
In addition to the above placebo-controlled trials, there have been at least 12 open trials
of imipramine, all of which have found the drug effective (Table 1.6). Comparisons of
imipramine with other drugs are summarized in Table 1.6. Perhaps of most interest is
the comparison with phenelzine (see Section 4.4.3) and with the benzodiazepines. Imip
ramine was more effective than chlordiazepoxide in reducing panic attacks (McNair and
Kahn, 1981) and anxiety symptoms (Kahn et al., 1986), although alprazolam and imip
ramine were equally effective in treating a range of anxiety symptoms, and alprazolam
had a more rapid onset of action (Rizley et al., 1986).
The main effect of imipramine in these studies was in the reduction in frequency or
severity of panic attacks. In some cases, this might occur at relatively low doses of the
drug, sometimes as low as 10 mg/day (Modigh, 1987), although doses in the range of
150 mg/day have more usually been used. The relationship between plasma levels of
imipramine and clinical response was unclear, although it was likely that a threshold
level was required for imipramine to be effective (see review by Lydiard and Ballenger,
1987). There also appeared to be an effect on phobic avoidance behavior. If it was
reported at all, this effect was slower in onset than the antipanic effect, and it appeared
that higher plasma levels of imipramine were needed to reduce avoidance behavior than
those that had an antipanic effect alone (Mavissakalian and Perel, 1985; McNair and
Kahn, 1981). This may reflect a nonpharmacological effect, such as exposure-related
desensitization secondary to the pharmacological reduction in panic frequency and
severity. Imipramine was also effective in nonpanic anxiety disorders, i.e., generalized
anxiety disorder (see below). After discontinuation of imipramine, rates of relapse do not
exceed 30% (Cohen et al., 1984; Mavissakalian and Michelson, 1986a; Zitrin et al.,
1983).
Other antidepressants have been used in panic disorder. Clomipramine has been com
pared with placebo in four studies and has been found effective in treating phobic and
panic symptoms in all (Table 1.7). In double-blind comparison with fluvoxamine, both
have been equally efiective (den Boer et al., 1987), and a comparison with imipramine
is described above (Cassano et al., 1988b). Comparisons with diazepam (AUsopp et al.,
TABLE 1.6. Trials of Imipramine in Panic Patients
Placebo-controlled
double-blind trials Open trials Double-blind comparisons
Klein, 1964, 1967 Klein, 1964 Sheehan et al., 1980 (phenelzine)

Zitrin, et al., 1978, 1980 Muskin andFyer, 1981 McNair and Kahn, 1981
Sheehan etal., 1980 Lieberman et al., 1983 (chlordiazepoxide)
Marks et al., 1983 Mavissakalian et al., 1983 Ko et al., 1983 (Clonidine)
Mavissakalian and Perel, 1985 Sweeney et al., 1983 Munjack et al., 1985 (propranolol)
Telch etal., 1985 Bueno et al., 1984 Evans et al., 1986 (zimelidine)
Evans etal., 1986 Cameron etal., 1984 Kahn etal., 1986
Kahn etal., 1986 Garakani et al., 1984 (chlordiazepoxide)
Mavissakalian and Michelson, Liebowitz et al., 1984 Rizley et al., 1986 (alprazolam)
1986b Chamey et al., 1986 Cassano et al., 1988b
Aronson, 1987 (clomipramine)
Nutt, 1989
TABLE 1.7. Trials of Clomipramine in Panic Patients
Placebo-controlled
double-blind trials Open trials Double-blind comparisons
Escobar and Colgan, 1975 den Boer et al., 1987 (fluvoxamine)

Undbloom, 1976 Marshall and Micev, 1975 Cassano et al., 1988b (imipramine)
Karabanow, 1977 Waxman, 1975 AUsopp et al., 1984 (diazepam)
Kahn etal., 1987 Hoes etal., 1980 Kahn et al., 1987 (5-hydroxytryptophan)
Johnstonetal., 1988 Glogeret al., 1981
Pecknold et al., 1982
Grunhaus et al., 1984
1984) and 5-hydroxytryptophan (Kahn et al., 1987) have shown clomipramine to be

more effective (Table 1.7). There are at least seven open trials of clomipramine that all
show a substantial antipanic effect (Table 1.7). It appears that the dosage of clomipra-
mine required for a therapeutic effect is similar to the dose range used for imipramine,
and these drugs appear to be effective against the same range of symptoms (Cassano et
al., 1988b). Although anecdotal reports suggest that clomipramine may be more potent
than imipramine (e.g., Modigh, 1987), there is no good evidence to support this. The
only comparison of the two drugs is an open trial that was flawed by a dropout rate
approaching 60% (Cassano et al., 1988b); this showed a more rapid onset of antiphobic
and antipanic actions for clomipramine in the ñrst 2 weeks of treatment. On the present
evidence, imipramine would have to be regarded as the tricyclic of choice in treating
panic disorder.
Some of the variability in the response rates and outcomes reported above may be due
to problems in design of studies, most notably issues of patient selection and diagnosis.
For example, some studies included patients with depression (Sheehan et al., 1980),
whereas others excluded them (Marks et al., 1983), and often no distinction was made
between symptomatology that might range from minor degrees of phobic anxiety to
severe illness requiring hospitalization. Despite this heterogeneity, the effectiveness of
imipramine and clomipramine in panic disorders is still evident.
4.4.2. Other Antidepressants

One study has compared the selective serotonin reuptake inhibitor fluvoxamine with
the norepinephrine reuptake inhibitor maprotiline in panic disorder (den Boer and Wes-
tenberg, 1988). Although these authors found fluvoxamine to be more effective, mapro-
tiUne has been shown to be effective in open studies (Lydiard, 1987a; Sheehan, 1982).
An open trial using zimelidine reported it had some antiphobic effects (Koczkas et al.,
1981). Trazodone has been used in a small number of panic patients with little effect
(Sheehan, 1982). There are isolated reports suggesting that amitriptyline (Davidson et
al., 1981; Jobson et al., 1978), nortriptyline (Muskin and Fyer, 1981), and desipramine
(Lydiard, 1987b; Rifkin et al., 1981) may be of beneñt in panic disorder. A recent trial
using dothiepin is marred by the inadequate dosage used (Tyrer et al., 1988).
4.4.3. MAOIs
Much less work has been carried out on the use of MAOIs in panic patients compared
with the recent increase in interest in the use of imipramine, despite the ñrst reports of
their effectiveness being published in the same year (MAOIs: King, 1962; Sargant and
Dally, 1962; imipramine: Klein and Fink, 1962). Phenelzine has been the MAOI most
studied to date. There have been a number of open trials of MAOIs, and at least seven
double-blind trials in which phenelzine or iproniazid have been shown to be more effec-
tive than placebo, or as effective as imipramine (Table 1.8). MAOI doses required for
antianxiety effects are comparable with tiiose used in treating depressed patients, and in
TABLE 1.8. Trials of MAOIs in Panic Patients
Placebo-controlled Double-blind
double-blind trials Open trials comparisons
Lipsedge et al., 1973 (iproniazid) King, 1962 Sheehan et al., 1980

Solyom et al., 1973, 1981 Sargant, 1962 (several MAOIs) (imipramine)
Tyrer et al., 1973a, b Sargant and Dally, 1962 (several MAOIs) Mountjoy et al., 1977
Mountjoy et al., 1977 Kline, 1967 (several MAOIs) (diazepam)
Sheehan et al., 1980 Kelly et al., 1970 (several MAOIs)
Sheehan et al., 1984 Buiges and Vallejo, 1987
All studies used phenelzine unless otherwise indicated.

MAOIs = monoamine oxidase inhibitors.
a double-blind, controlled trial using 30-mg or 60-mg doses of phenelzine, it was shown
that the higher dose was more effective (Ravaris et al., 1976). MAOIs are effective against
a similar range of symptoms as the tricyclics, although the antipanic effects have mostly
been demonstrated in open studies (Buiges and Vallejo, 1987; Kelly et al., 1970). Only
one controlled comparison of phenelzine and imipramine has been reported to date
(Sheehan et al., 1980), which found that both drugs were more effective than placebo
and that phenelzine was significantly more effective than imipramine on global ratings
of symptoms. This interesting finding has yet to be replicated. Although withdrawal
symptoms have been reported after cessation of phenelzine, it is difficult to estimate how
commonly these occur from published data (Dilsaver, 1988; Lydiard and Ballenger,
1987).
In practice, because of the dietary restrictions that MAOIs entail, imipramine would
be the antidepressant of choice when starting treatment, although MAOIs may give bet
ter initial compliance due to their being less anxiogenic (see below). However, with the
recent development of reversible MAOIs, it is possible that the problem of interactions
between these drugs and tyramine-containing foods may be overcome (for review see
Nutt and Glue, 1989). Patients who do not respond to one class of drugs should be
offered a trial of the other.
4.4.4. Combination Pharmacotherapy for Panic Disorder

All the drugs mentioned above for the treatment of panic disorder have drawbacks,
either provoking anxiety at the initiation of treatment (imipramine); having a slow onset
of action (imipramine, phenelzine); or having problems with development of tolerance,
and withdrawal and symptom relapse on cessation of treatment (alprazolam or other
benzodiazepines). It is likely that a combination of alprazolam and either imipramine or
phenelzine might overcome these problems, with a short course (2-3 weeks) of a reduc
ing dose of benzodiazepine giving rapid initial anxiolysis and covering any symptoms
that might be caused by starting imipramine. By the end of this short course of benzo
diazepine, an effective therapeutic dose of imipramine or phenelzine would have been
reached. The benzodiazepine could then be withdrawn without problems. No clinical
trials have been carried out using this combination regime, although it is alluded to in
one of the earlier psychopharmacology texts (Sargant and Slater, 1972).
4.4.5. Antidepressants in Generalized Anxiety Disorder and Social Phobia

To date, little work has been done in treating the nonpanic anxiety disorders, i.e., gen
eralized anxiety disorder. A recent study by Kahn et al. (1986) found that imipramine
had a substantial antianxiety effect, but that chlordiazepoxide had only limited and brief
beneficial effects. This is in contrast to the report that clorazepate provided sustained and
effective anxiolysis over 6 months (Rickels et al., 1988). There are also a few studies
looking at treatment of social phobia. A number of groups have reported some benefit
using phenelzine (Liebowitz et al., 1986; reviewed by Liebowitz et al., 1985), and there
are some case reports of successful treatment with alprazolam but not with tricyclics
(Lydiard et al., 1988), imipramine (Benca et al., 1986), and clomipramine (AUsopp et
al., 1984). Further studies are needed to see if there are treatment differences between
any of these groups and agoraphobics.
4.4.6. Other Drugs

Propranolol, a beta-adrenoceptor antagonist, has been found to be eflFective in treating
some peripheral manifestations of autonomic arousal in anxiety (e.g., tremor, palpita-
tions), but is of little use in treating the psychic component of anxiety (Granville-Gross-
man and Turner, 1966). In support of this is the finding that lactate-induced panic
attacks are not blocked by pretreatment with propranolol (Gorman et al., 1983). Case
reports suggest it may have some use in acute panic episodes, but it appears to be of little
use in chronic anxiety and for avoidance symptoms (Heiser and DeFrancisco, 1976), and
panic patients often find the sense of lethargy and slowness unpleasant (Tyrer, 1976). In
a short trial against diazepam, it had no effect on panic frequency or severity (Noyes et
al., 1984), and indeed diazepam has been shown to be more effective than propranolol
in treating somatic anxiety symptoms (Tyrer and Lader, 1974). Comparison with imip-
ramine showed that both drugs had similar effects on panic frequency, although the dura-
tion of treatment in this study was probably too brief to allow valid comparison (Mun-
jack et al., 1985). Shehi and Patterson (1984) suggested using a mixture of alprazolam
and propranolol in panic disorder, titrating the dose of each with respect to the propor-
tion of psychic and somatic anxiety symptoms, and this may be a more effective com-
bination than either drug alone (Hallstrom et al., 1981).
Clonidine, an a2-adrenoceptor agonist that is an effective antihypertensive, has also
been used to treat anxiety disorders. There is evidence to suggest NE dysfunction in panic
disorder (see Section 3.1), with paroxysmal excessive neuronal activity. Clonidine
switches off locus coeruleus activity by an action at the presynaptic autoreceptor (Cedar-
baum and Aghajanian, 1976), thus reducing output of NE (see later). However, clinical
studies with Clonidine have found it to be of limited benefit in patients with panic dis-
order or GAD, having most effect on symptoms of psychic rather than somatic anxiety
(Hoehn-Saric et al., 1981). This is in contrast to the significant anxiolytic effects noted
with Clonidine in withdrawing alcoholics (Glue and Nutt, 1987). The differences in these
clinical responses may support ideas of different abnormalities in aj-adrenoceptor func-
tion in these disorders, as has been demonstrated by different patterns of abnormality in
responses to challenge testing with Clonidine (see review by Glue and Nutt, 1988; also
Chamey and Heninger, 1986; Nutt, 1989). One case report suggests Clonidine may also
be effective in social phobia (Goldstein, 1987) and in benzodiazepine withdrawal (Kes-
havan and Crammer, 1985). The hypotensive actions of Clonidine can be a problem
when this drug is used in the normotensive psychiatric population.
More recent agents used in treating anxiety disorders include a group of primarily
serotonergic drugs, especially buspirone (Goa and Ward, 1986). Although it was initially
thought this drug mi¿it have weak neuroleptic properties, it now seems more likely that
it acts upon serotonin receptors as a 5-HTIA agonist (Peroutka, 1985). Another drug with
a similar pharmacological profile is ipsapirone, which has been claimed to also have anx-
iolytic and euphoric properties (Traber and Glaser, 1987). Clinically, buspirone has been
shown to have an anxiolytic effect (Goldberg and Finnerty, 1979; Rickels et al., 1982;
Schuckit, 1984), but this is partly influenced by previous treatment experience, as
patients who have previously received benzodiazepines report it as being less effective
than those who are benzodiazepine-naive (Lader and Olajide, 1987). Although, in gen-
eral, it seems to be somewhat less effective and slower in onset than the benzodiazepines,
it is active and has the advantage of not seeming to produce dependence or withdrawal
reactions (Riblet et al., 1982, 1984), and the risk for cross-dependence would also seem
to be low. Whether problems of this nature will develop after long-term use is yet to be
16 D . J. NUTT AND P. GLUE
established. The anxiolysis of buspirone is not the same as that of the benzodiazepines
in that buspirone tends to cause insomnia rather than sedation (Seidel et al., 1985). Inter
estingly, a preUminary study of buspirone in a small number of patients with panic dis
order showed it to be ineffective (Rickels et al., 1988), and it may provoke panic (Chig
non and Lepine, 1989).
The third member of this group of 5-HTIA agonists is gepirone (McMillen and Mat-
tiace, 1983). This is being developed as an antidepressant, but would appear equally as
likely to be anxiolytic, since it has a receptor-binding profile very similar to that of ips-
apirone. A preliminary report has suggested that gepirone is more effective than placebo
in the treatment of patients with GAD, and withdrawal symptoms are not seen after
abrupt cessation (Harto et al., 1988).
4.5. TRICYCLIC ANTIDEPRESSANTS AND ANXIETY PROVOCATION
Despite the original report of Klein and Fink (1962) who started their patients on 75
mg of imipramine per day and did not describe any problems early in treatment, it is
now well established that imipramine increases anxiety, worsens somatic symptoms, and
precipitates panic attacks. These effects occur after the first dose(s) and commonly lead
to patients dropping out of treatment early. Although described by a number of authors
(Aronson, 1987; Cassano et al., 1988a; Evans et al., 1986; Sheehan et al., 1980; Zitrin et
al., 1978), in general, these problems are mentioned infrequently or are ignored in most
of the studies to date. A comparison of starting dose of imipramine and percentage of
patients dropping out early from treatment because of drug-related problems shows a
high degree of correlation (Fig. 1.4) and clearly shows that the higher the initial dose, the
higher the rate of early dropouts. As a practical point, it would seem prudent to start
patients on the smallest possible dose of imipramine (10 mg daily) and to increase this
slowly and in small increments, to minimize this problem. We feel the high dropout rate
probably gives important information about the nature of anxiety in these patients. The
acute effect of imipramine is to exacerbate the symptoms of panic and even to precipitate
full-blown attacks. The most likely cause for this is that increased central and peripheral
availability of NE leads to increased central arousal and anxiety and may also give rise
to peripheral side effects (e.g., tremor) that are inteφreted as panic symptoms. This
would support theories of noradrenergic dysfunction in anxiety disorders (Chamey and
Heninger, 1986; Chamey et al., 1984; Heninger et al., 1988; Nutt, 1989), and contrasts
50H
30-
20-
10-
04
25 50 75 100
Starting dose off imipramine (mg/day)
FIG. 1.4. Relationship of starting dose of imipramine and percentage of early dropouts from treat
ment. Data from Aronson, 1987; Cassano et al., 1988b; Evans et al., 1986; Garakani et al., 1984;
Kahn et al., 1986; Middleton, Η. C , personal communication, February, 1989; Nutt and Glue,
1990; Zitrin et al., 1983.
Clinical phannacology of anxiolytics and antidepressants 17
with depression, in which worsening of symptoms after starting treatment with tricychcs
is not seen. These symptoms are rarely seen when using small initial doses of antide-
pressant and occur more commonly when using higher starting doses (Fig. 1.4). As well
as the increased noradrenergic activity, the anticholinergic side effects such as tremor and
dizziness might also contribute to high early dropout rates. Whether the use of very high
(and thus sedating) initial doses of tricyclics might overcome this problem is an interest-
ing possibility.
Similarly, high levels of treatment dropouts are seen with use of other tricyclics, includ-
ing the serotonin reuptake blockers. These are also due to side effects, especially nausea
and dizziness. These drugs may have an additional central serotonin-mediated anxi-
ogenic component, which is suggested by the induction of panic symptoms in normal
volunteers in challenge tests using the 5-HT agonist m-chlorophenyl piperazine (m-CPP)
(Chamey et al., 1987; Kahn et al., 1988). MAOIs do not seem to have this problem of
early exacerbation of panic and anxiety symptoms. This would fit in with their different
mode of action from the tricyclics, in that MAO inhibition occurs more slowly than
blockage of monoamine reuptake and would not give rise to abmpt increases in central
and peripheral monoamines.
4.6. W H Y D O TRICYCLICS WORK IN BOTH ANXIETY A N D DEPRESSIVE DISORDERS?
A number of suggestions have been made to explain the effectiveness of tricyclics in

both disorders. One possibility is that panic disorder is a form of atypical depression (for
reviews, see Gmnhaus, 1988; Lesser, 1988). Although both illnesses may overlap, this is
an oversimplification of the complex relationship between the disorders. Distinction can
be made on the basis of symptoms of either disorder worsening or ameliorating indepen-
dently of the other (Breier et al., 1984, 1986). In addition, some biological markers can
distinguish depressive and panic disorders (e.g., reduced REM latency in depressives and
normal latency in panic disorder [Kupfer et al., 1978; Rosa et al., 1983; see review by
Roy-Byme et al., 1988]); exaggerated BP fall after Clonidine in panic patients and normal
responses in depression (Checkley et al., 1981; Nutt, 1986a). There are also case reports
of patients with both disorders, in whom the panic disorder was responsive to treatment
with imipramine, while the depressive illness was resistant (Numberg and Coccaro,
1982). It has been suggested that tricyclics only work by ameliorating depressive elements
in panic patients (e.g., Marks et al., 1983). Althou¿i these dmgs clearly do improve
depressive symptoms, there are also obvious beneficial effects on anxiety, even in those
panic patients without depressive symptoms (Sheehan et al., 1980). One other possibility
is that depression and panic disorder are different expressions of a common neurochem-
ical disorder that is tricyclic-responsive.
It is most likely that both illnesses are neurochemically distinct and that antidepres-
sants normalize both. These points are discussed further in the next section.
4.7. W H Y ARE ANTIDEPRESSANTS BOTH ANXIOGENIC AND ANXIOLYTIC?
The above discussion has emphasized the paradox that tricyclics are capable of pro-
ducing both anxiogenic and anxiolytic effects. However, the time course of these effects
is different, with anxiogenic effects being early and anxiolytic effects, late. These phenom-
ena could be accounted for by changes in noradrenergic function produced by antide-
pressant dmgs, which are illustrated in Fig. 1.5. Acute administration would produce
increased synaptic availability of NE due to reuptake blockade and would lead to
increased central arousal and peripheral symptoms that might then lead to a panic attack
(Fig. 1.6a). After chronic administration, stabilization of noradrenergic activity would
occur, with improvement in symptoms (Fig. 1.6b).
While it is by no means certain that the actions that are important in treating depres-
sion are the same as those that produce the response in anxiety disorders, it is possible
i
3 (
i
\ stabilization / ^ rebound
O. í
i
I \\
i f \
¡ í % .
/
t
l
t
í
a
Ul
Θ Θ Θ
1 2 3 5 Time (weeks)
— Antidepressant
Start Stop
Panic Disorder
ζ ϊ ) Uptake Blockade: increased synaptic availability. Normal Subjects
( 2 ) Presynaptic Stabilization: decreased firing rate and Depression
? postsynaptic receptor down-regulation.
( 3 ) Rebound: decrease in alpha-2 inhibition and decreased

receptor number leading to increased presynaptic activity
FIG. 1.5. Antidepressant mechanisms: effects on noradrenergic functions.
that mechanisms of synaptic stabilization are involved in both conditions (Siever and
Davis, 1985; Nutt and Cowen, 1987). The differences in initial response to tricyclics
between anxious and depressed patients may be related to baseline noradrenergic activ
ity, with tonic overactivity in anxiety and subfunction in depression. Acute reuptake
blockade would lead to greater synaptic levels in anxious patients than in depressed
patients and could account for the exacerbation of anxiety seen in the former, but not
Increase in
• Noradrenergic-
Activity
Tricyclics Panic
(first doses)
Anticholinergic. ^ Cognitive
(a) Side Effects Misinterpretation
Stabilization of Reduced _
Noradrenergic- " Responsiveness
Activity
Tricyclics Reduced Panic
(chronic dose)
Reduced
, Habituation to
. Anticholinergic-
Side Effects
(b) Side Effects
FIG. 1.6(a). Anxiety provocation after initial doses of tricyclic antidepressants, (b). Anxiety reduc
tion after chronic tricyclic antidepressants.
in the latter, group. Acute withdrawal of tricyclic antidepressants is associated with anx
iety and insomnia (Chamey and Redmond, 1983), indicating disruption of antidepres-
sant-induced synaptic stabilization, which would be independent of pretreatment, tonic
noradrenergic activity.
It is not clear whether tricyclic administration leads to the same time course of neu
rotransmitter changes in animals, and thus care must be taken in ascribing any acute
effects seen to antidepressant action. However, there is an extensive literature on the
effects of tricyclics on monoamine function (for reviews see Green and Nutt, 1983,
1985), which would support the pattern of changes described above. Of particular inter
est are the effects of tricyclics on changes in noradrenergic cell firing, where there is an
initial depression of cell firing that then normalizes (Svensson, 1980), and on «j-adren-
oceptors (Green et al., 1982; Svensson and Usdin, 1978).
4.8. D R U G TREATMENT, PSYCHOLOGICAL TREATMENT, OR A COMBINATION?
4.8.1. Panic Disorder

Almost all the studies in panic patients mentioned above included some sort of psy
chological therapy (e.g., supportive psychotherapy or exposure therapy) or patient edu
cation, either directly, as part of a treatment package, or indirectly, as part of normal
doctor-patient clinical interaction. In order to see if psychological treatment or educa
tion was necessary for improvement, Garakani et al. (1984) carried out an open trial of
imipramine in panic patients, while specifically avoiding giving any instructions or sup
port, and found drug treatment alone was effective.
There have been several reports comparing treatment with imipramine alone with
imipramine and various behavioral or psychological therapies. Perhaps the most com
prehensive is that by Telch et al. (1985), who compared patients given imipramine and
instructions to avoid exposure for the first 8 weeks of treatment, another group given
imipramine and exposure avoidance instructions for 4 weeks and then intensive expo
sure training, and a third group given placebo and the same behavioral instructions as
the second group. Only the group given combined treatment had a reduction in panic
frequency, although the other two groups had improvements in some outcome measures.
Mavissakalian et al. (1983) compared imipramine alone with imipramine and exposure
practice and found the combination treatment was more effective than imipramine
alone, especially in improving phobic symptoms. This group also compared behavioral
treatment (self-instruction and exposure) and placebo with behavioral treatment and
imipramine, and again found the combined treatment to be significantly better (Mavis
sakalian and Michelson, 1986b). Marks et al. (1983) reported no difference in outcome
between groups of patients treated with behavior therapy and either imipramine or pla
cebo, although subsequent reevaluation of his data has revealed that some outcome mea
sures were better for those patients treated with the imipramine and behavior therapy
combination (Raskin, 1983). From the studies by Zitrin et al. (1978, 1980) it appears
that imipramine affects panic attacks initially and avoidance behavior later, while expo
sure affects avoidance but not panic frequency. Overall the impression is that combined
imipramine and behavioral treatment is better than either alone.
Four studies have looked at therapeutic outcome after combining psychological treat
ment with MAOIs. Solyom et al. (1973) reported that patients treated with phenelzine
and brief psychotherapy responded better on some outcome ratings than did those given
placebo and psychotherapy. Two groups (Lipsedge et al., 1973; Solyom et al., 1981)
found that a combination of MAOIs (iproniazid and phenelzine respectively) and behav
ior therapy was more effective than MAOI given alone. Finally, patients treated with
phenelzine and behavior therapy improved more than those treated with behavior ther
apy alone (Sheehan et al., 1980). Although the effect in each study is not strong, it sug
gests that, as with tricyclic antidepressants, combined drug and psychological treatment
is better than either alone.
Long-term treatment outcome is controversial. A recent review suggests that improve

ments after behavioral treatment are enduring and stable (Marks and O'Sullivan, 1988;
also see Marks, 1971; Mathews et al., 1976). Although relapse rates are undoubtedly
higher after drug treatment, their assessment of these is unduly pessimistic. After stop
ping imipramine, relapse rates 1-5 years later do not exceed 30% (Cohen et al., 1984;
Lydiard and Ballenger, 1987; Mavissakalian and Michelson, 1986a), and relapse rates
after stopping phenelzine are of similar magnitude (Kelly et al., 1970; Tyrer and Stein
berg, 1975). Cessation of alprazolam may lead to higher rates of relapse, although this is
complicated by withdrawal symptoms in one third of this population (Pecknold et al.,
1988) and has not been found with all studies (Nagy et al., 1989). The only prospective
comparison of behavioral and drug treatments showed no difference in outcome (Mav
issakalian and Michelson, 1986b). Clearly, this is an area of some controversy as high
lighted by a recent dialogue (Klein, 1988; Lelliott and Marks, 1988). Further prospective
studies using parallel group design are required to clarify this area.
Effective treatment for the range of symptoms seen in phobic anxiety patients must
contain a number of elements, both pharmacological and psychological. As well as drug
treatment, starting at an appropriate dose, some sort of supportive psychotherapy and
patient education will be required, as are specific instructions for dealing with panic epi
sodes. Indeed, in the earliest reports on the utility of antidepressants in anxiety states,
mention is made of the need for concurrent psychotherapy (Klein and Fink, 1962; Sar
gant and Dally, 1962). The latter study found that after successful treatment of panic
symptoms, patients still required additional psychotherapy to help overcome phobic
avoidance behavior. Significant degrees of personality disorder may affect response to
treatment or its outcome (Reich, 1988; Sargant and Dally, 1962).
4.8.2. Depression
There is less evidence that psychotherapeutic interventions are as complementary to
drug treatment in depression as they appear to be in anxiety disorders. Certainly, in the
case of severe depression, there is little evidence to suggest that psychological approaches
have any benefit, although supportive psychotherapy may be useful during recovery. In
less severe illnesses, there has been considerable interest in the reports that cognitive ther
apy may be equally efficacious with antidepressants (Beck et al., 1985b). In this study,
the combination of both treatments was not more eflfective than either treatment alone.
A recent multicenter, placebo-controlled study, comparing the effectiveness of imipra
mine, and cognitive and psychodynamic therapies in out-patient depression, found sig
nificant improvement on all treatments (Elkin et al., 1989). Imipramine generally was
ranked as the best in terms of outcome, and placebo, the worst. Clearly, pharmacother
apy is cheaper and easier to administer than cognitive therapy, although more likely to
produce side effects. It would be of interest to see if cognitive therapy produces biochem
ical changes similar to those seen after antidepressants.
5. CONCLUSIONS
Anxiety and depression are separate conditions that often coexist, especially in mild
forms. Anxiety may progress to depression as a result of psychological and neurochem
ical processes. Antidepressants work in both conditions. The time course of the treatment
responses is similar for both illnesses, although acute anxiogenic effects of tricyclic anti
depressants are seen only in anxious patients. We suggest a commonality of action of
tricycUc antidepressants in both disorders, i.e., a stabilization of noradrenergic function.
Differing basal levels of activity or responsiveness may explain some of the symptom
differences and acute responses to tricychcs. Future animal studies should consider these
observations and direct further work toward comparing the acute and chronic effects of
antidepressants in behavioral tests of anxiety and depression.
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(1991) Psychopharmacology ofAnxiolytics and Antidepressants.
Peifumon Press, Inc. (New York), pp. 29-55
CHAPTER 2
INTERACTIONS OF ANXIOLYTIC
AND ANTIDEPRESSANT DRUGS WITH HORMONES
OF THE HYPOTHALAMIC-PITUITARY-ADRENAL AXIS
SANDRA E . FILE
Psychopharmacology Research Unit, UMDS Division of Pharmacology, University of London, Guy's
Hospital, London, United Kingdom
1. INTRODUCTION
There are several neurochemical and endocrinological responses to stressful stimuli.
These include changes in central and peripheral catecholamines, endoφhins, the release
of glucocorticoids, prolactin, gonadal and growth hormones, and changes in glucose util
ization and gastric secretion. By far the most extensively studied are the hypothalamic-
pituitary-adrenal (ΗΡΑ) and the sympathetic-adrenomeduUary systems. The interrela
tionship between these two systems has been reviewed by Dunn and Kramarcy (1984).
The importance of changes in central monoamines in response to stressful situations has
been reviewed by Anisman and Zacharko (1990; see also Chapter 3), with particular
emphasis on the conditions (e.g., controllability of shock, genetic factors) that might be
relevant to the subsequent development of depression.
In response to stressful stimuli, corticotropin releasing hormone (CRH) is released
from the hypothalamus and acts on the anterior pituitary to release corticotropin
(ACTH), which in turns acts on the adrenal cortex to stimulate the synthesis and release
of glucocorticoids. The release of CRH is controlled by several neurotransmitter path
ways, e.g., serotonergic and choHnergic (which are stimulatory), and norepinephrinergic
and gabaergic (which are inhibitory), and is also modulated by opioid peptides (Buck
ingham, 1985). Therefore, many classes of drug are likely to influence its release. In addi
tion to its endocrine function, CRH seems to function as a neurotransmitter or neuro
modulator in extrahypothalamic sites. Immunohistochemical methods have visualized
CRH cell bodies in the paraventricular, supraoptic, suprachiasmatic, preoptic, periven
tricular, premamillary, and arcuate nuclei (Sawchenko et al., 1984). Fibers with CRH
immunoreactivity emanate from the paraventricular, periventricular, and supraoptic
nuclei and course toward the bed nucleus of the stria terminalis as well as to the median
eminence. A second collection of CRH cell bodies has been found in the central and
medial nuclei of the amygdala (Moga and Gray, 1985) and there is a third group of
scattered cells in the anterior hypothalamus, continuing as a band of concentrated cells
through the preoptic area, diagonal band, and septal area, and projecting to the nucleus
accumbens and olfactory tubercle (Petrusz et al., 1984). A fourth group of ceUs has been
found in the parabrachial nucleus of the pons and the locus coeruleus (Sawchenko and
Swanson, 1985) with fibers projecting both rostrally to the lateral hypothalamus and pos
teriorly to the medulla. Using the Palkovits punch technique and a radioimmunoassay
for CRH, Chappell et al. (1986) have determined the CRH concentrations in 32 nuclei
of the rat brain. This confirmed high concentrations in hypothalamic and amygdala
nuclei and also in dorsal and median raphe nuclei. Using autoradiographic and biochem
ical methods, CRH binding sites have been found in both hypothalamic and extrahy
pothalamic sites (DeSouza et al., 1984, 1985). These putative CRH receptors are coupled
to adenylate cyclase in the anterior pituitary and some brain regions.
Stress-induced release of ACTH from the pituitary is predominantly controlled by
29
30 S . Ε . FILE
CRH, but can also be influenced by vasopressin, oxytocin, angiotensin II, epinephrine,
and norepinephrine. In the anterior pituitary, ACTH is derived from the precursor pro
opiomelanocortin, which also gives rise to i3-lipotropin and a small amount of i8-endor-
phin (Hipper and Mains, 1980). There has long been evidence of ACTH in the brain and
cerebrospinal fluid (CSF) (Allen et al., 1974), although its source was not clear. There is
now considerable evidence for the transport of ACTH from the pituitary to the hypo
thalamus, through the median eminence region, which has no blood-brain barrier (Oli
ver et al., 1977; Mezey and Palkovits, 1982). In addition, histological studies using anti-
sera to ACTH have found cell bodies in the arcuate nucleus of the hypothalamus, with
axons projecting to the amygdala, septum, and locus coeruleus (Watson et al., 1978;
Bloch et al., 1979; O'Donohue et al., 1979). It is not known whether ACTH is released
from these extrapituitary sites during stress, nor is the physiological role of central ACTH
clearly established (see Dunn and Kramarcy, 1984).
The endocrine role of ACTH is to stimulate the synthesis and release of glucocorticoids
from the adrenal cortex. Other fragments of pro-opiomelanocortin also stimulate ste
roidogenesis (Shanker and Sharma 1979; Pedersen et al., 1980), as do low doses of nal
oxone (Lymangrover et al., 1981), although high doses of naloxone inhibit glucocorticoid
release. The ΗΡΑ system has several feedback mechanisms, a major one being the glu
cocorticoid inhibition of ACTH release at the level of the pituitary (de Wied, 1964). This
inhibition is powerfully mimicked by the synthetic steroid, dexamethasone. In addition
there are feedback loops onto the adrenocortical cells themselves (Carsia and Malamed,
1979) and onto steroid receptors in the brain (McEwen, 1979), which may in turn inhibit
CRH release (Rees and Gray, 1983). The extensive distribution of glucocorticoid recep
tors in brain regions such as the hippocampus, septum, and amygdala (Martin et al.,
1977) suggest that glucocorticoids may play a role in normal or pathological emotional
responses through an action at these sites.
Stress has been proposed to play a potent role in the genesis or aggravation of many
psychiatric and physical diseases, for example, schizophrenia, obesity (Sachar, 1970;
Antelman and Chiodo, 1984), and cardiac disease (Surwit et al., 1982). However, the
role of stress and, in particular, the hormones of the hypothalamic-pituitary-adrenal
axis have been most consistently implicated in the development of anxiety and depres
sive disorders. This will be the focus of the present review. The glucocorticoids (corti
costerone for the rat; Cortisol, which is 17-hydroxycorticosterone, for humans) have
either been measured directly in CSF or plasma or the glucocorticoid metabolites (17-
ketosteroids) have been measured in urine. Section 2 will review the changes in ΗΡΑ
hormones in patients suffering from anxiety and depressive disorders and the changes
that occur when animals are exposed to test situations that are used preclinically to
model or study anxiety or depression. Section 3 will discuss the effects of exogenous
administration of ΗΡΑ hormones, both clinically and in animal tests. Section 4 will
review the effects of clinically effective anxiolytics and antidepressants on hormones of
the ΗΡΑ axis. Section 5 will discuss the stress-induced changes that take place on CNS
receptor systems thought to be relevant to the neuropharmacological action of anxiolytic
and antidepressant drugs.
2. CHANGES IN ΗΡΑ HORMONES IN ANXIETY AND DEPRESSION

2.1. CLINICAL STUDIES
Depression is a pathological mood state and, unlike anxiety, is not seen in normal
individuals. This makes it harder to study experimentally with either human or animal
subjects. Several proposals have been made for the subclassiñcation of depression (see
Nutt and Glue, 1989 and also Chapter 1), but in general a distinction is made between
severe/endogenous/psychotic depression and the milder, neurotic depression. Psychotic
depression is further subdivided into unipolar, in which patients suffer only a depressed
Anxiolytics, antidepressants, and the Η Ρ Α axis 31
mood, and bipolar, in which there are mood swings between depression and mania.
There is good evidence that stressful life events cluster before the onset of a depressive
illness, i.e., they are seen as one precipitating factor (Paykel, 1982; Brown et al., 1973).
However, there is also evidence for a similar cluster prior to the onset of panic disorder
(Roy-Byrne et al., 1986).
There has been considerable recent psychiatric interest in the classification of anxiety
disorders, and since 1980 the American Psychiatric Association Diagnostic and Statis
tical Manual of Mental Disorders (DSM-III) made a clear distinction between panic dis
order, phobias, and generalized anxiety. However, this separation has been challenged,
particularly in the UK, because of the overlap between generalized anxiety disorder and
panic, and depression and panic (see Nutt and Glue, 1989; see also Chapter 1). It has
been suggested that the disorders should be combined and viewed as a general neurotic
disorder in which patients may exhibit one or more of the symptoms of anxiety, depres
sion, and panic (Tyrer, 1986). In measurements of anxiety, a distinction is usually made
between trait and state anxiety. Trait anxiety refers to a habitual tendency of an individ
ual, and those suffering from anxiety disorders will score high on trait anxiety. State anx
iety in humans refers to a temporary emotional state and may be either a normal or a
pathological response to a stressful event. Many of the experimental human endocrino
logical studies and almost all the animal studies have focused on state anxiety.
2ΛΛ. Anxiety States

Increased plasma Cortisol has been found in normal human infants after crying, as
compared with their concentrations during quiet wakefulness, and the difference
between the two states was as great in infants 1 to 5 weeks old as in infants 5 to 15 weeks,
suggesting early maturation of the ΗΡΑ system. Increased Cortisol concentrations have
been found in a wide range of real-life situations such as driving (Hill et al., 1956), flying
(Colehour and Graybiel, 1964), psychological tests (Korchin and Herz, 1960), exami
nations (Semple et al., 1988), psychiatric interviews (Hetzel et al., 1955; Oken et al.,
1960), combat (Elmadjian, 1955), and thoracic surgery (Price et al., 1957). The increased
Cortisol during surgery was shown to be at maximal level, due to increased synthesis and
the lack of normal feedback inhibition resulting from raised CRH concentrations (Kehlet
and Binder, 1973). Computerised psychological tests have also been shown to elevate
plasma ACTH and i8-endoφhin levels (Mutti et al., 1989).
In one study (Zuckerman et al., 1966), the increase in 17-ketosteroids during percep
tual deprivation was paralleled by self-ratings of anxiety, and in another, there was a
relationship between scores of state anxiety and plasma Cortisol concentrations (Ablanalp
et al., 1977). It has become increasingly clear that it is an individual's perception of the
situation that determines his emotional and endocrinological reaction to it, and various
coping strategies allow the individual to avoid being stressed when exposed to threatening
situations (for review, see Rose, 1984). Anticipation of threatening events such as surgery
or examinations was found to produce as marked an elevation in Cortisol as the event
itself (Czeisler et al., 1976; Mason, 1968). It is interesting to speculate that it may be
cognitive differences in their assessment of risk that may distinguish subjects who score
high on trait anxiety.
While a variety of physical stressors can evoke a response from the ΗΡΑ axis. Mason
(1975) pointed out that they shared one important characteristic, that of exposure to a
novel environment or stimulus. Most workers have found that, on reexposure to the
same situation, there is rapid habituation of this endocrine response. Thus, first-time
parachutists showed a marked elevation of Cortisol on their first jump, but most showed
no elevation on subsequent jumps (Ursin et al., 1978). Several theories have highlighted
the importance of chronic or repeated stress on the etiology of psychiatric disorders, and
it is therefore surprising that there are relatively few studies on the endocrinological
response to chronic stress. Bourne et al. (1967) found no rise in 17-hydroxycorticoster-
32 S. Ε. FILE
oids (mainly reflecting Cortisol) in helicopter medics on the days they were involved with
medical evacuation, compared with the days they stayed at base. D u t t o n et al. (1978)
found no difference in the urinary corticoids of ñreñghters and paramedics between their
working days and t h e i r days off, Gullen et al. (1979) found no difference in the Cortisol
concentrations of experienced truck drivers working for 11 hours on 4 successive days,
compared with nondriving days. However, pieceworkers had higher urinary c o r t i c o i d s
when they were paid on a piece work basis, compared with a period of guaranteed salary,
even though they were used to the former type of pay (Timio and Gentili, 1976; Timio
et al., 1977). This suggests that there might be some situations to which the endocrine
responses of the ΗΡΑ axis do not adapt, and perhaps a more detailed analysis of these
would be fruitful for predicting the development of psychopathology.
2.1.2. Anxiety Disorders

Bliss (1956) reported that anxious patients had raised plasma Cortisol and urinary 17-
hydroxycorticosteroids over a 4-day period, and Bridges and Jones (1968) found that
plasma Cortisol concentrations correlated with neuroticism scores on the Eysenck per
sonality inventory. Plasma Cortisol concentrations also correlated with anxiety scores on
the Taylor-Zuckerman and Baron tests (Persky, 1962).
Initial studies of patients with panic disorder r e p o r t e d normal Cortisol suppression to
dexamethasone ( C u r t i s et al., 1982; Lieberman et al., 1983; Sheehan et al., 1983) and
normal plasma Cortisol levels (Liebowitz et al., 1985). However, one study did report
lack of Cortisol suppression to dexamethasone, (Avery et al., 1985) and elevated after
noon Cortisol concentrations have been found in panic patients (Nesse et al., 1984). Roy-
Byme et al., (1986) found that patients with panic disorder had blunted ACTH and Cor
tisol responses to a challenge with CRH. The patients with panic disorder differed from
depressed patients and those with anorexia nervosa who also showed blunted responses
to CRH, in that the panic patients also exhibited raised ACTH levels prior to CRH infu
sion. It was not clear whether this was an enhanced response to the anticipation of the
infusion or whether raised ACTH levels are sustained in panic patients. An intriguing
possibility is that panic patients may more readily release CRH in response to environ
mental events. The behavioral effects of exogenously administered CRH will be discussed
in a later section. CRH increases locus coeruleus activity (Valentino et al., 1983) and
plasma norepinephrine concentrations (Brown et al., 1981), both of which have been
implicated in panic attacks. Furthermore, centrally administered CRH induces kindling
(Weiss et al., 1986), i.e., an augmentation of seizure activity with repeated presentations,
and in many respects, the development of panic attacks resembles the phenomenon of
kindling (see Nutt and Glue, 1989 and Chapter 1). Blunted ACTH response to CRH
challenge has been reported in patients suffering from posttraumatic stress disorder, even
those without accompanying depression (Smith et al., 1989). These patients showed
raised basal Cortisol concentrations, and there was a positive correlation between these
and the severity of their psychiatric symptoms.
Curtis et al. (1976) studied the plasma Cortisol responses of phobic patients confronted
with their phobic object during the course of therapy ("flooding"). Despite large increases
in self-ratings of anxiety, the patients failed to show any increased Cortisol during the
flooding session. This could be inteφreted as evidence for habituation of the ΗΡΑ
response as a result of repeated exposure to the phobic object, whereas the emotional
response to the object remained. It would therefore seem that an abnormal response of
the ΗΡΑ axis is unlikely to be causal in the maintenance of phobia.
2.1.3. Depression
There has been considerable interest in the regulation of the hypothalamic-pituitary
hormones in depression, and for a review of the roles of thyroid, gonadal and somato-
trophic hormones, see Rupprecht and Lesch (1989) and Rupprecht et al. (1989). An early
Anxiolytics, antidepressants, and the ΗΡΑ axis 33
report of a correlation between raised Cortisol levels and suicide (Bunney and Fawcett,
1965) was not replicated (Fink and Carpenter, 1976). However, raised Cortisol concen
trations have been found in unipolar and bipolar depression (Sachar et al., 1973; Carroll,
1978; Christensen et al., 1989), and in these patients normalization of their Cortisol
response is correlated with clinical improvement (Carroll, 1972; Carroll et al., 1976; Gre
den et al., 1980). An abnormal diurnal rhythm has been reported in depressed patients
(Sachar et al., 1973). Linkowski et al. (1987) confirmed raised 24-hour Cortisol concen
trations in depressed patients and an abnormal diurnal rhythm, with the nocturnal nadir
advanced by almost 3 hours. However, the balance of studies would indicate that depres
sion is better correlated with high Cortisol concentrations than with abnormalities in the
circadian rhythm (Rubin, 1989). High plasma Cortisol concentrations in the afternoon
seem to be related as much to the severity as to the type of depression (Christensen et
al., 1983). It is considered that the raised Cortisol is not a response to the stressful nature
of their illness, but reflects a basic biological defect in a subgroup of endogenously
depressed patients. No diflerences in Cortisol c o n c e n t r a t i o n s were found between
depressed adolescents and their age-matched controls (de Villiers et al., 1989), suggesting
that hormonal differences might develop with the chronicity of illness. In addition to
hormone levels, it is possible that an abnormality of glucocorticoid receptors could con
tribute to the abnormalities seen in depression. Excess glucocorticoid secretion decreases
the number of glucocorticoid receptors in both the hippocampus (Tomello et al., 1982)
and the pituitary (Svec and Rudis, 1981). Depressed patients have a decreased number
of receptor sites on lymphocytes (Schlechte and Sherman, 1985; Gormley et al., 1985;
Whalley et al., 1986). Moreover, dexamethasone decreases the lymphocyte cytoplasmic
receptor number only in Cortisol suppressors, with nonsuppressors showing no change
(Gormley et al., 1985).
Carroll et al. (1968) reported that patients suffering from endogenous depression
showed a reduced suppression of their plasma Cortisol levels following injection of dexa
methasone. The dexamethasone suppression test was claimed to be a highly specific diag
nostic indicator of endogenous depression and has been the subject of extensive inves
tigation. Unfortunately, the diagnostic specificity of the test was not confirmed to be
higher than 76%, with the additional problem of a sensitivity of 40% (for review, see
Berger and Klein, 1984). Several psychiatric groups have been reported to have abnormal
Cortisol suppression to dexamethasone, e.g., schizophrenics (Myers, 1984; Munro et al.,
1984), alcoholics (Kroll et al., 1983), and dements (Spar and Gemer, 1982). In addition,
several nonpsychiatric factors can influence dexamethasone suppression such as weight
loss or acute hospitalization (Berger and Klein, 1984). Indeed, it may be a response to
stress (Domisse et al., 1985). Investigations of the influence of severity of depression on
dexamethasone suppression have produced conflicting results. Stokes et al. (1975), Klein
et al. (1984), and Sangal et al. (1984) found positive correlations between postdexame-
thasone Cortisol levels and measures of depression, but others did not confirm these find
ings (Brown and Shuey, 1979; Saleem, 1984). In contrast to a reduced suppression of
Cortisol in response to a dexamethasone challenge, an exaggerated Cortisol response has
been reported to ACTH (Amsterdam et al., 1983).
Ettigi et al. (1983) found that an abnormal dexamethasone suppression in unipolar
depressed patients was associated with a better response to desmethylimipramine, and
Brown et al. (1980) found nonsuppressors responded better to desmethylimipramine or
imipramine than to amitriptyline or clomipramine. Ettigi et al. (1988) found nonsup
pressors responded better to imipramine than to alprazolam, whereas depressed patients
who had low Cortisol after dexamethasone improved better when treated with alprazo
lam. Thus the dexamethasone suppression test may reveal differences in types of depres
sion that are predictive of the type of drug treatment that is most appropriate, even
though it has failed to correlate with clinically defined subtypes of depression. At present,
it is at best a marker for the clinical state of some patients. There is some evidence that
Cortisol nonsuppression after a dexamethasone challenge might resuh from a reduced
34 S. Ε. FILE
bioavailability of dexamethasone (Berger et al., 1984; Arana et al., 1985; Morris et al.,
1986). This seemed to be an important factor only when serum dexamethasone concen
trations fell below a threshold level; the major factor determining Cortisol nonsuppres-
sion was the pretest Cortisol concentrations (Poland et al., 1987).
Several research groups have reported increased ACTH levels in patients who failed to
suppress their Cortisol (Reus et al., 1982; Nasr et al., 1983; Holsboer et al., 1984), but
others have failed to confirm this (Yerevanian et al., 1983). Some studies have found
elevated ACTH levels in depressed patients (Pfohl et al., 1985; Roy et al., 1986), but
again this has not always been confirmed (Fang et al., 1981; Linkowski et al., 1985).
Widerlow et al. (1986) found raised concentrations of CRH in the plasma of 10 drug-
free, depressed patients, compared with normal controls; however, this was not sup
ported by the study of Charlton et al. (1986). In two studies, Nemeroff and colleagues
(Nemeroff et al., 1984; Banki et al., 1987) found that CSF CRH concentrations were
significantly higher in depressed patients than in normal controls or in nondepressed
psychiatric patients (those suffering from schizophrenia or dementia). Interestingly, there
was a bimodal distribution of CRH values in the depressed patients, indicating that there
might be a subgroup with raised circulating CRH. Arato et al. (1986) found elevated CSF
CRH concentrations in postmortem depressed patients. A significant decrease in CRH
receptor density was found in the brains of suicide victims (Nemeroff, 1988).
When challenged with intravenous administration of CRH, patients with major
depression and elevated basal Cortisol levels showed a blunted ACTH response, yet a
nearly normal Cortisol response, when compared with normal controls (Gold et al., 1984;
Gold and Chrousos, 1985; Holsboer et al., 1984; Lesch et al., 1987, 1988; Muller et al.,
1986, 1988;Katholetal., 1989).
This blunted ACTH response could be the result of a negative feedback response to
high circulating levels of Cortisol and desensitization of CRH receptors on the cortico-
trophs of the pituitary. A normal Cortisol response, in the presence of a blunted ACTH
response, suggests the possibility of up-regulation of ACTH receptors on the adrenal cor
tex. Roy et al. (1987) have reported a significant correlation between scores in the dexa
methasone suppression test and the patients' plasma CRH concentrations. Thus, the
hypercortisolemia seen in the subgroup of depressive patients could be due to chronic
CRH hypersecretion, or it could result from an abnormality of glucocorticoid receptors.
However, as was seen for the other ΗΡΑ hormonal responses, a blunted ACTH response
to CRH is not unique to depressed patients. It has also been reported in patients with
anorexia nervosa (Hotta et al., 1986) and panic disorder (Roy-Byme et al., 1986).
2.2. ANIMAL STUDIES
Cognitive factors in potentially stressful situations are also important in determining

the ΗΡΑ response of animals. Thus, it will be seen that many animal tests of anxiety
employ novelty, and in tests of depression, one cmcial factor is the inability of the animal
to make a coping response.
2.2.1. Animal Tests of Anxiety

In some animal tests of anxiety, a particular response is punished (e.g., the Vogel pun
ished drinking test) or a particular stimulus (e.g., a tone) is specifically paired with a
shock (as in the conditioned emotional response test). It could therefore be argued that
these tests are generating a state more akin to fear than to anxiety. This question is dif
ficult to settle, but it is generally considered that the source of danger is more readily
identified in fear. For a recent review of this type of test of anxiety, see Pollard and How
ard (1990; see also Chapter 6).
In the Vogel punished drinking test, water-deprived rats are given a period of unpun
ished drinking and then receive a period in which licks are punished every 3 minutes or
20 licks. Rats exposed to this conflict test for 5 minutes showed elevations in plasma
corticosterone concentrations (Vogel et al., 1980). In a recent experiment, we gave 22-
hour water-deprived rats a 1-minute period of unpunished licking, followed by a 5-min-
ute period in which every 20th lick was punished by a foot shock of either 0.18 or 0.4
mA. In both cases, the groups tested under these conditions had plasma corticosterone
concentrations significantly higher than their home-cage controls, and the increase was
greater for the group that received the higher shock level (File et al., 1988 and see Fig.
2.1). The main factor in this test was not novelty either of the test apparatus or of the
deprivation state, since the rats had previously been deprived for 22 hours and then
allowed 15 minutes unpunished drinlcing in the same test box. However, exposure to
foot shock was not only a physical stressor, albeit very mild, it was also novel. In addi
tion, both the deprivation state and the conflict engendered by the punishment could
have contributed to the raised corticosterone concentrations. In a conditioned suppres
sion test in which a tone is paired with a shock, both monkeys and rats show elevated
plasma corticoid concentrations (Mason et al., 1957; Davis et al., 1978). Interestingly,
however, although this elevation was a corollary of behavioral suppression, it was not
necessary for the suppression to occur. Rats implanted with crystalline Cortisol (which
inhibits normal ΗΡΑ function) still acquired a conditioned suppression response (Davis
etal., 1978).
The elevated plus-maze is a test of anxiety in which rodents are exposed to the novel
stress of open, elevated arms (Pellow et al., 1985; Lister, 1987). In this test, animals are
given a free choice of two open, elevated arms or two enclosed arms. Undrugged rats
make more entries into, and spend more time in, enclosed arms than the open ones. In
this test, it is the elevation or openness of the arms, rather than the level of illumination,
that is the crucial factor (Pellow et al., 1985; Baldwin and File, 1986). Rats trapped on
the open arms for 5 minutes had significantly higher plasma corticosterone concentra
tions than rats trapped in the enclosed arms (Pellow et al., 1985). Rats tested for 5 min-
50
40
30
20
10
SOCIAL PLUS-MAZE VOGEL VOGEL

INTERACTION Ο.ΙβηιΑ 0.4IA
FIG. 2.1. Increase from home-cage levels in rat plasma corticosterone concentrations as a result
of exposure to animal tests of anxiety. S(X:iAL INTERACTION: Pairs of male rats were placed
into a low-lit, unfamiliar arena for 7.5 minutes. PLUS-MAZE: Rats were placed singly on the
maze and allowed free access to all arms for 5 minutes. VOGEL: For this punished-drinking test,
rats were water-deprived for 22 hours and then placed into the apparatus for 6 minutes. After a
1 minute unpunished period they received one 0.18- or 0.4-mA foot shock per 20 licks of the
water bottle. In all cases, after exposure to the test, rats were replaced into their home cages until
they were anesthetized with ether (this took place 30 minutes after being placed in the test). Blood
was removed by cardiac puncture and plasma corticosterone concentrations measured by RIA.
Values are mean ( ± SEM) plasma corticosterone concentrations (Mg/dL). Different from home
cage group: *P <0.05; • • P < 0 . 0 1 .
36 S. Ε. FILE
Utes in the elevated plus-maze had significantly higher plasma corticosterone concentra
tions than rats left in their home-cages (File et al., 1988). These rats were tested at the
same time as the rats tested in the Vogel test and came fi-om the same batch of rats. It is
therefore possible to make a comparison between the stress of the two tests. It can be
seen from Fig. 2.1 that the Vogel punished drinking test is the more stressful, as defined
by the elevation in the plasma corticosterone concentrations.
Rats exposed to a novel environment show elevations in plasma corticosterone, and
the elevation is greater the greater the difference between the novel environment and the
rats' housing conditions (Flaherty et al., 1986). In the social interaction test of anxiety,
pairs of male rats are placed in a wooden test arena that is either familiar or unfamiliar
to them and is lit by low or by high light. The time spent in active social interaction is
highest when the test arena is familiar to the rats and is lit by low light; social interaction
decreases when the rats are tested in bright light or in an unfamiliar arena. These
decreases are prevented by anxiolytic drugs, whereas anxiogenic drugs decrease the time
spent in social interaction (File, 1980, 1987, 1988). In this test, the novelty of the arena
is manipulated, but the test also confronts the rat with a novel partner. The interaction
with a novel partner is increased by housing the rats singly for 5 days prior to the social
interaction test. It should first be considered whether social isolation for this period of
time is stressful for the rat. The evidence is that it is not. Indeed, the plasma corticoste
rone concentrations of pair- or group-housed rats were significantly higher than for those
housed singly (File and Peet, 1980). There is also evidence from Benton and Brain (1981)
that even extended periods of isolation housing do not increase corticosterone concen
trations in mice. While Armario et al. (1983) found that social interaction was higher
when the rat had an unfamiliar partner, suφrisingly, perhaps, they found that plasma
corticosterone was higher when the partner was familiar. This suggests that social inter
action per se does not elevate corticosterone concentrations. File and Peet (1980) found
that the plasma corticosterone concentrations of rats exposed to a novel environment
were higher when they were tested alone than when they were tested with an unfamiliar
partner. This suggests that the presence of another rat reduces the stressful impact of an
unfamiliar environment.
Figure 2.1 shows the plasma corticosterone concentrations of pairs of rats given a 7.5-
minute trial in a low-light, unfamiliar, social-interaction test arena, tested at the same
time as the rats in the plus-maze and Vogel tests. It can be seen that this test condition
elicited a significant elevation in corticosterone, compared with the concentration in rats
that remained in their home cages. In a previous study, the effects of different levels of
illuminance and of an unfamiliar versus familiar test arena were examined (File and
Peet, 1980). The highest plasma corticosterone concentration was in the high-light, unfa
miliar condition, and the next highest was in the low-Ught, unfamihar condition. The
plasma corticosterone concentrations were significantly lower in both the familiar test
arenas, and under these test conditions, the level of illuminance did not affect the corti
costerone concentrations. The rats that were famihar with the arena had been placed
singly in the arena for two previous 10-minute trials, under the appropriate light level.
It therefore seems that the corticosterone response to bright Ught had habituated. There
is good evidence that the corticosterone response habituates on repeated exposure to test
arenas (File, 1982a). The fact that the corticosterone concentrations were lower in the
low-light, famihar group than in the low-Ught, unfamihar group is further evidence for
this habituation.
Another animal test of anxiety uses the social behavior of an isolated mouse when
confronted with an intruder into its home cage (Krsiak et al., 1984). Drug treatment is
given only to the isolated mouse. In this situation, the social interactions are agonistic
and comprised of offensive, defensive, and submissive components. Anxiolytic drugs, in
particular, reduced submissive behavior, reflected in attempts to escape, and defensive
behavior. Submissive behavior in rats intruding into a well-established colony has been
shown to be reduced by chlordiazepoxide (File, 1982b). While androgens play a major
role in offensive aggression, hormones of the ΗΡΑ axis have been implicated in submis
sive behavior (Leshner, 1981). The effects of exogenously administered ACTH and cor
ticosterone on submissive behavior will be discussed in the following section. In agonistic
encounters, it is the animal that is intruding into another's territory that is usually sub
missive, and in rats intruding into a foreign territory, there is an increased plasma cor
ticosterone concentration in the intruder, but not in the resident rats (File, 1984).
In a negative contrast test, rats that have been given a 32% sucrose solution to drink
are then given a 4% solution. These rats consume less of the 4% solution than do rats
that experienced only 4% sucrose, and anxiolytic drugs reduce this negative contrast
effect (Flaherty, 1990; see also Chapter 9). This negative contrast cannot be explained
solely as a response to a novel solution, since the addition of novel stimulus during the
postshift period reduced, rather than increased, the effect. The effects of anxiolytic drugs
is seen not on the ñrst postshift day, but on the second, which also argues against an
explanation solely in terms of novelty. Interestingly, plasma corticosterone concentra-
tions were elevated on the second, but not on the first, postshift day (Flaherty et al.,
1985). Furthermore, there was a correlation between the corticosterone concentrations
and the extent of response suppression.
The administration of drugs known to cause anxiety in man has also been used in
animals to study the neural basis of anxiety. Indeed, the administration of pentylenetet-
razol, the benzodiazepine inverse agonist FG 7142, yohimbine, and caffeine, all of which
provoke anxiety in man, also result in anxiogenic profiles in animal tests (for review, see
File, 1987). There is considerable evidence that these drugs also increase plasma corti-
costerone concentrations. Inverse agonists acting at the benzodiazepine receptor increase
corticosterone concentrations in unstressed and stressed rats (Pellow and File, 1985;
Eisenberg and Johnson, 1989) and raise Cortisol concentrations in primates (Insel et al.,
1984). The «z-receptor antagonist, yohimbine, on both acute and chronic administration
increases basal corticosterone concentrations in the rat (Johnston et al., 1988). The effect
of yohimbine on corticosterone concentrations of stressed rats has not yet been studied.
Caffeine further enhanced the plasma corticosterone concentrations of socially stressed
mice (Henry and Stephens, 1980).
Thus, anxiogenic drugs further increased plasma corticosterone concentrations in
stressed animals and they also increased levels in unstressed rats. The elevation in plasma
corticosterone could be the result of the novelty of the drug state, although not all drugs
have this effect, at doses that are, nonetheless, sufficient to produce a discriminable inter-
nal state. Alternatively, it could be secondary to increased anxiety, or the effects on cor-
ticosterone could be the direct result of the drugs' actions on, for example, CRH release.
The drugs that act at the gamma-amino butyric acid (GABA)-benzodiazepine receptor
complex might increase plasma corticosterone concentration by inhibiting GABA's
inhibitory action on CRH release. The effects of yohimbine would be consistent with an
action at a postsynaptic α-adrenoceptor to reduce the norepinephrinergic inhibition of
CRH release.
2.2.2. Tests of Mixed Anxiety and Depression

Just as in clinical states it is difficult to clearly separate anxiety and depression, there
are some animal tests in which both features may be important. As was discussed above,
when the benzodiazepine inverse agonist FG 7142 is given to normal volunteers or to
animals, there are behavioral indications of anxiety as well as an arousal of the ΗΡΑ
system. Another of the effects of FG 7142 suggests that anxiety might play an important
role in the development of "learned helplessness," which has been proposed as an animal
model of depression and reviewed by Willner (1985) and discussed fully by Anisman and
Zacharko (1990; see also Chapter 3). Learned helplessness refers to a pattern of behav
ioral deficits seen after an animal is exposed to inescapable shock. Administration of FG
7142 to a restrained rat had the same long-term consequences as restraining the rat and
38 S. Ε. FILE
giving unavoidable shock (Drugan et al, 1985). A dexamethasone challenge was given
to animals exposed to inescapable shock; those that displayed ''helpless behavior'' in
response to the shock had higher corticosterone responses (i.e., less suppression) than
those that did not develop "helpless behavior" or nonshocked controls (Greenberg et al.,
1989).
There is further evidence for a role of anxiety in the development of behavioral deficits
following inescapable shock. In the forced-swim test, prior shock has biphasic effects on
activity. Soon after the shock, mice show increased swimming compared with non-
shocked controls, and this was the effect, regardless of whether the shock was avoidable
or not. However, when the mice are tested 24 hours after the shock, there was increased
immobility in the swim test, but only in mice that were exposed to unavoidable shock
(Prince and Anisman, 1984). The motor excitation phase may be related to anxiety, in
that diazepam eliminates this response (Prince et al., 1986). If diazepam is administered
prior to the inescapable shock, then the state of learned helplessness does not develop.
However, when administered 24 hours after the shock it is antidepressant drugs, rather
than anxiolytic drugs, that change the immobility response. Once again, it seems as if
anxiety may be important in the acute response to the stress, but that the longer term
response may be more akin to a depressive state.
The impetus for mother-infant separation studies in animals came from Spitz's study
(1946) of anaclitic depression in separated human infants and Bowlby's formulation
(1969, 1973) of the mother-infant attachment process. Infant squirrel monkeys and their
mothers show elevated plasma Cortisol concentrations during separation, but this
response was of the same magnitude whether the infant was placed in a small cage adja
cent to the mother or whether it was removed to another building (Vogt and Levine,
1980). An important question is whether this was a response to maternal separation or
to being placed in a novel environment. Surrogate-reared squirrel monkeys removed
from their home environment showed an equally large elevation of plasma Cortisol as
did infants separated from their mother (Mendoza et al., 1978), suggesting that exposure
to a novel environment was a major factor. However, while mother-reared infants
showed raised plasma Cortisol to the removal of their mothers from the home cage, sur
rogate-reared infants did not respond to the removal of the surrogate (Hennessy et al.,
1979). Again, this could partially reflect the degree of environmental change in the two
situations, but it could also reflect a specific response to the loss of the mother. However,
these studies provide little more than further examples of an ΗΡΑ response to novelty
and, as such, really belong in the section of anxiety states. Most infants of most species
respond with agitation and protest to separation, but only some infants of some species
separated in certain environments become depressed under the circumstances (Suomi et
al., 1971, 1981). So far, there is little evidence that prolonged separation leads to changes
in the ΗΡΑ system. Kalin et al. (1987) found rises in plasma ACTH and Cortisol in infant
rhesus monkeys 1 hour after they were separated from their mothers and placed in
another room. Gunnar et al. (1981) found large rises in plasma Cortisol of separated
infant rhesus monkeys 30 minutes and 3 hours after removal of their monkeys, but by
24 hours and thereafter they returned to basal levels. Thus, it seems that one must look
elsewhere than the ΗΡΑ axis for long-term changes during separation.
Exposure of animals to repeated or chronic stress has been used as an animal model
of depression (e.g., Roth and Katz, 1979). Often the behaviors elicited by stressors in
animals bear Uttle resemblance to the symptoms of human depression (Willner, 1985),
and in some cases, treatments that are ineffective against clinical depression are able to
reduce the effects of stress in animals (see Zacharko and Anisman, 1989). Most of the
evidence suggests that on repeated exposure to stress the changes in the ΗΡΑ axis habit
uate (Armario et al., 1984; Alario et al., 1987; Keim and Sigg, 1977; File, 1982a; Mason,
1972). Armario et al. (1986) showed that the habituation is specific to the particular
stressor and that there is an undiminished response to a novel stressor. However, there
are some reports of failure to habituate (Hennessy et al., 1977; Kant et al., 1983; Kant
et al., 1989) or even of an enhanced response with repeated restraint stress (Baron and
Brush, 1979). The nature, intensity, duration, number, and frequency of repetitions of
the stressor are all factors that might be relevant to whether or not the corticosterone
response habituates. These have not been systematically studied, but the evidence is that
stress intensity might be important. Natelson et al. (1988) reported that habituation was
slower for more intense stressors and that another important factor was the rat's original
response, those with low responses showing no subsequent habituation. In rats repeatedly
exposed to restraint stress, the basal corticosterone concentrations rose (Pitman et al.,
1988), thus making the changes in stress-induced corticosterone difficult to interpret.
However, this increase in basal corticoid concentrations is similar to the hypercortisole-
mia seen in depressed patients. In another study, repeated exposure to the stress of
restraint plus light and noise led to a small increase in basal corticosterone and a sev
enfold increase in response to stress over 3 weeks (Vogel and Jensh, 1988). These authors
also commented on the marked individual differences in response to stress. Thus, it
seems that, at least for some individuals, repeated exposure to restraint stress (whether
because of its nature or severity) does lead to increasing levels of corticosterone, both in
response to the stress and in basal levels. However, it is only in these extreme conditions
that a rise in ΗΡΑ responses is seen, and other systems, for example central catechol
amines (Anisman and Zacharko, 1990) may show changes more relevant to the devel
opment of psychiatric pathology on exposure to chronic stress.
Olfactory bulbectomy in the rat has been proposed as an animal model of depression
(van Riezen and Leonard, 1990; see also Chapter 10), and initially it was reported that
following bulbectomy, rats had elevated plasma corticosterone concentrations that were
reduced by chronic antidepressant treatment (Caimcross et al., 1977, 1979). While Cat-
tarelli and Damael (1986) confirmed the corticosterone elevation postbulbectomy,
O'Connor et al. (1985) and Broekkamp et al. (1986) failed to find any changes. It seems
that the raised corticosterone may be the result of bulbectomy plus lack of handling and
that if rats are well-handled after surgery, this rise is not seen (see van Riezen and Leon
ard, 1990; see also Chapter 10).
3. EFFECTS OF EXOGENOUS ADMINISTRATION OF HORMONES OF THE

ΗΡΑ AXIS IN ANXIETY AND DEPRESSION
3.1. CLINICAL STUDIES
The studies of hormones exogenously administered to human subjects seem to yield

results apparently at variance with the studies of endogenous levels of these hormones in
anxiety and depression. On the whole, the pattern seen for endogenous hormonal levels
was one of raised concentrations correlating with anxiety and/or depression. However,
when these hormones are administered to human subjects the general pattern seen is of
reductions in anxiety or depression. When CRH was administered to depressed patients,
there was a significant improvement in mood and well-being 2 hours after an intravenous
infusion (Laux et al., 1988). Administration of ACTH analogs to normal volunteers was
found to decrease anxiety (Sandman et al., 1975; Miller et al., 1976) and to increase a
sense of well-being (Dombush et al., 1981). Following administration of a synthetic ana
log of ACTH, decreases in depression were found in patients with senile organic brain
syndrome (Branconnier et al., 1979), and decreases in both anxiety and depression were
found in normal aged subjects (Ferris et al., 1981).
It is possible that, because of the relatively long time between administration of ACTH
or CRH and the assessment of mood, the anxiolytic or mood-elevating effects were actu
ally the result of the increased Cortisol production in response to the hormone challenge.
Glucocorticoids in relatively high doses are used as anti-inflammatory agents and initial
reactions of euphoria have been noted. However, with prolonged treatment, there is an
increased risk of depression. Thus, while it seems that the acute effects of glucocorticoids
40 S. Ε. FILE
might be anxiolytic and mood-elevating, some other change occurs in response to pro
longed high levels and this, in turn, leads to increased depression.
3.2. ANIMAL STUDIES
Intraventricular administration of CRH has anxiogenic effects in the Geller-Seifter

conflict test (Thatcher-Britton et al., 1983), on food consumption in a novel environment
(Britton, 1984), in the social interaction test (Dunn and File, 1987), and in the elevated
plus-maze (File et al., 1988). The effects of CRH in these tests were antagonized by
administration of chlordiazepoxide. However, this reversal could be functional (i.e., an
anxiolytic reversing the effects of an anxiogenic agent) rather than pharmacological,
because the benzodiazepine antagonist, flumazenil, did not reverse the effects of CRH in
the plus-maze or the social interaction test (File et al., 1988), although it did reverse the
effects in the Geller-Seifter conflict test (Thatcher-Britton et al., 1983). Central admin
istration of CRH increased the isolation-induced ultrasonic calls of rat pups, thought to
reflect distress (Insel and Harbaugh, 1989) and the administration of a CRH antagonist
decreased these calls. In infant monkeys briefly separated from their mothers, intracer-
ebroventricular administration of CRH increased the separation-induced inactivity, but
not the vocalizations; it also raised the ACTH and corticosterone concentrations (Kalin
et al., 1989). In neither the rat pup nor the monkey study did peripherally administered
CRH have any effects.
Intraperitoneal administration of ACTH had anxiogenic effects in the social interac
tion test and these effects were antagonized by chlordiazepoxide (File and Vellucci,
1978). This behavioral effect was not secondary to the release of corticosterone since the
shorter fragment, ACTH4_,o, which has no steroid-releasing properties, also had an anx
iogenic effect after intraperitoneal, intracerebroventricular, and intraseptal administra
tion (File, 1979; File and Clarke, 1980; Clarke and File, 1983). Further evidence that the
anxiogenic effects of CRH and ACTH are not due to the release of corticosterone comes
from the effects of corticosterone itself While the effects of this hormone are complex,
depending on dose and time of testing, the changes were in the opposite direction (i.e.,
it tended to have an anxiolytic effect in the social interaction test [File et al., 1979]).
If the effects of corticosterone are anxiolytic and mood-elevating, this could explain
the apparent discrepancy between the effects of CRH and the ACTH in animal tests and
those seen when these hormones are given to man. Unless the time interval between
administration of the hormone and the assessment of mood were short, sufficient Cortisol
would have been released to have made a contribution to, if not dominated, the final
effect. For example, in the study by Laux et al. (1988), the anxiolytic and mood-elevating
effects of CRH were seen 2 hours after intravenous administration, by which time CRH
in plasma was close to zero.
Distinctions between the effects of ACTH and corticosterone can also be seen in
aggressive behavior. Chronic treatment with ACTH decreases offensive behavior and this
has been shown to be a direct effect of ACTH (Leshner et al., 1973). Leshner et al. (1980)
showed that while only a very high dose of corticosterone increased submissiveness if
given before the first test, the combination of experience of defeat and even a low dose
of corticosterone greatly enhanced submissiveness on a second test. This hormonal effect
on submissive behavior has been shown to be independent of ACTH (Leshner, 1981).
Shock-elicited fighting is usually considered to reflect defensive behavior, and Heller
(1979) has concluded that this behavior is mediated by corticosterone and is independent
of ACTH. While roles for corticosterone in enhancing submissive and defensive behav
iors do not seem incompatible, it is harder to reconcile the report that acute elevations
in ΗΡΑ hormones leads to increased offensive aggression and this also appears to be due
to the action of corticosterone, rather than of ACTH (Brain and Evans, 1973). However,
although hormones of the ΗΡΑ axis might play a role in various types of aggressive
behavior, it is clear that their role in offensive aggression is very minor compared with
that of the androgens (for review, see Miczek, 1987), whereas they have the more impor
tant role in mediating submissive behavior (Leshner, 1981).
4. EFFECTS OF ANXIOLYTICS AND ANTIDEPRESSANTS ON HORMONES OF

THE ΗΡΑ AXIS
Because of the eflfects of the benzodiazepines, it has been suggested that changes in the
rat corticosterone stress response (Lahti and Barsuhn, 1974; Le Fur et al., 1979) or in
human Cortisol levels (Gram and Christensen, 1986) might serve as a useful screen for
anxiolytic drug action. The effects on hormones of the ΗΡΑ axis of benzodiazepines and
of other clinically effective anxiolytics and antidepressants will therefore be reviewed in
detail.
4.1. EFFECTS ON BASAL CORTICOSTERONE CONCENTRATIONS
In high doses, benzodiazepines elevate the basal corticosterone concentrations (i.e., in

unstressed rats left in their home cages). Thus, acute administration of diazepam (10
mg/kg) to unstressed rats increased plasma corticosterone concentrations (Barlow et al.,
1979; Keim and Sigg, 1977; Lahti and Barsuhn, 1974; Marc and MorseUi, 1969; Poho-
recky et al., 1988) as did chlordiazepoxide (12.5 mg/kg) (de Boer et al., 1988). There is
some discrepancy in the results reported following chronic treatment. Lahti and Barsuhn
(1974) found tolerance to the corticosterone-elevating effects of diazepam after 4 days of
treatment, but Marc and MorselU (1969) found that the increase was maintained for 8
days of treatment. After 10 days of diazepam (5 and 10 mg/kg), Chabot et al. (1982)
found elevations in the corticosterone concentrations of unstressed rats, but the samples
were drawn 24 hours after the last dose of diazepam. It is therefore possible that the
elevations reflected a withdrawal response from the benzodiazepines.
The increase of corticosterone induced by benzodiazepines is not the result of a direct
action on the adrenal gland, but seems to be mediated by ACTH release from the pitu
itary, since hypophysectomy aboUshed this response (Chabot et al., 1982) and acute
administration of a triazolobenzodiazepine elevates hypothalamic CRH (Owens et al.,
1988). Benzodiazepines reduce norepinephrine turnover (Wise et al., 1972), and since
norepinephrine is inhibitory to ACTH release, this has been proposed as the underlying
mechanism (Chabot et al., 1982). It is thought that norepinephrine acts at postsynaptic
a2-adrenoceptors to inhibit the release of CRH (and therefore also of ACTH), and thus
it is consistent that Clonidine, an «j-adrenoceptor agonist, can counteract the diazepam-
induced rise in basal corticosterone concentrations (Pericic et al., 1984). Although de
Boer et al. (1988) found that the dose of chlordiazepoxide that raised basal corticosterone
concentrations was without effect on plasma epinephrine and norepinephrine concen
trations, this should not necessarily be inteφreted as evidence against the «j-receptor
mediation, since it is central, not peripheral, norepinephrine that is relevant. De Boer et
al. (1988) also reported that the benzodiazepine antagonist, flumazenil, failed to antag
onize the chlordiazepoxide-induced increase in basal corticosterone concentrations. The
dose of flumazenil used (5 mg/kg) should have been quite high enough to antagonize the
dose of chlordiazepoxide (12.5 mg/kg). This suggests that the corticosterone-elevating
effects of benzodiazepines might not be mediated through the presently known benzo
diazepine receptors in the CNS. Some benzodiazepines also act at peripheral-type ben
zodiazepine receptors, but neither flumazenil nor chlordiazepoxide has appreciable activ
ity at these receptors, and hence this possible site of action seems unUkely. However, the
possibility that the peripheral-type benzodiazepine receptors can mediate changes in cor
ticosterone cannot be excluded, since ligands (Ro 5-4864 and PK 11195) for these recep
tors increased both basal and stress-induced corticosterone concentrations (File & Pel-
low, 1985). But since these drugs have opposite effects on the peripheral-type receptors
and since relatively high doses were used, it is more likely that the elevations in corti-
42 S. Ε. FILE
costerone were mediated at central sites. Even the proposed action via a2-adrenoceptor
sites would involve an initial action of the benzodiazepines at a benzodiazepine receptor.
Clearly, this area requires further experimentation.
The physiological role of extrahypothalamic CRH receptors is unknown, but they have
been implicated in anxiety and depression. Chronic administration of imipramine sig
nificantly increased CRH binding in the brain stem, suggesting that the drug may have
reduced CRH secretion in the locus coeruleus, resulting in the up-regulation of binding
sites (Grigoriadis et al., 1989). In contrast, chronic administration of diazepam and tria-
zolobenzodiazepines led to a significant decrease in CRH binding in frontal cortex and
hippocampus (Grigoriadis et al., 1989).
Brick et al. (1984) found that acute administration of ethanol increased plasma corti
costerone concentrations at 6 A.M, 6 P.M., and 12 A.M. but not at 12 P.M., suggesting a
diurnal variation in sensitivity to ethanol. While acute administration of ethanol was
found to increase corticosterone concentrations in rats left in their home cages, chronic
ethanol treatment was without effect (Guaza et al., 1983).
Buspirone is a serotonin,^ agonist with clinical anxiolytic and antidepressant actions
(Lader, 1988), and a related compound, gepirone, has similar actions. Both of these com
pounds elevate basal corticosterone concentrations (Koenig et al., 1986; Matheson et al.,
1988, 1989; Urban et al., 1986), as does ipsapirone, another serotonin,A agonist (see Fig.
2.2 and File et al., 1988). Chronic antidepressant treatment in rats did not modify the
serotonin,A-induced rise in corticosterone (Aulakh et al., 1988). These compounds also
increase ACTH secretion (Gilbert et al., 1987, 1988), probably as a resuh of stimulating
CRH release by acting at a postsynaptic serotonin,A receptor. Serotonin,A agonists have
also been reported to increase plasma ACTH concentrations in humans (Koenig et al.,
1985).
Ritanserin, a serotonin2 receptor antagonist with reported clinical efficacy as an anx
iolytic (Ceulemans et al., 1985) is without effect on basal corticosterone concentrations
(see Fig. 2.2), and a similar compound, ketanserin, failed to block the effects on basal
corticosterone of serotonin,A agonists (Gilbert et al., 1988). Thus, the increase in ΗΡΑ
axis activity seems selective to stimulation of postsynaptic serotonin,A receptors (see Gil-
50
40
§ ^ 3 0
20
10
4 10 2.5 5 0.25 10 •g/kg

QUIPAZINE IPSAPIRONE RITANSERIN
FIG. 2.2. The effect of quipazine (4 and 10 mg/kg), ipsapirone (2.5 and 5 mg/kg) and ritanserin
( 0 . 2 5 and 10 mg/kg) on plasma corticosterone concentrations of rats injected and replaced into
their home cages for 6 0 minutes before removal of blood. Blood samples were taken from a small
cut made at the tip of the tail. Plasma concentrations of corticosterone were measured using an
RIA for ^H-corticosterone. Values are presented as increases above the level of control-injected
rats in mean (±SEM) plasma corticosterone concentrations (Mg/dL). */* < 0 . 0 5 , compared with
control-injected group.
bert et al., 1988), but of course nonselective serotonin agonists such as quipazine also
have the same effect (see Fig. 2.2 and Johnston, 1988).
Finally, there is cUnical evidence that benzodiazepines reduce the plasma Cortisol con
centrations in normal volunteers (Gram et al., 1984), poor sleepers (Adam et al., 1984),
and depressed patients (Gram and Christensen, 1986; Christensen et al., 1989), and a
variety of antidepressants were reported to decrease the plasma Cortisol of depressed
patients (Christensen et al., 1989). However, it is less certain in these human studies that
there was no stressful component in the experimental conditions and, certainly, Petraglia
et al. (1986) found no changes in unstressed plasma Cortisol levels after diazepam.
4.2. EFFECTS ON STRESS-INDUCED RISE
Petraglia et al. (1986) found that benzodiazepines significantly reduced the rise in
plasma Cortisol caused by the stress of hypoglycemia. When normal volunteers were
exposed to a brief, painful electrical stimulus, diazepam prevented the rise in plasma
Cortisol, although ACTH concentrations were unchanged (Roy-Byme et al., 1988). It is
Ukely that the failure to find changes in ACTH was due to the time at which the plasma
samples were collected.
In animal studies, the antistress effect of chlordiazepoxide is found with a wide range
of stressors. After both acute and 5 days' administration, it significantly reduces the rise
in plasma corticosterone concentrations that result from exposure to a holeboard, to star
tle stimuU, to cold restraint (File, 1982a), and when intmding into other rats' territory
(File, 1984). There have been several other reports of benzodiazepine reduction of the
corticosterone response to noise (Barlow et al., 1979; Laurent et al., 1981) electric shock
(Kmlik and Cemey, 1971), or novelty (Seggie and Brown, 1975), and this effect seems
to be mediated by central benzodiazepine receptors, since it is blocked by the benzodi
azepine receptor antagonist, flumazenil (de Boer et al., 1988). Chlordiazepoxide, but not
ethanol, reduced the rise in plasma corticosterone concentrations that occurred during
the Vogel punished drinking test (Vogel et al., 1980). Diazepam blocked the rise in
plasma corticosterone concentrations seen in rats exposed to an elevated plus-maze (Bal
four et al., 1986). The eflfects of chlordiazepoxide on the plasma corticosterone response
was examined in the four test conditions of the social interaction test of anxiety. It mark
edly reduced the corticosterone response in the rats tested in an unfamiliar arena, but
was without eflfect in the groups tested in the less stressful familiar arena (File and Peet,
1980). Diazepam blocked the rise in ACTH and Cortisol seen in infant rhesus monkeys
separated from their mothers (Kalin et al., 1987). This eflfect also seemed to be mediated
through the benzodiazepine receptors, since it was blocked by the benzodiazepine antag
onist, flumazenil (Kalin et al., 1987).
The antistress eflfect of benzodiazepines is thought to be caused by an inhibition of
ACTH or CRH release. Benzodiazepines reduce the stress-induced rise in ACTH (Bmni
et al., 1980) and are without eflfect on the stress-induced rise in plasma corticosterone in
either hypophysectomized or dexamethasone-treated rats (Le Fur et al., 1979).
Evidence that the benzodiazepines reduce stress-induced rises of plasma corticosterone
through an action at the benzodiazepine binding site comes from the reversal of this
eflfect by the benzodiazepine antagonist, flumazenil (Bizzi et al., 1984; Mormede et al.,
1984; Kalin et al., 1987; De Boer et al., 1988). Picrotoxin, which acts at a site associated
with the chloride ionophore on the GABA-benzodiazepine complex also prevents the
benzodiazepine eflfect on stress-induced rise in corticosterone (Pericic et al., 1984). These
reversals may, however, be functional rather than pharmacological, since there is evi
dence that both flumazenil and Picrotoxin themselves increase plasma corticosterone
concentrations. Pellow and File (1985) and de Boer et al. (1988) found that flumazenil
enhanced the stress-induced rise in plasma corticosterone, as did the atypical benzodi
azepine, Ro 5-4864, at doses at which it acts at the Picrotoxin binding site (File and
Pellow, 1985). The failure to find an antistress eflfect with CL 218,872 (McElroy et al..
44 S. Ε. FILE
1 9 8 7 ) , a triazolopyridazine that acts only at the type 1 benzodiazepine receptors, indi

cates that these receptors are not involvol in mediating the antistress effect of benzodi
azepines and therefore suggests that it is the benzodiazepine, type 2 central receptors that
are involved.
In some studies, the decrease in the corticosterone response to stress was observed with
benzodiazepines and other minor tranquilizers and not with a wide range of other psy
chotropic agents (including neuroleptics, psychostimulants, antidepressants, and anal
gesics). However, in other studies, the corticosterone stress response has been reduced by
antidepressants (Keim and Sigg, 1977; Pekkarinen et al., 1 9 6 1 ; Pekkarinen, 1970; Kour-
koubasetal., 1988).
Two studies investigated the effects of buspirone on corticosterone plasma concentra
tions that were elevated following stress induced by a conditioned fear paradigm (Urban
et al., 1 9 8 6 ) or by angular rotation (Matheson et al., 1 9 8 8 ) . Urban et al. ( 1 9 8 6 ) reported
that the stress-induced increase in corticosterone secretion was inhibited by a low dose
of buspirone (0.5 mg/kg). However, this conclusion is questionable, since the reduction
in corticosterone plasma concentrations was not significant. In fact, a higher dose of bus
pirone ( 2 mg/kg) produced a significant enhancement of the stress-induced rise in cor
ticosterone concentrations. Matheson et al. ( 1 9 8 8 ) also reported that buspirone increased
concentrations of corticosterone following stress, and File et al. ( 1 9 8 8 ) reported that ip
sapirone, which also acts at 5-ΗΤ,Λ sites further increased plasma corticosterone concen
trations in rats stressed by exposure to a novel, brightly lit test arena. These results could
prove crucial in separating the mechanisms for anxiety reduction and reduction of Η Ρ Α
system activity. However, before it can be concluded that it is possible to achieve a clin
ically effective anxiolytic activity, despite Η Ρ Α activation, it is necessary to determine
the effects of chronically administered buspirone on the Η Ρ Α axis. Buspirone is clinically
ineffective as either an anxiolytic or an antidepressant before 2 weeks of treatment, and
its effects on the Η Ρ Α system are not known at this time.
Ritanserin is a 5-HT2 receptor antagonist that has been reported to have anxiolytic
activity in one clinical trial (Ceulemans et al., 1 9 8 5 ) , and it decreased the rise in corti
costerone concentrations induced by placing rats in a novel environment (File et al.,
1988).
5. CHANGES IN NEUROTRANSMITTER SYSTEMS IN RESPONSE TO STRESS

5 . 1 . CHANGES IN CENTRAL AMINE PATHWAYS
There are marked changes in norepinephrine, epinephrine, and dopamine pathways

in response to stressful stimuli and, more recently, changes in serotonin and acetylcho
line systems have also been reported. These changes, and their possible importance to
the neurochemical actions of antidepressant drugs, have been the topic of a recent review
by Anisman and Zarcharko ( 1 9 9 0 ; see also Chapter 3 ) and will not be discussed further
here.
5.2. CHANGES IN THE GABA-BENZODIAZEPINE RECEPTOR COMPLEX
In cerebral cortical membranes taken from rats first exposed to handling stress, there
was a decreased number of low affinity G A B A A binding sites, compared with membranes
from rats habituated to handhng. In the latter group, benzodiazepine receptor inverse
agonists (such as FG 7 1 4 2 ) decreased the number of G A B A A binding sites to the level of
the rats stressed by handling (Biggio et al., 1 9 8 4 ) . In rats first exposed to handling stress
there was a significant decrease in the uptake of ' * C - G A B A into the hippocampus and
frontal cortex (File et al., 1 9 9 0 ) and a significant increase in '^C-GABA release from
hippocampal and frontal cortical slices, compared with rats habituated to handling (see
Table 2.1). Furthermore, an acute administration of chlordiazepoxide to the unhandled
rats reduced the G A B A release. These rapid changes in G A B A release might be mediated
TABLE 2,1. Effects of Handling Stress on GABA Release in Previously

Unhandled Rats and in Handling Habituated Rats
Hippocampus Cortex
Handled Unhandled Handled Unhandled
20 mM 2.8 6.4 3.3 5.2

±0.2 ±1.4 ±0.3 ±2.0
40 mM 7.3 12.7 7.4 12.4
±1.1 ±2.1 ±1.1 ±2.4
Mean ( ± S E M ) **C-GABA release from slices from hippocampus and frontal cortex
in handling habituated (handled) and previously unhandled rats. Release was stimu
lated by 20 and 40 mM KCl and is expressed in the increase in fractional rate coeffi
cients above baseline. Effects of handling: Ρ < 0.001, analysis of variance.
by CRH as it has been shown to stimulate in vivo GABA release from the striatum and
globus pallidus (Sirinathsinghi and Heavens, 1989). Trullas et al. (1987) found increases
in chloride-stimulated benzodiazepine binding and in the binding sites associated with
the chloride ionophore in membranes taken from the cerebral cortex or hippocampus of
rats stressed by removing them from their home cages. These changes occurred within
15 seconds of removal from the home cage, before there were significant elevations of
ACTH or corticosterone, although changes in CRH would have probably occurred by
this time.
Havoundjian et al. (1986a, 1986b) reported that cerebral cortical membranes from rats
exposed to a brief, ambient-temperature swim stress showed an increase in both the
number and affinity of binding sites associated with the chloride ionophore on the
GABA-benzodiazepine receptor complex. There was also an increase in the chloride
enhancement of benzodiazepine binding, although no change in the basal binding or in
GABA-stimulated benzodiazepine binding. In rats exposed to inescapable shock and
then displaying deficits in an avoidance task (learned helplessness), there was a significant
decrease in muscimol-stimulated ^Cl uptake in synaptoneurosomes from the cerebral
cortex, compared with naive controls (Drugan et al., 1989).
Several studies have investigated the effects of adrenalectomy on the GABA-benzo
diazepine receptor complex. Majewska et al. (1985) found that adrenalectomy decreased
binding to hi¿i-affinity GABA receptors in the cortex, cerebellum, thalamus, and hip-
pocampus, whereas Kendall et al. (1982) found increased binding to low-affinity GABA
receptors in midbrain and striatum. Schwartz et al. (1987) found that swim stress
enhanced muscimol-stimulated chloride uptake in synaptosomes of cerebral cortex and
hippocampus, but that this effect was not seen in adrenalectomised rats. Adrenalectomy
had complex effects on the muscimol enhancement of benzodiazepine binding, enhanc-
ing it in the hypothalamus and striatum and decreasing it in the hippocampus (Goeders
et al., 1986); the changes in the striatum were reversed by dexamethasone. DeSouza et
al. (1986) have reported increases in the number of benzodiazepine receptors in the hip-
pocampus, striatum, and hypothalamus after adrenalectomy, but no changes in the cor-
tex, olfactory bulb, and cerebellum. The changes in binding were reversed by dexameth-
asone replacement therapy. Further evidence for an interaction between corticoid
hormones and the GABA-benzodiazepine receptor complex comes from the report by
Majewska et al. (1986) that tetrahydrodeoxycorticosterone inhibits binding to the chlo-
ride ionophore and enhances benzodiazepine binding. This is the profile seen with bar-
biturates, and tetrahydrodeoxycorticosterone has also been shown to have anxiolytic
activity in mice (Crawley et al., 1986). Tetrahydrodeoxycorticosterone is a naturally
occurring mineralocorticoid, which is a precursor of aldosterone, but whose secretion is
under control of ACTH (Schambelan and Bigüeri, 1976).
Early studies on benzodiazepine binding after stress found small and inconsistent
changes (Lippa et al., 1978; Braestrup et al., 1979; Grimm and Hershkowitz, 1981; Soub-
46 S . Ε . FILE
ríe et al., 1980; Lane et al., 1982). However, recent studies of in vivo receptor occupancy
have found changes in flumazenil binding in response to stress (Deutsch et al., 1988).
Following the mild stress of a 2- or 10-minute ambient temperature swim, there was an
increase in flumazenil binding in all brain regions examined. However, the more severe
stress of 10-minute intermittent foot shock (10 seconds of 2 mA every 30 seconds)
resulted in decreased binding. There is some evidence that at least the decreases in some
brain regions were not the result of the physical effects of electric shock. Similar decreases
in flumazenil binding were found in the hippocampus and hypothalamus of animals
exposed either to electroconvulsive shock or to the control procedure that involved all
aspects except delivery of shock. Decreased flumazenil binding was also found in the
cerebral cortex, hippocampus, hypothalamus, and striatum (but not in the cerebellum,
pons, medulla, or midbrain) in mice 24 hours after the last of seven consecutive, daily
swim stresses. There is evidence that some of these changes may occur in response to the
physical, rather than psychological, impact of the stress. In rats that were exposed to an
inescapable tail shock, some displayed impairments in a subsequent escape task, that is,
the phenomenon of learned helplessness, whereas some showed acquisition of the escape,
that is, the "copers". In the group showing learned helplessness, there was decreased flu
mazenil binding in several brain regions, but even in the group of copers there was
decreased binding in the hypothalamus and cerebellum. Very similar results were
reported by Drugan et al. (1989). They found that a group showing learned helplessness
had a decreased flumazenil binding in cerebral cortex, hippocampus, and striatum, com
pared with naive controls; however, both this group and the group of copers had
decreased flumazenil binding in the cerebellum and hypothalamus, compared with naive
controls. Thus, the results from animal tests suggest that there might be some changes in
the functioning of the GABA-benzodiazepine receptor complex in states of depression
as well as in anxiety. In agreement with this, a decreased number of benzodiazepine
binding sites has been reported in the frontal cortex of depressed suicide victims, an effect
that seemed to be independent of any drug effects (Cheetham et al., 1988).
Thus, in summary, there are changes in binding of GABA, benzodiazepines, and
ligands for the chloride channel that occur rapidly in response to an acute stress. The
GABA-benzodiazepine receptor complex, like the hormones of the ΗΡΑ axis, is sensi
tive to acute stressors. Whether the changes in the former are caused by the release of
ΗΡΑ hormones has yet to be established, but in view of the rapid changes reported, CRH
would be the most likely candidate. However, in addition, there is evidence for changes
in the GABA-benzodiazepine receptor complex following chronic stress and persisting
after exposure to inescapable shock. It is therefore possible that this receptor complex
also plays a role in the pathological changes that occur during the development of depres
sive disorders.
6. CONCLUSIONS
Exposure to anxiogenic or stressful situations leads to activation of the ΗΡΑ axis and
to changes in the GABA-benzodiazepine receptor complex. Administration of anxi
ogenic drugs activates the ΗΡΑ axis, and benzodiazepines can antagonize this and the
stress-induced activation of the ΗΡΑ axis. Additionally, exogenous administration of
CRH and ACTH, but not corticosterone, leads to anxiogenic effects in animal tests.
However, one cannot infer causal Unks from these results. Not all cUnically effective
anxiolytics reduce the stress-induced rise in ΗΡΑ hormones, and some may even exac
erbate it. Further, several drugs that are not anxiogenic, including the benzodiazepines
themselves at higher doses, also activate the ΗΡΑ system. Therefore, a reduction in ΗΡΑ
activity is not essential to anxiolytic efficacy. It is Ukely, therefore, that the hormones of
the ΗΡΑ system are simply one of several systems that modulate anxiety and, indeed,
the different ΗΡΑ hormones may modulate anxiety in different directions (CRH and
ACTH increasing, and glucocorticoids decreasing, anxiety).
Changes in the ΗΡΑ axis seem even less likely to be causal to the development of
depressive disorder. The ΗΡΑ changes are most likely to reflect a primary disorder, for
example, in central catecholamine pathways.
Thus, in summary, while changes in ΗΡΑ hormones may provide useful clinical mark
ers of anxiety or depressive disorders, they are Ukely at best to represent correlational
changes. There is little evidence to date for a causal connection with these disorders.
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Pttgsmon Press, Inc. (New York), pp. 57-82
CHAPTER 3
MULTIPLE NEUROCHEMICAL
AND BEHAVIORAL CONSEQUENCES OF STRESSORS:
IMPLICATIONS FOR DEPRESSION
HYMIE ANISMAN AND ROBERT M. ZACHARKO
Psychology Department, Unit for Behavioral Medicine and Pharmacology, Carleton University, Ottawa,
Ontario, Canada
1. INTRODUCTION
The notion that aversive events may provoke or exacerbate clinical depression in
humans is an intuitively appealing one. Indeed, both human and animal experimenta
tion has provided evidence commensurate with this position, although it may be pre
mature to dismiss the proposition that the reaction to a stressor is symptomatic of an
already existent depression. Despite the appreciable data assessing the stressor-depres-
sion topography, several issues have received only scant attention. Among other things,
it ought to be considered that (1) those events that are perceived as being stressful to one
individual may not be stressful to another, (2) the behavioral, emotional, and even the
neurochemical impact provoked by stressors may be different across individuals; (3) even
if a depressive syndrome is evident, the symptoms associated with the depression may
vary widely across individuals; (4) heterogeneity may exist with respect to the neuro
chemical substrates of depression; and perhaps as a result (5) variability exists with
respect to the treatments effective in alleviating depression.
Few would disagree that the development of an adequate animal model of human
depression is exceedingly difficult. Often the stressor-induced behaviors examined in ani
mals have borne little resemblance to the symptoms typically associated with human
depression (see Willner, 1985), and in some instances, treatments that are ineffective in
alleviating the symptoms of depression in humans are effective in eliminating the behav
ioral effects associated with stressors in rats and mice (see Zacharko and Anisman, 1989).
Even if one were to accept that a particular behavior was suitable as a model of human
depression, it is still necessary to consider that considerable variability exists in response
to stressors, symptoms of depression, neurochemical concomitants of the illness, and the
effectiveness of treatments. Fortunately, the interindividual variability that is evident in
the human condition is also present in animal research, and could potentially be used as
a tool to identify some of the organismic, experiential, and environmental factors that
subserve depressive-like behaviors.
2. CENTRAL NEUROTRANSMITTER CHANGES ASSOCIATED WITH

STRESSORS
We have described on several occasions the neurochemical concomitants associated
with stressors (Anisman, 1984; Anisman and Zacharko, 1982a; Zacharko and Anisman,
1989). Accordingly, a detailed account of this literature will not be provided at this point,
but rather only some of the more pertinent data will be discussed. Moreover, although
stressors have been shown to induce variations of a number of neurotransmitters and
hormones, the present discussion will be limited to those that are thought to be most
closely aligned with depression.
57
58 Η. ANISMAN AND R. M. ZACHARKO
It has been our position that acute exposure to environmental insults will lead to neu
rochemical changes that may be of adaptive value. As the stressor continues, further
variations may occur, presumably to meet environmental demands. Such alterations
may occur within a transmitter system (e.g., a compensatory increase in the synthesis of
a transmitter to assure adequate amine supplies or receptor regulation thereby increasing
or decreasing efficiency) or between systems (i.e., variations within a particular trans
mitter system may lead to compensatory changes of a second transmitter). However,
under certain conditions, the system may become overly taxed or, alternatively, the
within- or between-system adaptations do not occur, and hence vulnerability to pathol
ogy will be increased (Anisman and Zacharko, 1982a; Zacharko and Anisman, 1989).
The effects of stressors on norepinephrine (NE) activity have received greater attention
than any of the other transmitter systems. Figure 3.1 displays a schematic representation
of the NE variations that occur under various stressor conditions. It is well established
that under stressful conditions NE utiHzation and synthesis are increased. If the stressor
is sufficiently severe or behavioral methods of coping are unavailable, then utilization of
the transmitter may come to exceed synthesis and consequently concentrations of the
amine decline (Anisman, 1984; Anisman et al., 1980a; Tsuda and Tanaka, 1985; Weiss
et al., 1976). Not suφrisingly, stressor severity influences the variations of NE turnover
and levels, and it appears that the amine variations are provoked more readily in some
brain regions than in others (Tanaka et al., 1982). In addition, organismic factors such
Synthesis
MNd or Controlable Stress Severe or UncontroNable Stress Repeated Uncontrollat>le Stress
Ptwsel Pt^se I
RBoeplor ainreüMiy
Re-exposure to Re-exposure to Re-exposure to
(incontrolable Stress UncontroNable Stress Uncontrollable Stress
FIG. 3.1. Schematic representation of norepinephrine (NE) variations that occur under various
stressor conditions. When the stressor is mild or controllable, NE utilization is increased, and this
is accompanied by increased synthesis. Accordingly, the concentration of the amine is unaltered.
However, if the stressor is uncontrollable, further increases of utilization may occur, ultimately
exceeding synthesis, and resulting in a net decline of amine concentrations. Following a chronic
stressor regimen, a compensatory increase of synthesis may be engendered, and amine concen-
trations may equal or exceed control values. Additionally, under such conditions, subsensitivity
of /3-NE receptors may occur, and NE-stimulated adenylate cyclase (cAMP) activity may be
reduced. The lower portion of the ñgure depicts the effects of stressor reexposure as a function of
the organism's prior stressor history. In mildly stressed animals, reexposure to the stressor pro-
vokes a modest increase of amine or amine turnover, however, in animals previously exposed to
a traumatic stressor, subsequent reexposure to the environmental insult may provoke a marked
NE release, which may exceed synthesis, thereby resulting in reduced NE levels. Indeed, reex-
posure to cues previously associated with a stressor may augment NE turnover. In animals that
had been exposed to a chronic stressor that resulted in neurochemical adaptation, reexposure to
the stressor may provoke elevated NE concentrations, presumably owing to a rapid and marked
increase of NE synthesis. From Anisman and Zacharko, 1986, reprinted with permission.
Neurochemical consequences of stressors 59
as the age of the animal appear to be fundamental in determining the NE alterations,

with older animals exhibiting greater vulnerability and more persistent decreases of NE
than younger animals (Ida et al., 1982; Ritter and Pelzer, 1978).
While the amine variations are relatively transient, persisting for as little as a few min
utes to as long as 72 hours, depending on the stressor severity or the brain region exam
ined (Weiss et al., 1981), aversive events have also been shown to proactively influence
the response to subsequently applied stressors. In particular, in animals that had previ
ously been exposed to an acute stressor, subsequent reexposure to cues that had been
associated with the stressor come to increase the utilization of NE (Cassens et al., 1980;
Irwin et al., 1986a), and reexposure to the stressor will more readily reinduce NE reduc
tions (Anisman and Sklar, 1979; Irwin et al., 1986a). More recently, stressors have also
been shown to increase unit activity in the locus coeruleus of cats (Abercrombie et al.,
1986; Morilak et al., 1986; Rasmussen and Jacobs, 1986). Moreover, the locus coeruleus
unit activity was also enhanced in response to stimuli previously paired with a stressor
(Rasmussen et al., 1986). Such effects were not apparent in response to cues that previ
ously signaled reward or to neutral cues. Interestingly, when these cats were confronted
with potentially arousing stimuli (e.g., rats), overt signs of behavioral excitation were
elicited, but no significant deviations from baseline activity were noted from the locus
coeruleus (Abercrombie et al., 1986).
Following repeated exposure to a stressor or during the course of protracted exposure
to a continuous stressor (e.g., restraint), a further series of adaptive changes occurs. The
synthesis of NE increases appreciably, and as a result, the reduction in transmitter levels
is precluded (Irwin et al., 1986a; Kvetnansky et al., 1977; Roth et al., 1982). The
enhanced synthesis may persist for some time after stressor termination, and concentra
tions of the amine may actually come to exceed those of nonstressed animals (Anisman
et al., 1986; Irwin et al,, 1986b; Roth et al., 1982). Presumably, the enhanced synthesis
is essential for the organism to deal effectively with the ongoing stressor. The fact that
the increased synthesis persists after stressor termination may be essential in assuring that
amine supplies will be sufficient to deal with impending stressors.
Just as the acute effects of stressors (e.g., the enhanced utilization) may be influenced
by cues that had been associated with the stressor, it seems that the enhanced synthesis
associated with a chronic stressor may be subject to conditioning-like effects (see Fig.
3.2). In particular, having been exposed to a repeated stressor that resulted in the neu
rochemical adaptation, later exposure to cues associated with the stressor resulted in
increased accumulation of the NE metabolite, as well as increased concentrations of NE,
suggesting that exposure to the stressor-related cues provoked a marked and rapid
increase of NE synthesis (see Irwin et al., 1986a).
The conditions that delay or preclude the neurochemical adaptation have not been
extensively evaluated. It was demonstrated that when the stressor is applied at unpre
dictable times, the adaptation progresses more slowly than when the stressor is presented
on a predictable basis (Anisman et al., 1986). It might be supposed that still slower adap
tation occurs when the stressor regimen involves different types of stressors (as opposed
to a single type of stressor applied repeatedly). It is our contention that a stressor regimen
that does not favor the development of adaptation is more Ukely to be associated with
depressive symptoms than a chronic, predictable stressor regimen. Likewise, in those
organisms in which the neurochemical adaptation does not develop readily (as a result
of genetic or experiential variables), stressor-related depressive symptoms will be more
likely to develop.
The effects of chronic stressors are not limited to alternations in the synthesis and
concentrations of NE. In a series of reports. Stone and his associates (Stone, 1979, 1983;
Stone and Herrera, 1986; Stone and Piatt, 1982; Stone et al., 1984, 1986; see review in
Stone, 1987), as well as other investigators (e.g., Nomura et al., 1981; U'Pritchard and
Kvetnansky, 1980), have demonstrated that following a chronic stressor regimen the sen
sitivity of jS-NE receptors is reduced, as is the sensitivity of the cyclic adenosine mono-
I2(t
1001- -
140h
I20h
NO SHOCK ACUTE CHRONIC

PRETREATMENT
FIG. 3.2. Mean ( ± S E M ) norepinephrine (NE) and 3-methoxy-4-hydroxyphenylethylene glycol

(MHPG) concentrations in hypothalamus of mice that received either no shock, a single session
of 360 shocks (2-second duration, 150 μΑ) (acute), or 14 sessions of 360 shocks on consecutive
days (chronic). Fourteen days afterward, mice were either placed in the apparatus and not
shocked (open bars) or were exposed to 60 shocks (2-second duration) (closed bars). It will be
noted that in the acutely stressed mice, the shock reexposure treatment provoked a reduction of
NE concentrations, while the same treatment applied to mice that had previously been exposed
to a chronic stressor regimen resulted in enhanced NE concentrations. From Irwin et al., 1986a,
reprinted with permission.
phosphate (cAMP) response to catecholamines. In contrast, when the chronic regimen

involved a series of different stressors applied on successive days, then an up-regulation
of ß'NE receptor activity occurred (Molina et al., 1990). Inasmuch as chronic antide
pressants result in a ß-NE down-regulation, Stone offered the view that the reduced sen
sitivity of the NE receptors and of the cAMP response associated with chronic stressors,
like the response to antidepressant drugs, may reflect an adaptive change that limits the
development of depressive symptoms (Stone, 1983). In accordance with this view, it was
demonstrated that behavioral depression (i.e., immobility in a forced swim test and
anorexia) was reduced in parallel with the development of the receptor subsensitivity
(Piatt and Stone, 1982; Stone and Piatt, 1982).
It might be noted at this juncture that the reduction of ß-NE receptor sensitivity and
the down-regulation of the cAMP response associated with chronic stressor application
may be subserved by independent mechanisms. In particular, although chronic stressors
have been shown to reduce the density of ß-NE receptors in rat cortex, this effect was
relatively transient, being absent 24 hours following stressor termination (U'Pritchard
and Kvetnansky, 1980; see also Stone et al., 1984). In contrast, the cAMP response is
reduced to a comparable degree immediately and 24 hours after a chronic stressor regi
men (Stone et al., 1984). This does not necessarily imply that the mechanisms governing
the response to a chronic stressor are different from those mediating the response to anti
depressants, since some antidepressant agents have also been shown to reduce the N E -
cAMP response without affecting the density of /8-NE receptors (Mishra et al., 1980).
In considering the NE subsensitivity and the cAMP down-regulation associated with

antidepressant therapy, it should be made clear that such effects are probably not sec
ondary to the blockade of NE reuptake, since these effects are evident using atypical
antidepressants (see review in McNeal and Cimbolic, 1986). It does seem, however, that
there is a role for serotonin (5-HT) in the provocation of the receptor subsensitivity asso
ciated with antidepressant treatment. In particular, intracerebral administration of the 5-
HT neurotoxin, 5,7 dihydroxytryptamine, resulted in the eUmination of the jS-NE sub
sensitivity associated with desmethyUmipramine treatment, but did not influence the
down-regulated cAMP response (see Janowsky and Sulser, 1987). Interestingly, chronic
administration of desmethylimipramine in conjunction with the 5-HT reuptake inhibi
tor, fluoxetine, induced a more rapid development of the /3-NE receptor sensitivity than
treatment with desemethyUmipramine alone (Baron et al., 1988). The mechanisms
underlying the chronic stressor provoked jS-NE subsensitivity have yet to be elucidated,
although it does appear that, in rat hypothalamus, phospholipid metabolism may be
involved (Torda et al., 1981). It remains to be determined, however, whether 5-HT activ
ity plays a role in the development of the stressor induced NE receptor subsensitivity.
The dopamine (DA) variations associated with stressors are less widespread than those
of NE. In contrast to early reports that revealed increases or no change of DA concen
trations in large tissue samples, later reports revealed marked reductions of DA and
increases of utilization in certain brain regions. Reports by Kvetnansky et al. (1976) and
Kobayashi et al. (1976) indicated that restraint stress produced marked DA reductions
that were restricted to the arcuate nucleus of the hypothalamus. In other hypothalamic
nuclei, DA concentrations were either unaffected or were increased by the stressor. It was
subsequently determined, as well, that environmental insults profoundly influenced
mesoUmbic and mesocortical DA activity, but did not affect nigrostriatal DA
activity (Deutch et al., 1985; Herman et al., 1982; Herve et al., 1979; Thierry et al.,
1976). For instance, several forms of stressors have been shown to increase 3,4-di-
hydroxyphenylacetic acid (DOPAC) accumulation in the mesocortex and the nucleus
accumbens, without appreciably influencing DOPAC accumulation in the substantia
nigra (see Fig. 3.3). It was recently reported that using in vivo microdialysis, the stressor-
DOPAC HVA
FIG. 3.3. Dopamine (DA), 3,4KÍihydroxyphenylacetic acid (DOPAC), and homovanillic acid
(HVA) as a percent of control values (±SEM) in hypothalamus (Hypo), frontal cortex (FC),
nucleus accumbens (Nas), caudate, substantia nigra (SN), and ventral tegmentum (VTA) of mice
that had received either 30 or 360 foot shocks (150 μΑ, 2.second duration). From Anisman and
Zacharko, 1986, reprinted with permission.
62 Η . ANISMAN A N D R. M . ZACHARKO
induced enhancement of extracellular DA was more pronounced in the mesocortex than

in the nucleus accumbens. However, within 15 minutes after stressor termination, extra
cellular DA increases were also observed in the striatum (Abercrombie et al., 1989).
Thus, while these data confirm the previous regional differences with respect to the
stressor-provoked DA alterations, they suggest that aversive events may influence striatal
DA activity as well.
In addition to the immediate effects of stressors on mesocortical DA activity, it appears
that aversive events may also proactively influence amine utilization. Indeed, it was dem
onstrated that in the mesocortex, DA utilization may be enhanced by cues that had been
associated with a previously applied stressor (Herman et al., 1982; see also Deutch et al.,
1985). Additionally, it has been reported that the enhanced mesocortical DOPAC accu
mulation associated with an acute stressor may be prevented by pretreatment with the
anxiolytic, diazepam, or by treatment with the ß-NE inhibitor, chloφropanol, which acts
as an anxiolytic, but without primary sedative action (Fadda et al., 1978; Fekete et al.,
1981). Thus, it is conceivable that anxiety induced by the stressor is fundamental in pro
voking the mesolimbic/mesocortical DA changes.
Following a chronic stressor regimen, the mesocortical DA reductions ordinarily asso
ciated with an acute stressor may be absent. Unlike the enhanced synthesis that is respon
sible for the increased NE associated with repeated stress, there is some evidence sug
gesting that the DA adaptation is determined, at least in part, by moderation of the
excessive DA utilization (Anisman and Zacharko, 1986; Herman et al., 1984). That is,
although a chronic stressor enhances DA utilization, the DOPAC accumulation after
such treatment is less pronounced than after an acute stressor. The relative decline of
DA utilization may even be evident within a protracted stressor session. It was reported,
for instance, that in the mesocortex the accumulation of DOPAC is enhanced within 10
minutes of the commencement of restraint, peaks at approximately 20 minutes, and
declines toward control levels thereafter. Interestingly, in the nucleus accumbens,
DOPAC accumulation does not increase until 20 minutes after the restraint session com
mences, but does not decline over the course of a 120-minute session. Thus, while a
pronounced adaptation of DA utilization may be evident within a session, such an effect
is dependent on the brain region being examined (Roth et al., 1988). In accordance with
the data indicating that DA mesocortical neurons are particularly responsive to stressors,
Kramarcy et al. (1984) reported that the enhanced DOPAC accumulation associated
with acute foot shock was also accompanied by enhanced DA synthesis, as determined
from the accumulation of [Ή]-ΟΑ foUowing incubation with [^H]-tyrosine. Curiously,
following repeated stressor application, the enhanced DA synthesis associated with acute
shock was absent. Unfortunately, the latter effect was exhibited in a separate experiment
in which the effects of acute shock were not monitored. The absence of the necessary
comparison group in this experiment prevents a strong conclusion from being drawn.
There are data available suggesting that aversive environmental events will influence
DA receptor sensitivity. In particular, it has been demonstrated that like the progressive
enhancement of the behavioral effects associated with repeated treatment with amphet
amine, exposure to a stressor may augment the behavioral response ordinarily elicited
by amphetamine (Anisman et al., 1985; Robinson et al., 1985), and conversely, amphet
amine treatment may augment the behavioral effects of a mild stressor (Antelman et al.,
1980). Based on their work assessing electrophysiological recordings from DA neurons,
Antelman and Chiodo (1983) suggested that repeated treatment with amphetamine (as
well as repeated electroconvulsive shock) resulted in a time-dependent subsensitivity of
DA autoreceptors (as gauged by the response to low doses of apomoφhine). Presumably,
the subsensitivity of autoreceptors would result in increased DA release ordinarily pro
voked by a stimulant.
The data concerning the effects of stressors on serotonergic activity are less extensive
than those concerning NE and DA, and there appears to be greater inconsistency, as well.
Several investigators reported that stressful events may influence the turnover and/or
concentrations of 5-HT (Joseph and Kennett, 1981; Kennett and Joseph, 1981). It has
been demonstrated, for instance, that stressors will increase the utilization of 5-HT; how-
ever, it appears that the severity of the stressor required to alter 5-HT utilization is greater
than that necessary to elicit the alterations of NE activity (Thierry et al., 1968). Other
investigators (Hellhammer et al, 1983) have reported that stressors increase 5-HT con-
centrations in some brain regions (e.g., pons/meduUa, septum, striatum), but decrease
concentrations of the amine in other areas (midbrain, posterior hypothalamus). In the
posterior hypothalamus, the reduced 5-HT concentrations were accompanied by
reduced levels of 5-hydroxyindoleacetic acid (5-HIAA). More recently, Dunn (1988)
reported that a 15-minute session of foot shock resulted in increased accumulation of 5-
HIAA and reduced concentrations of 5-HT in the prefrontal cortex and in the hypo-
thalamus in CD-I mice. With a longer stressor period (30 minutes), amine utilization
increased still further, as reflected by greater accumulation of 5-HIAA; however, follow-
ing such a stressor regimen 5-HT levels were comparable to those of nonstressed animals,
while brain tryptophan levels increased appreciably. Possibly, the increased availability
of tryptophan resulted in increased synthesis of 5-HT, thus replenishing the amine stores.
Paralleling some of these ñndings, Adell et al. (1988) reported that following chronic
restraint, 5-HT levels increased in several brain regions. These increases were accompa-
nied by increased accumulation of 5-HIAA, suggesting that the enhanced 5-HT concen-
trations likely stemmed from a compensatory increase in synthesis. Indeed, it has been
reported that while acute sound stress provoked an increase of cortical tryptophan activ-
ity that persisted for less than an hour, following a chronic stressor regimen, the increased
trypophan hydroxylase activity was more pronounced and persisted for at least 24 hours
(Boadle-Biber et al., 1989). In our laboratory, we observed (Shanks et al., 1988) profound
alterations of 5-HT and 5-HIAA following application of foot shock (360 2-second
shocks, applied over 1.1 hours) in the mesolimbic cortex in ñve of six strains of mice
tested (A/J, BALB/cByJ, C57BL/6J, DBA/2J, C3H/HeJ, but not in CD-I), while in
hypothalamus there were no such reductions. Of course, as suggested by the data
reported by Dunn (1988), a lesser amount of shock may have permitted expression of
the 5-HT reductions in the hypothalamus. In both the mesocortex and hypothalamus,
5-HIAA accumulation was increased by the stressor treatment, but again this effect was
strain specific. Thus, some of the divergent effects reported in the literature may be a
reflection of the amount of stress applied or the strain of animal examined.
Scant information is available concerning the contribution of coping factors to the
alterations of 5-HT activity. It has been reported that uncontrollable shock induced 5-
HT reductions more readily than did controllable shock, but this effect was evident only
within the lateral septum (Sherman and Petty, 1980). In fact, Weiss et al. (1981) reported
that in other brain areas, controllable shock was more likely to influence 5-HT concen-
trations, possibly suggesting that the 5-HT changes reflected the effort associated with the
act of emitting an escape response.
A Umited number of studies have been performed assessing the effects of stressors on
acetylchoUne (ACh) concentrations and activity. It was demonstrated that the duration
of the stressor session influenced ACh levels in the hippocampus by affecting chohne
uptake mechanisms. Following a stressor of short duration (10 minutes of restraint), cho-
line uptake was augmented and ACh release was increased. With a stressor of longer
duration (3 hours), choUne uptake was reduced, while repeated exposure to a stressor (20
minutes for 5 days) resulted in reduced choline uptake, enhanced ACh release, and an
up-regulation of muscarinic binding. The changes of ACh activity and binding following
the chronic regimen required as long as 7 days to return to control values (Finkelstein et
al., 1985). These investigators suggested that the enhanced transmitter release, coupled
with reduced synthesis of ACh following a chronic stressor resulted in diminished ACh
levels and the up-regulation of muscarinic receptors. Indeed, following 1 or 24 hours of
cold or 20 minutes of swim stress, Fatranska et al. (1987) observed that hippocampal
ACh levels were reduced, probably owing to enhanced release of the transmitter. Unlike
the supposition of Finkelstein et al. (1985), there was no evidence of diminished ACh
synthesis. However, such effects may have been related to the nature or duration of the
stressor employed. Using an intermediate stressor session (45 minutes of restraint) Lai et
al. (1986) reported that restraint stress reduced choline uptake in both the hippocampus
and the frontal cortex. In the former, but not the latter, region this effect was prevented
by pretreatment with the opiate antagonist, naltrexone. Thus, it was suggested that the
hippocampal ACh alterations may stem from a stressor-provoked opioid change. Finally,
in contrast to the previous reports, Mizukawa et al. (1989) reported that 30 minutes and
3 hours of restraint did not affect ACh levels, but increased muscarinic binding in the
hippocampus. Following the shorter stressor session, the increased muscarinic binding
was evident in the CAl, CA2, and dentate gyrus, while the longer session resulted in
increased binding only in the dentate gyrus. It is not clear what factors might have been
responsible for the differential results observed between studies, although different sexes
and strains of rats were used.
In addition to the amine variations associated with stressors, it has been reported that
aversive events may influence opiate peptide activity. Opiate peptides and receptors have
been identified in the central nervous system (Mansour et al., 1988), as well as in neu
roendocrine systems, endocrine glands, and plasma (Owens and Smith, 1987). i3-endor-
phin has been found to be stored with corticotropin (ACTH) in the pituitary and Met-
enkephalin appears to be localized with catecholamines in the adrenal medulla (Owens
and Smith, 1987). While the role of opiate peptides in mediating specific behaviors has,
of course, not been fully elucidated, it was suggested (Swanson, 1983) that the presence
of synaptic "cocktails" favors multiple receptor activation and the development of com
plex behavioral states.
Acute stressors have been shown to reduce pituitary i8-endoφhin, while increasing
plasma levels of this opiate peptide in rats (Guillemin et al., 1977; Knepel et al., 1983;
Madden et al., 1977). The latter effect has also been demonstrated in humans (Owens
and Smith, 1987). The altered plasma endoφhin levels have been observed following a
variety of stressors, including surgery (Owens and Smith, 1987), childbirth (Pancheri et
al., 1985), and electric foot shock (Knepel et al., 1983). Following chronic foot shock
application, increases of i8-endoφhin may not be evident, and may even be reduced,
possibly owing to accelerated degradation of the neuropeptide (Young et al., 1989). In
addition to accumulating evidence that stressors activate peripheral opioid mechanisms,
there is evidence indicating that aversive events may exert potent central opiate effects
(Pert, 1982). It was demonstrated that an acute session of foot shock decreased enkeph
alin availability in the hypothalamus (Fratta et al., 1977; Rossier et al., 1978). Other
stressors such as food deprivation, mild tail pinch, and insulin-induced hypoglycemia
decreased dynoφhin activity in the cerebral cortex, while restraint or swim stress were
ineffective in this respect. Cold exposure, however, produced a significant decrease in
hypothalamic dynoφhin activity (Morley et al., 1982). It might be noted at this juncture
that there is also reason to believe that opiate peptide activity may be subject to condi
tioning-like effects much like those described for NE and DA. In particular. Chance et
al., (1978) observed that cues associated with a stressor resulted in decreased binding of
[^]-A2-Leu-enkephalin.
Although there is considerable evidence to suggest that stressors may produce altera
tions of opioid activity in cortical and hypothalamic sites, more recent data suggest that
opioid variations are associated with the site of origin of the major ascending noradren
ergic and dopaminergic pathways. In particular, Jacobs and his associates (Abercrombie
and Jacobs, 1988; Abercrombie et al., 1988; Morilak et al., 1987a, 1987b) observed that
the stressor-induced conditioning of locus coeruleus unit activity is profoundly influ
enced by opioid manipulation. These findings have been substantiated by demonstration
of considerable opioid immunoreactivity in and around the area of the locus coeruleus
(Finley et al., 1981; Leger et al., 1983), a dense distribution of opioid receptors in the
locus coeruleus (Atweh and Kuhar, 1977; Lewis et al., 1985) and inhibition of locus
coeruleus unit activity by central opioid administration (Bird and Kuhar, 1977; Guyenet
and Aghajanian, 1979). Abercrombie and Jacobs (1988) have argued that stressors acti-
vate the locus coeruleus NE system, which results in fear and/or anxiety. The coactiva-
tion of an endogenous opioid system, which functions to inhibit the activity of the locus
coeruleus, would favor adaptive behavioral coping.
In addition to the potential modulation influence of opioids on central NE activity, it
has also been demonstrated that stressor-induced alterations of central DA activity can
be influenced by opioid administration. In particular, both Met- and Leu-enkephalin, as
well as their respective mu and delta receptors, have been identiñed in the ventral teg-
mental held (i.e., the VTA, the A10 region of the DA containing mesoUmbic/mesocor-
tical system). Uncontrollable stressors have been shown to augment endogenous enkeph-
alin activity in the VTA and to induce accelerated DA turnover in mesoUmbic/
mesocortical sites. Exogenous administration of enkephalin analogs into the tegmentum,
in turn, has been shown to increase stressor-induced DA turnover in both the nucleus
accumbens and in the mesoUmbic frontal cortex (Kalivas et al., 1983). Administration
of the opioid antagonist, naloxone, prevents the stressor-induced variations of DA activ-
ity from the mesocortex (Miller et al., 1984). These results have recently been repUcated
by Kalivas and Abhold (1987), employing the shorter acting opiate antagonist, naltrex-
one. It was revealed that direct application of naltrexone into the A10 region attenuated
the elevation of DA metaboUsm normally evident after foot shock. It has also been
reported that reductions of the neuropeptide, substance P, which appears to be localized
in the tegmentum, attenuates mesocortical DA turnover in response to uncontrollable
foot shock (Bannon et al., 1983). There is also reason to suspect that sensitization/con-
ditioning may be involved in enkephalin/neuropeptide interactions with central DA
activity. For example, exposure to inescapable foot shock potentiated the augmented DA
turnover elicited by intracerebral administration of the enkephaUn analog, D-Ala^-Met^-
enkephalinamide (DALA) into the VTA, while naltrexone blocked this augmented neu-
rochemical response. Moreover, prior administration of DALA has also been noted to
sensitize animals to the subsequent effects of stressor application (Kalivas and Richard-
son-Carlson, 1986). In accordance with these ñndings, Cabib et al. (1984) demonstrated
that repeated immobilization stress increased sensitivity to DA agonists, an effect that
was prevented by prior administration of naloxone. It was concluded that chronic stres-
sors may sensitize central DA systems, owing to excessive production of central endog-
enous opioids. Together, these data suggest that although stressors appear to influence
DA activity, such alterations may in turn be regulated by endogenous opiate systems and
psychological variables associated with the stressor may come to influence the expression
of neurochemical processes.
In addition to alterations of central opioid concentrations, it appears that stressors may
provoke alterations of central opiate receptors. For example, Nabeshima et al. (1985)
reported that uncontrollable foot shock engendered conformational changes of central
opioid receptors, as evidenced by increased binding at antagonist receptor sites and
decreased binding at agonist receptor sites. Furthermore, Sirakova et al. (1988) repoilpd
that immobilization stress (i.e., 24 hours coupled with food and water deprivation)
enhanced delta receptor binding, with little if any effect on mu receptor binding in rat
whole brain. It is unclear whether these alterations stemmed from the immobilization
procedure, the deprivation schedule imposed, or a combination of the two. However, it
will be noted that fasted rats exhibited decreased levels of i8-endoφhin in the hypothal
amus (Gambert et al., 1981).
3. BEHAVIORAL CHANGES ASSOCIATED WITH STRESSORS

As indicated earlier, considerable attention has been devoted to the analysis of stressors
on subsequent behavioral impairments. By far the greatest emphasis has been placed on
the analysis of controUable and uncontrollable stressors on shuttle escape performance.
Specifically, it was demonstrated that following exposure to escapable shock, shuttle

escape performance is typically unaffected; however, in animals that were exposed to an
identical amount of shock, applied in a yoked paradigm, severe disturbances of escape
performance were apparent. The initial studies, conducted in dogs, demonstrated this
effect to be relatively transient, disappearing within about 72 hours of stressor termina
tion. These animals were described as being passive in the face of the stressor, without
apparent attempts to escape from the aversive stimuli (Maier and Seligman, 1976). Later
studies conducted in rats or mice provided a somewhat different behavioral profile.
While inescapable shock reliably disrupted subsequent escape performance, the behav
ioral deficits were most apparent when the response was either associatively or motori-
cally demanding (Anisman et al., 1978; Maier et al., 1973). Typically, escape deficits
were evident when animals were required to take several seconds of shock before escape
was possible (see Fig. 3.4). Furthermore, unlike the description provided in studies in
dogs, in mice and rats, shock onset almost invariably led to heightened motor activity,
which decayed rapidly as the shock continued. The initial response excitation seen upon
shock onset was evident irrespective of whether animals had initially been exposed to
escapable or inescapable shock, whereas the decline in shock-elicited motor activity was
particularly marked in mice that initially had been exposed to uncontrollable foot shock
or tail shock. Thus, it was suggested that the response excitation seen upon shock onset
favored proficient responding in tasks that permitted immediate escape. However, the
delay in active responding in mice previously exposed to uncontrollable shock favored
disturbances of successful performance. While not necessarily denying a role for other
factors in subserving the escape interference, the view was offered that the escape distur
bances could be accommodated on the basis of a deficiency in the organism's ability to
sustain an active response for protracted periods (Anisman et al., 1978).
Unlike the time course for the interference effect seen in dogs, long-term behavioral
disturbances could be provoked in rats and mice. The behavioral profile, however, was
dependent upon the specific stressor parameters that had been employed during inescap
able shock. Studies conducted by Glazer and Weiss (1976a, 1976b) and subsequently
N O PRESHOCK 6 SEC S H O C K S 12 SEC SHOCKS
16
A
14 ESCAPE DELAY
— 0 X
12 - 2
o-o 6
^101
^ 8|
1 2 3 4 5 1 2 3 4 5
BLOCKS OF FIVE TRIALS
FIG. 3.4 Mean (±SEM) escape latencies in a shuttle task over blocks of five trials (calculated
from the time of gate opening) as a function of the preshock treatment and duration of escape
delay. Note that the inescapable shock treatment did not affect escape latencies when escape was
possible soon after shock onset (0 or 2 seconds), but when escape was briefly prevented (4- or 6-
second delay) marked deficits of performance were evident. From Anisman et al., 1979b,
reprinted with permission.
confirmed in our laboratory (Anisman et al., 1978) indicated that if animals were
exposed to uncontrollable shock of relatively high intensity and short duration, then a
marked, but transient, shuttle performance disturbance was apparent. In contrast, if the
initial stressor treatment involved moderate-intensity, long-duration shock, then long-
term deficits were evident. Curiously, under these conditions, shuttle performance
immediately after the shock treatment was hardly affected.
Glazer and Weiss (1976a, 1976b) attributed the short-term behavioral disturbance
associated with high intensity shock to a motor activation deficit stemming from reduc
tions of NE. In accordance with this position, it was demonstrated that (1) Uke the
increased NE ordinarily associated with a chronic stressor regimen, the escape deficits
were not evident in mice that received repeated exposure to the stressor (Weiss et al.,
1976); (2) pharmacological compounds that reduced DA and/or NE or blocked DA
receptors (e.g., a-MpT, FLA-63, tetrabenazine, haloperidol, and pimozide) mimicked
the effects of inescapable shock (see Fig. 3.5) (Anisman and Zacharko, 1982b; Anisman
et al., 1979b; Weiss et al., 1976); and (3) drugs that increased catecholamine activity
eliminated the effects of inescapable shock (see Fig. 3.6) (Anisman et al., 1979b, 1980b,
1981b). To account for the long-term deficits of escape performance associated with
long-duration shock of moderate severity, it was suggested that these stressor parameters
permitted the development of learned competing motor responses (e.g., learned inactiv
ity) that interfered with the expression of the active response necessary for succcessful
escape (Glazer and Weiss, 1976a, 1976b).
While not taking issue with the formulations adopted by Weiss et al. (1976), the view
was expressed that the behavioral disturbances were not subserved by NE alterations
alone. The fact that DA antagonists mimicked the escape interference, and DA agonists
acted either as a prophylactic (i.e., drug applied before inescapable shock) or as a thera
peutic (i.e., drug administered after inescapable shock but before test) implicated a role
for DA (Anisman et al., 1979a, 1979b). Furthermore, the long-term behavioral distur
bances associated with inescapable shock could be attributed to conditioned neurochem
ical changes, such that the amine alterations in previously stressed animals could again
be engendered during the first few trials of escape training (Anisman and Sklar, 1979).
Moreover, a potential role for ACh in mediating the escape interference was suggested
by the findings that the choUnesterase inhibitor, physostigmine, provoked escape distur
bances Uke those elicited by inescapable shock, while the antichoUnergic (antimusca-
rinic), scopolamine, antagonized the escape interference produced by the uncontrollable
stressor (Anisman et al., 1981a, 1981b). Parenthetically, the effects of the anticholinergic
could be distinguished from those of catecholamine agonists, in that scopolamine was
effective in eUminating the escape interference provoked by previously applied inescap
able shock, but was ineffective in modifying escape performance if the drug was applied
prior to inescapable shock (Anisman et al., 1981a).
To account for the absence of the escape interference soon after inescapable shock of
moderate severity, several modifications of the initial neurochemical hypothesis were
necessary. As indicated earlier, the NE and DA variations associated with a stressor vary
across brain regions, becoming apparent in some areas only after relatively intense foot
shock. Likewise, the stressor severity necessary to induce a 5-HT change is appreciably
greater than that necessary to promote the NE variations. Accordingly, the magnitude of
the interference effect should increase with stressor severity. Yet, inescapable shock also
elicits increased arousal or anxiety and, hence, reactivity, which, in some tasks, may
favor successful escape (i.e., in tasks where the response requirement is fairly simple or
is a reflexive one). With relatively severe stressors, the multiple neurochemical changes
influencing motor activity may ultimately come to override the stressor-provoked
arousal and hence lead to an interference of escape performance even at brief intervals
after inescapable shock.
In accordance with this proposition, it was demonstrated that in a forced swim task,
inescapable shock had biphasic effects on activity. Soon after a stressor session, mice
VEHCLE 40mg/kg FLA-63 60mg/KO FLA-63
12
VEHICLE 04mgA(g PIMQZIDE 0.8mg/hg PIMOZlOE
21
15
12
p4
3 4 5 1 2 3 4 5
FIG. 3.5. Mean ( ± SEM) escape latencies in a shuttle task where escape was possible either imme
diately upon shock onset or after a delay of 2 , 4 , or 6 seconds. Note that treatment with a-MpT,
FLA-63, and pimozide, like inescapable shock, did not affect responding when escape was pos
sible immediately upon shock onset but became progressively more pronounced with longer
escape delays. From Anisman et al., 1979b, reprinted with permission.
exhibited invigorated swimming relative to nonshocked animals. Moreover, this was the
case irrespective of whether the stressor was controllable or uncontrollable. In mice tested
24 hours after inescapable shock, decreased swimming (increased floating) was evident,
but this effect was apparent only in those mice that had received inescapable shock
(Prince and Anisman, 1984). Commensurate with the view that the motor excitation was
related to increased anxiety, diazepam was found to eliminate the initial response invig-
4 5 1 2
FIG. 3.6. Mean ( ± SEM) escape latencies in mice that received either inescapable shock or no
shock 2 4 hours prior to shuttle escape testing. Treatment with L - D O P A prior to the test session
eliminated the escape deficits associated with the inescapable shock treatment. From Anisman et
al., 1979b, reprinted with permission.
oration (Prince et al., 1986). Moreover, it did not appear that this effect was secondary
to motoric (or sedative) effects of the drug. Specifically, in addition to the invigorated
swimming, mice placed in a water maze tended to approach an illuminated area (in con
trast to the preference for dark in a dry chamber). This prepotent defensive style was
further exaggerated in mice that had recently been exposed to inescapable shock (Szostak
and Anisman, 1985). It was suggested that upon exposure to a stressor, the ensuing
arousal increases the animal's propensity to emit highly prepared responses or to direct
their responses toward those stimuli that might be associated with escape. This stimulus
perseveration, which is independent of motor activity, was reduced by diazepam, just as
the motor response invigoration was eliminated by this treatment (Prince et al., 1986).
Generally, limited data are available concerning the effects of anxiolytics on the behav
ioral depression associated with an uncontrollable stressor. The few studies that have
been published suggested that anxiolytics are ineffective in antagonizing the conse
quences of previously administered inescapable shock. However, it has been reported
that if the drug is administered prior to inescapable shock, then the behavioral impair
ments that would otherwise be evident are prevented (see Drugan et al., 1984). Thus, the
position was offered that anxiety may be fundamental for the development of the behav
ioral deficits (and possibly for the catecholamine variations that subserve these deficits),
but that once these stressor-provoked behavioral or neurochemical states have been
established, anxiolytics are not effective in eliminating them.
In their analysis of the mechanisms underlying stressor-provoked behavioral distur
bances, Weiss and his associates (Simson et al., 1986a, 1986b; Weiss and Simson, 1984;
Weiss et al., 1986) suggested that the NE variations within the locus coeruleus are par
ticularly important in determining behavioral disturbances. In particular, it was sug
gested that large reductions of NE in the locus coeruleus would reduce the availability of
NE to stimulate inhibitory «j-receptors on these cells. This would result in increased
firing of locus coeruleus cells and increased NE activity in terminal regions. In effect, the
behavioral disturbances associated with acute stressors may be linked to the excessive
NE activity present in forebrain terminal regions, and the behavioral adaptation seen
after a chronic stressor may be a result of receptor subsensitivity as suggested by Stone
(1987). In accordance with this view, it was demonstrated that infusion of a2-antagonists
effectively produced behavioral depression in rats, while a2-agonists eliminated the

behavioral disturbances associated with an uncontrollable stressor (Simson et al., 1986a,
1986b; Weiss et al., 1986). It might be noted at this juncture that Weiss and Simson
(1984) suggested that this model of depression might be most closely aligned with that
form of clinical depression in which anxiety is an associated symptom. Indeed, the view
expressed by Weiss and Simson (1984) is commensurate with work suggesting that dis-
inhibition of the locus coeruleus may serve to influence the processing of external sensory
stimuH (Aston-Jones and Bloom, 1981; Aston-Jones et al., 1984; Foote et al., 1983). That
is, exposure to a stressor or cues associated with a stressor will engender activation (dis-
inhibition) of the locus coeruleus, which will result in suppression of irrelevant environ
mental stimuli, while increasing attention to those stimuli that are relevant to the current
environmental demands. In effect, following uncontrollable stressors, animals will be
more vigilant to specific environmental cues, which may, in a sense, be a collateral or
causative factor in provoking increased anxiety (see Redmond, 1981; Redmond and
Huang, 1979).
In addition to cognitive disturbances, Maier and Seligman (1976) proposed that ines
capable shock may hinder an animal's ability to form response-outcome associations.
In accordance with this view, Jackson et al. (1980) demonstrated that in a Y-maze dis
crimination task in which rats were required to turn right at the choice area, inescapable
shock retarded acquisition. In subsequent experiments from this same laboratory, diffi
culties were encountered in observing this effect unless extraneous cues were present
(Minor et al., 1984). Thus, these investigators suggested that inescapable shock may have
disrupted the organism's ability to attend to relevant cues or may have interfered with
the animal's ability to differentiate between relevant and irrelevant stimuli. It will be
recognized that this view is diametrically opposed to that proposed by Weiss and Simson
(1984) and is predicated on reports which imply that dorsal bundle lesions provoked by
6-hydroxydopamine (6-OHDA) may resuh in attentional disturbances (Mason, 1979).
Even if it were assumed that 6-OHDA lesions of the dorsal bundle lead to such atten
tional disturbances, one would be hard pressed to draw a parallel between the data of
Minor et al. (1984) and those of Mason (1979), since in the latter studies, the extent of
the NE depletions provoked by 6-OHDA are in the magnitude of 95%, while in the for
mer studies, where NE alterations were not determined, it is probably safe to say that NE
reductions did not approach this magnitude.
In our laboratory we found that inescapable shock did not influence cue discrimina
tion performance in a Y-maze escape task, nor did this treatment influence response-
choice discrimination performance in two different paradigms (Anisman et al., 1984). In
one of these paradigms, only a right turn led to shock offset, so that when an incorrect
response was emitted (i.e., a left turn), shock was not terminated until the animal made
another response of turning left. In the second paradigm, shock offset occurred when the
animals entered the arm that was in the right-hand position relative to the initial start
arm; thus, a right turn again resulted in shock termination, but if a left turn was made,
then a second left turn was necessary to reach the correct arm. While inescapable shock
did not influence acquisition of these responses, the uncontrollable stressor was observed
to retard acquisition of a reversal response in one of these paradigms. Specifically, in the
response-choice (positional) paradigm, a right turn (or two consecutive left turns)
resulted in shock termination. In the reversal task, in which a correct response required
a left turn or two consecutive right turns, animals that had not been exposed to inescap
able shock readily acquired the response, whereas the stressed animals persisted in adopt
ing the previously established response of turning right (twice successively), even though
a single left turn would have terminated the shock. It seems that the inescapably shocked
animals perseverated in adopting a previously estabUshed response, so long as this
response was ultimately effective in terminating shock. It will be recalled that these find
ings are consistent with those reported by Prince and Anisman (1984) and by Szostak
and Anisman (1985), showing that in a swim test, stressors increase the animal's propen-
sity to attend, or to respond, to those stimuli to which they are already predisposed. In
effect, our position is commensurate with that advanced by Weiss and Simson (1984),
except that we maintain that the uncontroUability of the stressor may not be essential
for the induction of this vigilance or anxiety.
As indicated earlier, we have maintained that one of the problems associated with
many of the animal models of depression is that the behavior examined often bears little,
if any, apparent relationship to the symptoms that characterize depression in humans.
Ultimately, investigation of shuttle escape performance deficits induced by inescapable
shock may contribute to the analysis of stressor-induced depression, but on the surface
it is difficult to see the relationship between this behavior and the symptom profile which
characterizes the human disorder. To be sure, it has been demonstrated that the shuttle
escape deficits, like the symptoms of depression are modified by repeated administration
of agents (e.g., desmethyUmipramine) that are effective antidepressants in humans (see
Sherman and Petty, 1980). Yet, the shuttle escape deficits are also modified by drugs that
are ineffective as antidepressants, including L-dopa and apomoφhine (Anisman et al.,
1979b, 1980b).
In light of some of the shortcomings of the typical approaches to the analysis of stressor
effects in animals as they relate to a model of clinical depression, it has been suggested
that it is necessary to assess a series of different behaviors, which as a group may be
reflective (or at least reminiscent) of some of the symptoms of depression (Weiss and
Simson, 1984; Zacharko and Anisman, 1989). While not discarding the analysis of the
shuttle escape deficits induced by inescapable shock, we have devoted considerable atten
tion, among other things, to the analysis of stressor effects on responding for electrical
brain stimulation. This behavior may provide an index of reductions of either motiva
tional or reward processes engendered by the stressor and, hence, might be taken to
approximate one of the key symptoms of depression, namely that of anhedonia. In our
initial studies, we examined the effects of escapable and inescapable foot shock on
responding for self-stimulation from the nucleus accumbens (Nas), medial forebrain
bundle (MFB), and the substantia nigra (SN) (Zacharko et al., 1983a). As seen in Fig.
3.7, escapable shock was without effect on responding for brain stimulation, regardless
of the site of electrode placements. In contrast, responding for electrical brain stimulation
from both the Nas and MFB was markedly reduced in animals that had been exposed
to inescapable shock, whereas this treatment did not affect responding from the SN. It
wiU be recalled that DA activity is affected by stressors in the Nas, while DA activity in
the SN appears not to be affected by stressors. Parenthetically, the decUne of responding
in self-stimulation should be distinguished from the shuttle deficits in a fundamental
way. In previously trained animals, inescapable shock was found not to disrupt shuttle
escape performance. That is, initial training "immunizes" the animals against subse
quent stressor-provoked behavioral impairments (Maier and Seligman, 1976). In the self-
stimulation paradigm the shock treatment disrupted performance, even though the
response had been weU established.
The disruptive effects of inescapable shock on responding for brain stimulation were
robust and persistent. When stressed animals were placed in the self-stimulation cham
ber, they typically commenced responding for a brief period of time, after which,
responding decayed and finally ceased entirely. Once an animal exhibited the decline in
responding, these performance deficits were evident even when mice were tested at fairly
lengthy intervals after the initial stressor session. In these animals, it was difiicult to rees-
tabUsh responding even when a priming procedure was employed. Fundamental to the
long-term performance reduction, however, was that animals initially be tested soon after
the stressor session. If the first self-stimulation test was conducted 1 week after the ines
capable shock session, then the performance deficit was not evident (provided that rein
statement shocks were not employed). Presumably, the performance disturbances were
related to the stressor provoked neurochemical changes (e.g., a decUne in the rewarding
value of stimulation owing to reduced DA activity). Once the neurochemical activity
72 Η. ANISMAN A N D R. M . ZACHARKO
NO SHOCK
1200 eSCAPABLE
YOKED INESCAPABLE
1000
800
600 MEDIAL FOREBRAIN BUNDLE
400
200
Í
500
I-.. J
400
1 i
300
NUCLEUS ACCUMBENS
200
100
BASELINE 0 24
TIME(hrs)
FIG. 3.7. Mean (±SEM) rates of responding for brain stimulation from either the medial fore-
brain bundle, nucleus accumbens, or substantia nigra. Following the establishment of baseline
rates of responding mice received either escapable shock, yoked inescapable shock, or no shock
and were tested in the self-stimulation task immediately thereafter and again 24 and 168 hours
afterwards. Note that performance deficits in intracranial self-stimulation (ICSS) only appeared
from those regions innervated by the mesolimbic system (nucleus accumbens) or areas through
which the ascending dopamine (DA) pathway courses (medial forebrain bundle). Nigrostriatal
l e s s was unaffected by the uncontrollable stressor. From Zacharko et al., 1983a, reprinted with
permission.
reverted to basal levels, performance deficits were absent. However, in animals that were
initially tested soon after the inescapable shock, thus leading to an extinction-like effect,
the performance deficits persisted upon retesting 1 week later.
These particular effects were not related to the initial rate of responding and were also
observed in a current intensity paradigm (Bowers et al., 1987). Moreover, it was not the
case that the difference between the stressor effects on responding from the Nas and SN
stemmed from the latter region being a site of cell bodies and the former being a terminal
region, since the same outcome was observed in subsequent studies in which responding
was assessed from the VTA (Kasian et al., 1987). It is of particular significance that foot
shock differentially influenced self-stimulation from different aspects of the VTA.
Whereas marked disturbances of performance were evident from the dorsal tegmentum,
responding was unaffected from the ventral portion (Kasian et al., 1987). These data are
of interest in light of the neurochemical and electrophysiological delineation of the VTA
provided by Deutch et al. (1985), which suggests that the tegmental field is not uniformly
affected by stressors. In effect, the findings concerning self-stimulation provide a func
tional differentiation of different portions of the VTA, commensurate with the view that
the midbrain tegmental area is not a homogeneous structure.
Paralleling the NE and DA variations associated with a chronic stressor regimen, it
was observed that the reduction of responding for brain stimulation from the Nas ordi-
3 SAL. - N.S.
• SAL - 180 X 6
3 D.M.I. - N.S.
•D.M.I. -180 X 6
ζ 500
400^
LU
< 300
GC
UJ
ζ 200
o
ü.
ω
100
BASELINE 0.0 24 168
TIME (HRS.]
FiG. 3.8. Mean ( ± SEM) response rates for electrical stimulation from the nucleus accumbens at
three intervals following a single session of inescapable shock (180 shocks of 6-seconds duration)
or no shock (NS). Prior to the shock session, mice had received administration of desmethyl
imipramine (D.M.L) on 15 consecutive days (5 mg/kg twice per day) or were treated with saline
(SAL). In the drug-treated mice, the disruption of responding ordinarily provoked by inescapable
shock was reduced immediately after the stressor session and entirely eliminated at the 24- and
168-hour intervals. From Zacharko et al., 1984b, reprinted with permission.
narily provoked by an acute stressor was absent in animals that had received repeated
exposure to inescapable foot shock (Zacharko et al., 1984a). Like Weiss et al. (1976),
who found a similar adaptation with respect to shuttle escape, we suggested that these
findings were incompatible with a learned helplessness position. After all, such a hypoth
esis would have predicted that with a greater number of stressor sessions the extent of
the behavioral disturbances should have been increased rather than decreased. Whether
the behavioral adaptation is related to the enhanced DA/NE levels or to alterations of
receptor sensitivity associated with the chronic stressor regimen is unclear.
In accordance with the view that responding for self-stimulation after inescapable foot
shock may provide an adequate model of clinical depression, it was demonstrated (as
seen in Fig. 3.8) that repeated treatment with desmethylimipramine eliminated the
stressor-induced reduction of responding for brain stimulation from the Nas (Zacharko
et al., 1984b). Interestingly, in a study in which the behavioral profiles of individual ani
mals were assessed, it was observed that following a chronic stressor regimen, some ani
mals exhibited behavioral adaptation (i.e., with the initial shock sessions, responding
declined, but increased with further shock exposure) while others did not. In those ani
mals in which the adaptation did not develop, repeated treatment with desmethylimip
ramine reestablished responding. In contrast, in animals that showed the behavioral
adaptation, treatment with desmethylimipramine provoked behavioral impairment
(Zacharko et al., 1983b). In effect, it seemed as if the antidepressant served to reestablish
an amine balance that had been disrupted by the stressor. If this equilibrium was rees
tablished through endogenous processes, administration of the drug served to disrupt this
balance, hence leading to behavioral impairment.
4. GENETIC AND INDIVIDUAL DIFFERENCES IN RESPONSE TO STRESSORS

As discussed earlier, one of the problems with current animal models of depression is
that they do not address the question of the vast interindividual differences that exist in
humans with respect to the symptom profile of depression, as well as the effectiveness of
various antidepressants in ameliorating these symptoms. The view had been offered that
several biochemical subtypes of depression exist, including those in which either NE or
5-HT were fundamental to the illness (Schildkraut, 1978; van Praag, 1984) or that in
which NE subserves depression, but when accompanied by 5-HT alterations, an anxious
form of depression emerges (van Praag et al., 1987). Additionally, increasingly greater
attention has been devoted to the potential contribution of DA, ACh, and endoφhins to
the expression of depressive symptoms (see Berger and Nemeroff, 1987; Janowsky and
Risch, 1987; Jimerson, 1987). While the diverse symptom profiles of depression may be
related to situational or experiential factors, it is also possible that the symptomatology
may be related to the particular transmitter disturbances that may exist and to the spe
cific brain regions in which these occur.
In an attempt to address the issue of interindividual differences in response to stressors,
we opted to examine the effects of environmental insults on a series of different strains
of mice that had been previously shown to display a range of behavioral differences, as
well as basal neurochemical variations. In our laboratory, these strains exhibited different
basal levels of NE, DA, 5-HT, and their respective metabolites and also were found to
be differentially affected by foot shock stress. Indeed, the effectiveness of a stressor in
modifying amine levels or turnover varied not only with the strain of mouse examined,
but also with the brain region and the particular transmitter considered (Shanks et al.,
1988).
A behavioral analysis of the effects of stressors confirmed that these strains differed in
response to environmental insults; however, whether a behavioral disturbance was evi
dent in any particular strain was dependent on the behavior being examined. Indeed,
strain-specific performance disturbances might be evident in one task, whereas perfor
mance was unaffected when a second task was examined (Shanks and Anisman, 1988).
For instance, as seen in Fig. 3.9, exposure to inescapable shock differentially affected
A/J .BALB/cByJ C3H/HeJ

20
10
LU
O
, C57BL/6J _ CD-I DBA/2J
ω 20
S
10
3 4 1 2 3 4 5 1 2 3
Blocks of Five Trials
FIG. 3.9. Mean (±SEM) latencies to escape over blocks of five trials as a function of the shock
treatment (S = inescapable shock and NS = no shock) and mouse strain in testing with a 2-
second gate delay conducted immediately after exposure to inescapable shock. From Shanks and
Anisman, 1988. Copyright ®I988 by the American Psychological Association. Reprinted with
permission.
escape performance across strains of mice. In the DBA/2J strain, performance was unaf
fected by inescapable shock, in the C57BL/6J and CD-I strains, a moderate disturbance
was evident, while in the BALB/cByJ and C3H/HeJ mice pronounced escape distur
bances were evident. The A/J strain was repeatedly found to exhibit a curious response
profile, wherein performance was initially disrupted after inescapable shock, but
improved thereafter. In nonshocked A/J mice, performance decayed over training
(which incidentally is common to most mice) such that the performance of nonshocked
mice was actually inferior to that of shocked mice in later trials. In contrast to the strain
profile seen in the shuttle task, in a forced swim test, BALB/cByJ mice that had been
exposed to inescapable shock exhibited invigorated swimming, while C57BL/6J mice
displayed behavioral suppression (both immediately and 24 hours after inescapable
shock). In CD-I and DBA/2J mice, the response invigoration was evident immediately
after inescapable shock, but within 24 hours, performance did not differ as a function of
the shock treatment. Finally, when the effects of inescapable shock were examined on
self-stimulation responding from the Nas, it was found that performance was retarded in
DBA/2J mice, unaffected in the C57BL/6J strain, and enhanced in BALB/cByJ mice
(Zacharko et al., 1987) (see Fig. 3.10).
Clearly, inescapable shock may disrupt performance in an assortment of tasks; how
ever, the emergence of a behavioral deficit varies as a function of the Strain X Task
interaction. Inescapable shock does not cause a general performance deterioration, and
it is thus inappropriate to assume that a single mechanism accounts for behavioral dis
turbances engendered by inescapable shock or that some animals were exceptionally vul
nerable to behavioral disturbances, while other animals are relatively invulnerable. For
DBA/2J o- — o N o Shock
600
· — · Shock
400
...i
c 200 -5
•H
Ε
0
in C57BL/6J
600
\
LU 400
Η
< 200
liJ 0
ω BALB/cByJ
ζ
o 800
Q.
en
LU
GC 600
400
200
-L
Baseline 0 24 1ΒΘ
T I M E ( h r s . )
FIG. 3.10. Mean (±SEM) rate of responding for electrical brain stimulation from the nucleus
accumbens in three strains of mice. Mice received a 15-minute test session 24 hours prior to
treatment (baseline) and again immediately, 24 hours, and 168 hours following exposure to either
inescapable shock (360 shocks of 2-seconds duration, 150 μΑ) or no shock. Note that the effects
of the uncontrollable stressor on intracranial self-stimulation (ICSS) were strain specific. In
DBA/2J mice, the stressor engendered reduced responding for ICSS; in BALB/cByJ mice,
responding actually increased; while in C57BL/6J mice, performance was unaffected by stressor
application. From Zacharko et al., 1987, reprinted with permission.
example, if conclusions were drawn from the shuttle test alone, one would assume that
the DBA/2J mice are relatively hardy and unaffected by the stressor, while the BALE/
cByJ mice are particularly vulnerable to stressor effects. Analysis of the strain profiles in
the self-stimulation test, however, clearly contradicts this conclusion. In effect, just as in
the human condition where the symptoms of depression vary considerably across indi
viduals, the effects of stressors in the different strains of mice varied across the different
tasks in which mice were tested.
The findings that drugs effective as antidepressants in humans also eliminated behav
ioral disturbances engendered by inescapable shock in animals has been taken as support
for the contention that these paradigms may be useful as animal analogs of the human
disorder. We have taken exception to this, as indicated earlier, on the basis that drugs
that are ineffective as antidepressants may have a therapeutic effect in the animal models
(Zacharko and Anisman, 1989). Furthermore, in evaluating the efficacy of antidepres
sants in animals, it should be considered that these compounds are not always effective
in humans. It seems that the effectiveness of different compounds (e.g., those agents that
primarily block NE reuptake versus those that also effect 5-HT uptake) may vary across
individuals, and this finding has been taken to support the contention that depression
may be a biochemically heterogeneous disorder.
Our analysis of the effects of antidepressants in different strains of mice has, in fact,
revealed that the effectiveness of antidepressants varies across strains of mice and with
the task being examined (Shanks and Anisman, 1989). The shuttle escape deficits pro
duced by inescapable shock in CD-I mice were unaffected by treatment with desmeth
ylimipramine (5 mg/kg, administered twice per day over 15 days). Likewise, desmeth
ylimipramine did not appear to affect the performance of the C57BL/6J or the Β ALB/
cByJ mice. In contrast, desmethyUmipramine enhanced the performance of inescapably
shocked A/J mice. The DA reuptake blocker, bupropion, when applied over 15 days
appeared to have a moderate effect in C57BL/6J mice, while not affecting performance
in the remaining strains. Finally, amitriptyline, which affects the reuptake of 5-HT as
well as that of NE, influenced performance in each of the strains. Curiously, this was the
case irrespective of whether the drug was applied acutely or chronically. The more selec
tive 5-HT uptake blocker, fluvoxamine, produced comparable results, suggesting that the
observed effect was not exclusive to amitriptyline and probably reflects the effects of
blockade of 5-HT reuptake.
In contrast to the effects of desmethylimipramine on escape deficits induced by ines
capable shock, we observed that chronic administration of the drug effectively eliminated
the disturbances of self-stimulation responding from the Nas in CD-I mice (Zacharko et
al., 1984b). It will be recalled that this treatment was ineffective in antagonizing the
escape disturbances in this strain. Thus, it appears that the CD-I mouse is not insensitive
to desmethylimipramine, but rather the ameliorative effects of this antidepressant may
be evident only in some behavioral paradigms. In effect, it appears that the antidepres
sant does not act on the stressor-induced behavioral syndrome, but rather eliminates
specific symptoms of the stressor-induced depression.
5. SUMMARY
Stressors will induce a wide variety of central neurotransmitter alterations. The specific
transmitter variations and the magnitude of these effects are determined, of course, by
factors related to the stressor itself (e.g., severity, controllability), as well as to experiential
factors. In particular, neurochemical adaptation typically occurs following a chronic
stressor regimen. However, in some animals, this adaptation does not readily occur, and
it has been our suggestion that vulnerability to behavioral pathology will be greatest in
these animals. In addition to these variables, stressor-provoked transmitter disturbances
may also be determined by organismic factors such as the age and strain of an animal.
In some strains of mice, for instance, specific amine reductions are readily engendered,
while in others the amine variations may not be provoked by the same stressors. The
nature of the behavioral impairments that are associated with aversive events will pre
sumably be a reflection of the specific transmitter changes induced, the magnitude of
these effects, and the brain regions in which they occur. Similarly the symptoms of
depression vary across individuals, and the profile presented might conceivably reflect
the nature of the endogenous neurochemical disturbances that exist.
Just as the symptom profile of depression differs across individuals, considerable vari
ability exists with respect to the ameliorative effects of antidepressant compounds. Sim
ilarly, the effects of antidepressants in mice may vary appreciably across strains. It is of
particular note, however, that these strain differences may not necessarily reflect drug
insensitivity in some strains, but rather, a particular antidepressant may antagonize only
some of the behavioral disturbances associated with a stressor. In effect, an antidepres
sant may be acting on a particular symptom of depression, presumably by influencing
the transmitter subserving this behavioral impairment. Thus, in the analysis of drug
effects on depression, it might be advantageous to consider specific behavioral character
istics of the disorder, rather than the syndrome itself This would clearly require that
animal models of the disorder evaluate a wide range of stressor-related behavioral dis
turbances in genetically different animals.
Acknowledgments—SuppoTteá by grants MT-6486 and MA-8130 from the Medical Research Council of
Canada.
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Peiâmon Press, Inc. (New York), pp. 83-105
CHAPTER 4
PRIMATE SOCIAL BEHAVIOR—ANXIETY OR DEPRESSION?

SANDRA V. VELLUCCI
Department of Anatomy, University of Cambridge, Cambridge, United Kingdom
1. PRIMATE SOCIAL BEHAVIOR

1.1. BACKGROUND
Numerous studies on the biology of anxiety and depression have been carried out
using singly tested animals, predominantly rodents. Such paradigms have provided a
great deal of useful information concerning the neurochemical aspects of these disorders
and have been of use for predicting whether or not a given drug may be clinically effective
in their treatment. However, single-animal models have limitations if one wishes to gain
insight into the more subtle aspects of normal and abnormal human physiology and
behavior and the way in which they may be affected by drugs. Indeed, psychiatric dis
orders such as anxiety and depression are, at least in part, disorders of social behavior
expressed in social settings. This problem may be overcome by utilizing various tech
niques that enable the study and quantitation of the behavior of nonhuman primates
living in social groups similar to those that are normally characteristic of the particular
species in the wild.
Studies of social behavior in nonhuman primates, in which the findings have been
inteφreted as being directly homologous with those in man, have left many questions
unanswered. However, if one were to consider the findings within a broad context (e.g.,
behaviors with similar functions), then a direct analogy between man and nonhuman
primates could be drawn. As will become evident, such models have proved to be
extremely useful because they provide information that cannot be obtained by studying
animals in nonsocial situations. There are several reasons why nonhuman primates are
the best available animal subjects for this type of research. Phylogenetically, monkeys,
apes, and human beings have evolved from a common ancestor for at least 30 million
years after they had separated from the other mammals. Moφholόgically and function
ally, nonhuman primates possess a very elaborate central nervous system that, in many
ways, is similar to that of man. Many species of nonhuman primates live in structurally
organized social groups, exhibiting a range of behaviors involving dynamic interactions
with other group members, which may be directly comparable with those of humans
(Sassenrath and Chapman, 1976; Raleigh and McGuire, 1980). Although the hierarchies
and attachment bonds that are formed remain relatively constant, they can be manipu
lated by various means, for example, by altering group structure (by removing or adding
members), by manipulating animal behavior pharmacologically (by treatment with hor
mones or psychoactive drugs), by the removal of endocrine organs, by the placement of
lesions in specific regions of the central nervous system, or by changing the environmen
tal conditions of the animals. As in the case of humans, the interactions between the
social position of a nonhuman primate and its response to a drug treatment may be
complex and may influence the efficacy of a given treatment (see later). Such drug effects
cannot be studied satisfactorily in animals that are not socially living. This type of model
is thus potentially useful for human research and, indeed, has had a long association with
clinical research (McKinney and Bunney, 1969). Various types of nonhuman primate
models have been developed involving observations of the behavioral interactions occur
ring within social groups or the behavior occurring following mother-infant or peer-peer
83
84 S . V . VELLUCCI
separation and the effects of drugs thereon, which are considered to be of relevance to
human anxiety and depression. However, before relevant data obtained using these mod
els are described, some mention will be made concerning the social behavior and social
structure of some of the nonhuman primate species that have been studied, along with
some of the problems that may occur in the inteφretation of data obtained from such
studies.
Unlike many mammals that exhibit social behavior only at certain times of the year,
primates live permanently in social groups. This can generate stress between group mem
bers (e.g., as far as access to food, mates, and preferred position are concerned), but can
also prove to be a useful means whereby external stressors (e.g., predators) are dealt with
satisfactorily. The most important property of a social group is that it has a well-defined
structure, that is, the relationships that exist between the different members of a given
group are not merely random or equal (Hinde, 1974).
The relationships thus formed can be divided into various classes, each of which have
specific behavioral features (e.g., mother-infant relationships, interjuvenile interactions,
or sexual consortships). Between-class interactions can occur, for example, an adult male
is likely to respond differently to a juvenile animal than to another adult male. The sec
ond type of interaction is between the members of each class, for example, the adult
males of social groups have an overall distinct pattern of behavior, but between individ
uals there is another set of relationships that are often based on aggressive interactions.
This determines not only their response to each other but also their access to favored
objects and how they will react to a common stressor from either within or outside the
group. The latter will also determine the responses they make to each other, as well as
who has priority of access to favored objects (e.g., choicest food, sleeping places, and
receptive females).
Even from early infancy, when a young monkey can understand and is concerned with
only a very small part of its social group, any event that affects the group as a whole will
also affect the infant, either directly or indirectly via an effect on its mother or peers.
These early social experiences, especially peer interactions, are essential for the normal
emotional and behavioral development of the animal and will greatly influence the way
in which an animal relates to the other members of its social group when it becomes an
adult. For example, Harlow and Harlow (1969) have shown that monkeys raised with
peers, but in the absence of a mother, grow up to be normal adults, whereas monkeys
raised with only a mother show little play and no sexual behavior when adult. On the
other hand, infants deprived of both maternal and peer-peer interactions become behav-
iorally abnormal, with mating behavior being absent and social interaction being greatly
reduced in the presence of normal monkeys. Biochemical and neuroendocrine function
is also altered in these animals.
1.2. THE CONCEPT OF DOMINANCE
As most socially living primates form dominance hierarchies, some discussion of this
concept is considered relevant at this point. The problems this may impose upon the
interpretation of data in a given species and the comparison of data between species will
be discussed. In addition, it is important to remember that the way in which a group
member may respond to treatment with psychoactive drugs will be influenced by the
rank or position held by that animal within its social group.
Much social stress in animals and humans is derived from, or expressed through,
aggressive interactions. Aggression per se is not a unitary behavior but nearly always
occurs in association with other behavioral processes. Socially living nonhuman primates
form dominance hierarchies that may be defined in terms of the outcome of aggressive
interactions. Thus, if animal A attacks animal B, and Β fails to either retaliate or initiate
aggression towards A, then A is said to be dominant to B. If animal Β attacks C and C
attacks D , a linear hierarchy is said to exist in which A is the dominant animal. It does
Primate social behavior—anxiety or depression? 85
not necessarily follow from this that animal A is the most aggressive within the group;
that is, dominance is deñned on the basis of the direction of the aggression and not nec-
essarily on the total amount of aggression given. In well-established social groups of mon-
keys, instances of overt aggression may be rare, and the dominant male is often able to
deal with any conflict or potential conflict that has arisen by a change in facial expression
or posture. Although it is possible to establish the relative position or rank of a monkey
within a social group by observing the behavioral interactions that occur within that
group, it is not possible, by studying a monkey in complete isolation, to ascribe to it the
rank that it will achieve when it is placed in a new group.
1.3. How M A Y DOMINANT BEHAVIOR B E RELATED TO INTERNAL/PHYSIOLOGICAL

FUNCTION?
In addition to there being clearly deñned differences in the types of behavior exhibited
by socially living nonhuman primates according to the rank that they hold, there is also
a difference in the endocrine status of the various group members. Thus, a number of
studies have shown that dominance status can influence circulating levels of adrenal and
gonadal hormones in several primate species (Candland and Leshner, 1974; Rose et al.,
1974; Chamove and Bowman, 1976; Coe et al., 1979; Mendoza et al., 1979; Dixson,
1980; Keveme et al., 1982), although some authors have failed to support these findings
(Eaton and Resko, 1974). Some of the differences between these studies may be
explained as being due to factors known to influence dominance rank. For example, in
the case of gonadal hormones in the male, dominance may have a more marked asso-
ciation with hormonal output during the mating season, and the failure of Eaton and
Resko (1974) to obtain a correlation between testosterone concentrations and rank in
Japanese macaques may have been due to the fact that these animals were not studied
during mating season. The effect of rank on hormone levels is also more marked in com-
petitive situations (e.g., competition for food, space, or attractive females) and following
agonistic encounters such as are seen during new group formation (Bernstein et al.,
1979b). In the talapoin monkey, Keveme (1979) demonstrated that on moving into a
new social group, the monkeys that became dominant showed an initial increase in Cor-
tisol and testosterone but not in prolactin, whereas those that became subordinate
showed increases in Cortisol and prolactin but no increase in testosterone levels. Once
the social group became established, there was little further change in testosterone levels,
but Cortisol was found to increase still fiirther in the subordinates and to decrease in the
dominants. In this species, an increased stress response appears to develop despite the
fact that, at this stage, overt aggression is low or nonexistent. Hence, the association
between (plasma) hormone levels and behavior may be less obvious in well-established
and stable social groups (Green et al., 1972; Bernstein et al., 1979a; Gordon et al., 1979).
Various authors have reported the existence of increased adrenal cortical activity in sub-
ordinate animals (Sassenrath, 1970; Chamove and Bowman, 1978), which agrees well
with observations made in rodents, whereas other studies have reported that dominant
animals can have increased glucocorticoid activity (Candland and Leshner, 1974; Coe et
al., 1979). On examining the information obtained, it is clear that one must take into
account the precise social situation under which the animals were investigated. One must
distinguish, for example, social stress and overcrowding, to which subordinate animals
are very susceptible, from conditions in which dominant animals may be more greatly
affected such as exposure to a novel environment, the introduction of a strange male, or
the introduction of attractive females. The relatively low levels of glucocorticoid concen-
trations seen in dominant males in a stable hierarchy may be inteφreted as reflecting a
lack of stressfiilness for that position. Conversely, the increase in plasma Cortisol concen
trations noted during social instability indicates the increased stressfulness of that posi
tion. As stated previously, although elevated concentrations of glucocorticoids are essen
tial for the adaptation to acute stress, prolonged hypersecretion of Cortisol is pathogenic
86 S. V . VELLUCCI
(Krieger, 1982; Munck et al, 1984). Indeed, it has been shown in subordinate wild
baboons that chronically elevated plasma Cortisol concentrations give rise to suppression
of high-density lipoprotein cholesterol concentrations and of lymphocyte counts (Sapol
sky and Mott, 1987). Thus, when studying the effects of socially induced stress, it can be
seen that the susceptibility of the different ranks will vary depending on the precise con
ditions under which the animals are studied. This is important when data from different,
or even the same, sources are being compared and also, as we shall see later, when inves
tigating the effects of psychoactive drugs on primate social behavior.
In this section, it may also be useful to consider the concept of "control'' or *'control-
lability" and the potential effects this may have on the hypothalamic-pituitary-adrenal
(ΗΡΑ) system. It is well established that organisms deprived of glucocorticoids are unable
to deal effectively with even minor stresses, such as water or salt restriction, that would
normally be of little consequence to an intact organism. Conversely, chronic glucocor
ticoid hypersecretion can lead to suppression of the immune system, behavioral distur
bances and has detrimental effects on the cardiovascular and digestive systems. It is clear,
therefore, that in order to survive normally animals must have evolved a mechanism
whereby they can either regulate or modulate any potentially large disturbances in ΗΡΑ
activity. These mechanisms have been extensively studied by Levine and co-workers (see
Levine et al., 1989, for an excellent review). These authors believe that many of the
mechanisms are essentially psychological, the most important of which being the dimen
sion of "control," which has been defined as the capacity to make active responses during
the presence of an aversive stimulus (Levine et al., 1989). The responses may then be
effective in allowing the animal to avoid or escape from the stimulus or may enable the
animal to change from one set of stimulus conditions to another. Experiments relating
to control and controllability have been carried out in various species, including non-
human primates. Hanson et al. (1976) studied groups of rhesus monkeys exposed to an
aversive loud noise. One group of animals was able to control the duration of the noxious
stimulus by pressing a lever to terminate the noise. A second group was exposed to an
identical amount of noise, but the group members were unable to regulate its duration.
It was found that the animals that were able to exert control over the noise stimulus had
plasma Cortisol levels that did not differ significantly from those of controls not exposed
to any noise, whereas their "yoked" counteφarts had greatly elevated plasma Cortisol
concentrations.
While studying baboons in the wild, Sapolsky and Ray (1989) made the interesting
observation that low basal plasma Cortisol concentrations are not generalized indicators
of social dominance per se. Instead, they are only observed in dominant males that
exhibit a certain style of dominance involving a degree of controllability. Thus, they
noted that lower basal Cortisol concentrations were present in those males that exhibited
any of the following behaviors: a marked ability to distinguish between threatening and
neutral interactions with rivals, and in the former case, the greatest likelihood of actually
initiating a fight; and skill at distinguishing between winning and losing a fight, and in
the latter case, the greatest UkeUhood of displacing the aggression onto a third party.
Overall these behaviors indicate the existence of a high degree of skillfulness, control,
and predictability over social contingencies, all of which are recognized as psychological
features that minimize the pathophysiological impact of stress. Dominant males that
lacked these behavioral strategies, however, had plasma Cortisol concentrations that were
comparable with those of subordinate animals. It has thus been demonstrated that in the
absence of control or predictability, an elevation in plasma Cortisol was evoked, whereas
the presence of both factors prevented, or greatly minimized, the stressful effect, as did
the ability to exert control over stressful stimuli even in the absence of predictability.
Studies that may be of possible relevance to human anxiety and depression have been
carried out in various species of socially housed nonhuman primates (e.g., talapoin and
rhesus monkeys), and some of these studies, along with any relevant details relating to
the group structure and the neurochemical and neuroendocrine profiles of the different
ranks, will now be reviewed.
1.4. STUDIES IN DIFFERENT SPECIES OF NONHUMAN PRIMATES
A brief overview of relevant studies relating to dominance that have been carried out
in different species of nonhuman primate will be presented.
1.4.1. Talapoin Monkeys

I shall start with the talapoin monkey, as this is the species we have used in our studies
of the effects of anxiolytic and anxiogenic drugs on social behavior and neuroendocrine
function (Vellucci et al., 1986). As mentioned earlier, established social groups of tala
poin monkeys (the smallest species of Old World monkey) show a well-defined linear
dominance hierarchy among both males and females. Thus, there are rank-related dif
ferences in aggressive, sexual, and social behaviors, in visual monitoring (Dixson and
Herbert, 1977; Everitt et al., 1981), and cage position, as well as in endocrine and central
nervous system (CNS) monoamine profiles (Keveme, 1979), with animals of high rank
receiving little, or no, aggression and exhibiting more sexual and social (affiliative) behav
ior and less visual monitoring than the corresponding subordinate animals. On the other
hand, individuals of low rank receive aggression from higher ranking animals and show
little, or no, social and sexual behavior, but high levels of visual monitoring. The domi
nant male is not necessarily the most aggressive animal within the group. However, as
stated previously, dominant males receive the least aggression, whereas subordinates
receive the most. It has been shown that dominant talapoin monkeys are able to keep
subordinate animals under control by the use of intermediate-ranking animals. This
strategy frees the dominant male from continuous monitoring of all males in the group,
and aggression can be exerted toward the subordinates through the hierarchical chain of
command. Such intimidation of subordinates puts them under considerable stress and
it is not surprising that, once the social hierarchy within a group has become firmly estab
lished, the levels of "stress" hormones will decrease in dominants and increase in sub
ordinates. In situations in which males and females are caged next to each other, but are
only allowed to interact freely at specific times, concentrations of Cortisol and the 5-HT
metabolite 5-hydroxyindoleacetic acid (5-HIAA) in plasma and cerebrospinal fluid (CSF)
are higher in subordinate male talapoins, whereas plasma testosterone concentrations are
lower than in other mature males housed in heterosexual social groups (Eberhart et al.,
1985; Yodyingyuad et al., 1985). An increase in the concentration of 5-HIAA in the CSF
is usually indicative of increased release of 5-HT such as is seen in conditions of chronic
stress (Curzon and Green, 1969; Kennett and Joseph, 1981; Joseph and Kennett, 1983)
or in anxiety (Stein et al., 1973; File and Vellucci, 1978). These clear, rank-related hor
monal differences are not present in stable groups of male talapoin monkeys that are
housed in large cages, receive adequate amounts of food, and do not have access to
females (i.e., in situations where the occurrence or need for overt aggression is mini
mized; Vellucci and Humby, unpublished observation).
In the case of females, aggressive behavior is rare, and the strategy that dominant
females adopt is quite different. Solicits from the highest ranking females toward males
are not only more frequent than from lower-ranking females, but are more likely to lead
to mounting. Studies have shown that when a female solicits a male of low rank, this
male receives increased aggression from the other males of the group. However, the most
likely behaviors to follow soHcits by low-ranking females are solicits by high-ranking
females. Thus, the strategies that have been adopted to restrict sexual activity in low
ranking nonhuman primate species such as the talapoin differ markedly between the
sexes. Males are more likely to intimidate subordinates by the threat of impending
aggression, preferably via an intermediate animal, whereas females are more likely to
prevent subordinate females from being mounted, by sexually soliciting the male's atten
tion themselves. Thus, in certain situations, a subordinate talapoin is under persistent
threat of aggression from higher ranking animals and is constrained behaviorally by their
constant presence. Furthermore, the subordinate animal cannot escape because of the
spatial confines of the cage in which he is housed. Thus, any neuroendocrine differences
88 S. V. VELLUCCI
that exist between dominant and subordinate animals may be considered to reflect the
degree of stress to which an animal is exposed; that is, a subordinate animal in this sit
uation is exposed to chronic inescapable stress, whereas a dominant animal will only
experience stress more acutely (e.g., during active aggressive encounters). In any event,
the dominant animal will generally be able to control the outcome of a potentially stress
ful situation, whereas a subordinate animal is unable to exert any control over such a
situation. If infrequent, sociosexual activity in subordinate males is a reflection of the
fact that these animals are under the constant threat of, or receipt of, aggression, then it
might be thought that removing all other males from a heterosexual social group would
enable the subordinate to exhibit increased sexual activity. This was tested by Eberhart
et al. (1983), who found that subordinate males in this situation failed to exhibit sexual
behavior. This cannot be accounted for in terms of the direct receipt of aggression, nor
can the differences in behavior between high- and low-ranking males be due to differ
ences in female sexual initiative, since females solicited the subordinates in this situation
to a greater extent than dominants. Thus, it is considered that the state of subordination
produces a long-term effect on behavior that is not readily reversible in the short term,
following removal of the dominant animal(s).
A brief overview of the social structure(s) and rank-related neuroendocrine differences
seen between members of other species of group-living, nonhuman primates will now be
presented.
1.4.2. Squirrel Monkeys

The relationship between dominance hierarchy and hormone levels has been exam
ined in this species (Vogt et al., 1981; Coe et al., 1982). Higher levels of plasma Cortisol
are found in high-ranking males compared with subordinates. Social manipulations such
as new group formation, the introduction of new females into a group, and the occur
rence (especially in large groups) of frequent aggressive interactions may account for
these results and may be related to the dominant monkeys being in a more vigilant and
aroused state. However, if the social conditions are altered such that the subordinates are
forced to continually avoid dominant animals, then plasma Cortisol concentrations
become higher in the subordinates, that is, comparable with the neuroendocrine differ
ences observed between dominant and subordinate talapoin monkeys.
1.4.3. Vervet Monkeys

In the wild, this species of Old World monkey is often found in multimale groups, and
although the dominant male from a social group can be easily identified, the ranking of
subordinates is less clear-cut and their behaviors are not consistently ordered in a linear
manner across a variety of measures. Thus, unlike the adult males of other species of
Old World monkeys such as the talapoin monkey, adult male vervet monkeys appear to
exhibit a binary, rather than a linear, dominance hierarchy. Although there is no clear
evidence for a correlation between social status and plasma Cortisol concentrations in
stable groups of vervet monkeys (McGuire et al., 1986), there appears to be a tendency
for dominant males to have the highest Cortisol levels, although the ranges for dominant
and subordinate animals overlap considerably. Furthermore, some reports clearly indi
cate that plasma Cortisol levels are highest in animals that are competing for dominance
status, especially in those animals that eventually became dominant (McGuire et al.,
1986). However, the dominant animals that were used for this work were isolated over
night prior to blood sampling and, therefore, the direct relevance of these values in
reflecting dominance status in a group situation is questionable.
1.4.4. Macaques
There is evidence for a relationship between plasma testosterone concentration and
dominance in male rhesus monkeys. Following aggressive encounters, those males that
were defeated showed depressed testosterone levels, whereas increases occurred in the
dominant male (Bernstein et al., 1974). Unlike other species of macaque, stumptail
macaques maintain a relatively constant pattern of mating behavior during the year and
do not conform to the well-defined seasonal pattern characteristic of many other
macaques. They also exhibit a clearly defined dominance hierarchy that remains stable
over time. The behavior of macaques has been extensively studied and well documented,
and this species has also been used for psychopharmacological research (Bernstein, 1980;
Bernstein and Gilloud, 1965; Boelkins, 1967; Gouzoules, 1975; Rhine, 1972, 1973;
Rhine and Kronenwetter, 1972).
1.4.5. Baboons
The physiological characteristics of dominant and subordinate wild olive baboons
(Papio anubis) have been extensively studied (Sapolsky, 1982, 1983; Sapolsky and Ray,
1989). Dominant males in stable hierarchies were consistently shown to have low basal
circulating concentrations of glucocorticoids. However, during exposure to stressful stim
uli, plasma Cortisol concentrations rise to similar levels in both dominant and subordi
nate animals (Sapolsky, 1982).
1.5. EFFECTS OF SOCIAL VARIABLES ON H P A FUNCTION
The effects of social variables on HPA function have been extensively investigated in
nonhuman primates, and it has been demonstrated that, at almost every stage of devel
opment, these variables can exert a modulatory influence on endocrine activity, both in
terms of increased hormone release and/or inhibition of endocrine responses to environ
mental disturbances. Disruption of social relationships is a potent stressor giving rise to
activation of the HPA axis, as indicated by increased excretion of urinary 17-hydroxy-
corticosteroids (Rose et al., 1969) and increases in plasma Cortisol in infant rhesus mon
keys (Hill et al., 1973; Meyer etal., 1975) or squirrel monkeys (Coe etal., 1978; Mendoza
et al, 1978) in response to separation. Conversely, the presence of social partners can
reduce or completely eliminate the HPA response that would have occurred in their
absence. Hence, although it can be stressful living in a social group, the presence of group
members can actually reduce the neuroendocrine response to external stimuU that nor
mally evoke marked HPA activation in singly housed animals (Mineka et al., 1980). For
example, Vogt et al. (1981) exposed squirrel monkeys to a live snake, either while they
were housed in established social groups or when they were individually housed. All of
the monkeys were found to show increased vigilance, agitation, and total avoidance of
the snake in both the group and individual conditions. However, on examining the adre
nal responses, it was found that there was only evidence of increased adrenal activity in
the singly housed animals. Thus, it can be seen that social factors such as predictability
of social interaction occurring in a group of familiar conspecifics are able to modulate
the pituitary-adrenal responses to fear-eliciting stimuU.
1.6. STRESS-INDUCED REPRODUCTIVE SUPPRESSION
This is another important feature exhibited by some members of primate social

groups. It has been shown in many species of socially living, nonhuman primates in
which the groups contain more than one male, that reproductive activity is closely
related to the dominance hierarchy. Dominant males copulate more frequently and sire
more young than those lower in the hierarchy. Several mechanisms are believed to con
tribute to the reduced breeding potential of subordinate animals. They may be attacked
by dominant animals if they attempt to mate, the females may show a lack of preference
for them, and the levels of plasma testosterone in the subordinate males may be signifi
cantly lower than those in the dominant animals (Eberhart et al., 1980; Rose et al., 1975).
However, even if subordinates are treated with testosterone supplements, they may stiU
fail to exhibit sexual behavior, thus indicating that factors other than hormonal defi
ciency may be of importance (Dixson and Herbert, 1977; Eberhart et al., 1985).
90 S. V. VELLUCCI
Attempts have been made to estabUsh the nature of these factors, for example, whether
the animal's social rank is important or whether the amount or nature of aggressive inter
actions are more important. For example, it has recently been shown in talapoin mon
keys that there is a negative correlation between mean plasma testosterone concentra
tions and aggression received from other males (Martensz et al., 1987). However, it is
clear that, despite the existence of some degree of steroid dependence of primate sexual
behavior, other factors must also be taken into account such as the evolutionary change
in the complexity of the primate brain, which provides far more flexibility in the expres
sion and hormonal regulation of these behaviors. The structure of the social group in
which the animal lives is also of fundamental importance.
In man, social stress, as well as chronic exposure to fear-provoking situations, can also
resuh in low plasma testosterone levels (McGrady, 1984), whereas socially dominant
men, who may not necessarily be aggressive, have higher serum testosterone levels
(Ehrenkranz et al, 1974). Furthermore, in nonhuman primates, if stress is applied from
outside the group, then the effects on the testosterone concentrations of the group mem
bers will depend on their rank. Plasma testosterone concentrations of dominant monkeys
may be elevated, whereas those of subordinates are lowered (Eberhart et al., 1980; Sapol
sky, 1986). The female reproductive system is also affected by social stress; for example,
subordinate female talapoin monkeys ovulate less frequently than dominant ones, and
rank is positively correlated with fertility (see Keveme and Vellucci, 1988 for review).
To some extent, comparable effects are observed in human females; for example, psy
chological stress, anxiety, or depression are all factors that can lead to reproductive sup
pression. In experimental animals, including rodents, stresses such as social stress cause
increased prolactin release. Furthermore, it is known that, in humans, prolactin has an
antifertility role, thus suggesting a possible mechanism whereby social stress can inhibit
reproductive function, and also suggesting some degree of similarity between human and
nonhuman primates with respect to the reproductive inhibitory effects imposed by social
stress, which in humans we know to be accompanied by the symptoms of anxiety or
depression. In practice, few studies have actually been carried out in order to ascertain
whether or not there are elevated prolactin levels in humans during stress. Acute stress
was found not to elevate plasma prolactin levels (Nesse et al., 1980), whereas more
chronic stress evoked an increase (Herbert et al., 1986). On the basis of the suggestion
that impotence in men is a consequence of raised prolactin levels, Everitt et al. (1981)
investigated the effects of dopamine antagonists (i.e., substances that can cause hyper-
prolactinemia) on the sexual behavior of male rhesus monkeys. However, the induction
of hypeφrolactinemia failed to produce behavioral deficits in male rhesus monkeys that
could correspond with those described clinically in men. Thus, although reproductive
suppression is observed in subordinate monkeys, as well as in humans that are anxious
or depressed, the underlying neuroendocrine mechanism(s) responsible for this have not
yet been fully elucidated and there is much scope for further work.
2. PHARMACOLOGICAL STUDIES IN NONHUMAN PRIMATES

2.1. INTRODUCTION
Investigations of the behavioral responses of animals following manipulation with

pharmacological agents are important because: (1) The animal's response is viewed as
being potentially analogous to the human response; and (2) Pharmacologically induced
changes in behavior are used to make inferences conceming normal species-typical
behavior. Such stuides have provided insight into human behavior and the possible
underlying causes of psychiatric disorders such as anxiety and depression. The two basic
approaches may be extended by emphasizing the interrelationship that exists between
socially induced variables and dmg effects on behavior, and the data thus obtained can
be applied to psychiatric research. As disorders such as anxiety and depression are partly
disorders of social behavior expressed in social settings, it is pertinent to study the drugs
that are of potential use in the treatment of these conditions on animal social behavior,
rather than in singly housed animals. Conversely, it may also be useful to study the effects
on social behavior (and neuroendocrine parameters) of agents that are believed to mimic
the biochemical deficits seen in these disorders. The following section will deal with stud
ies on primate social behavior and neuroendocrine function, involving drugs that are
used clinically in the treatment of anxiety or depression, and will also include data
obtained from studies that have involved the use of drugs or manipulations that have
evoked changes in CNS function that may be considered to be analogous with those
occurring during anxiety or depression.
2.2. ANXIETY, ANXIOLYTIC, A N D ANXIOGENIC DRUGS
Although several authors have attempted to study the behavioral effects produced in
monkeys following pharmacological manipulation with drugs that might, for example,
be expected to increase or decrease anxiety by acting at benzodiazepine binding sites,
most of these studies have made use of classical animal models of anxiety such as the
conflict test or have utilized chair-restrained animals (Sepinwall et al., 1978; Ninan et
al., 1982; Crawley et al., 1985). The effects of such drugs on the behavior of monkeys in
heterosexual social groups have not been previously studied in detail. As far as talapoin
monkeys are concerned, it may be considered that, as a consequence of the social con
straints imposed by the chronic receipt or constant threat of aggression, subordinate tal
apoins housed isosexually, but in visual, auditory, and olfactory contact with females and
allowed to interact together for a fixed period of time daily are more chronically stressed
than the corresponding dominant animals, and this is reflected in the behavioral and
endocrine differences observed between the two ranks. Thus, a subordinate animal is
exposed to a situation over which it has no control and which may have unpredictable
consequences. The chronic exposure to such an environment could ultimately lead to a
situation in which the subordinate animal huddles in a comer, moves very little, shows
high levels of visual monitoring, does not eat, and ultimately succumbs to infection and
dies. Therefore, it may be suggested that a subordinate male talapoin could be chroni
cally "anxious," compared with the corresponding dominant animal, and that perhaps
a subordinate animal would be more susceptible to the pharmacological effects of an
anxiolytic compound. The behavioral pattem exhibited by a subordinate talapoin mon
key may also be analogous with the leamed helplessness paradigm of depression
described in the rat (Seligman, 1975). However, although studies with anxiolytic and
anxiogenic compounds have been carried out in talapoin monkeys (see below), detailed
studies with antidepressant dmgs have not yet been carried out.
Thus, the behavioral and neuroendocrine profiles of dominant and subordinate male
talapoin monkeys have been examined following treatment with either an anxiogenic
dmg (iS-carboUne-carboxylic acid ethyl ester, ß-CCE) or an anxiolytic dmg (the short-
acting benzodiazepine derivative, midazolam) (Vellucci et al., 1986). ß-CCE and related
esters (such as FG 7142) have been shown to exhibit anxiogenic effects in rats (Lal and
Shearman, 1980; Corda et al., 1983; File and Lister, 1983; Vellucci et al., 1988), rhesus
monkeys (Ninan et al., 1982; Crawley etal., 1985; Petersen & Jensen, 1984), and humans
(Dorow et al., 1983). When used in humans, in doses that give rise to occupation of brain
benzodiazepine receptors, FG 7142 has been shown to elicit severe attacks of anxiety,
not accompanied by preconvulsive EEG changes. These symptoms were rapidly reversed
by the IV administration of the benzodiazepine derivative, lorazepam (Dorow et al.,
1983). ß-CCE can be a potent convulsant in monkeys (Skolnick et al., 1983; Vellucci et
al., unpublished observation). Thus the compound was administered in much lower
doses than those normally employed for comparable studies in the rat. The affinity of ß-
carboline derivatives for central benzodiazepine receptors is similar in both rats and
monkeys. The apparently greater sensitivity of monkeys to ß-CCE may be explained
92 S. V. VELLUCCI
largely on the basis of pharmacokinetic factors, as it has been demonstrated that, in the
rat, ß'CCE is rapidly degraded by plasma esterases, whereas in monkeys plasma degra
dation proceeds very slowly (Skolnick et al., 1983). The study of Vellucci et al. (1986)
involved the use of captive talapoin monkeys that had lived in well-established social
groups in the laboratory for 2.5 years. Four social groups were used each consisting of
three to four males and three to four females. The animals were housed in large cages,
each of which was divided into three sections. The males normally live in two thirds of
the cage with the females partitioned off in the remaining one third. At the same time
each day, the partitions between the two parts of the cages were removed, the males and
• untreated
• vehicle
Hß-CCE (375 gg/kg)
Aggression received
(a)
4(
ß-CCE administered to dominant ß-CCE administered to sutwrdinate
24
3
20
1
•o 16
c
12
0^
Aggression given
(b)
24
3 20
16
12
I
3^
JL
FIG. 4 . 1 . Aggression received (a) and given (b), and visual monitoring (c) in untreated dominant
and subordinate male talapoin monkeys and following treatment of either the dominant or sub
ordinate with ß'CCE ( 3 7 5 Mg/kg, I M ) or its vehicle. Each value is the mean ( ± SD) of six to eight
observations. Significantly different from vehicle treatment: * Ρ < 0 . 0 5 ; ** Ρ < 0 . 0 0 1 .
females were allowed to interact for a period of 50 minutes (in order to maximize the
amount of interaction), and their behavior was recorded using a computer-linked
keyboard.
In dominant male talapoins, jS-CCE altered behavior in such a way that the animals
exhibited an increase in aggression, reduced levels of sexual behavior, and increased lev
els of visual monitoring. Treatment of the corresponding subordinate males served only
to increase their levels of visual monitoring and had no significant effect on other behav
ioral parameters. Conversely, treatment with the benzodiazepine derivative midazolam,
using a dose that did not elicit sedation, produced behavioral effects that were essentially
opposite to those evoked by ß-CCE. Thus, when midazolam was given to the dominant
animal, it produced a reduction in aggressive behavior, an increase in sexual behavior,
and a decrease in visual monitoring. However, when the same dose of midazolam was
administered to subordinate males, no significant changes were noted in their behavior.
Thus, contrary to expectation, acute treatment with an anxiogenic (ß-CCE) or an anxio
lytic (midazolam) drug was found to affect the behavior of dominant animals, but not
that of subordinates (see Figs. 4.1[a-c] and 4.2 [a-e]). Possible reasons for this observa
tion are discussed in detail later in this section.
Social status has previously been shown to be an important determinant of drug effects
in monkeys, with the dominant and subordinate animals exhibiting quantitatively dif
ferent responses to the same drug and, in some cases, opposite effects. For example, low
doses of ethanol have been shown to increase the aggressive behavior of dominant squir
rel monkeys without affecting that of the subordinates (Winslow and Miczek, 1985). Sta
tus-dependent effects have also been noted with amphetamine, which has been shown to
have no significant effects on the low levels of aggressive behavior of subordinate squirrel
monkeys, but markedly reduces the aggressive behavior shown by dominant monkeys
(Miczek and Gold, 1983). Furthermore, the same dose of amphetamine decreased loco
motor activity in dominant animals, while increasing it in the subordinates. Other status-
dependent effects of amphetamine have also been reported (Haber et al., 1981; Schlem
mer and Davis, 1981, 1983). Such status-related differences in behavior could reflect
actual differences in responsiveness to the drug between animals of different rank. Alter
natively, it is possible that dominants and subordinates are equally sensitive to centrally
Visual monitprinq
(c)
ß-CCE administered to dominant ß-CCE administered to sutnrdinate

100
~ 60
40
I
20
S D
• untreated
^Vehicle
• ß - C C E (373 ug/kg)
FIG. 4.1. (Continued)
94 S. V . VELLUCCI
Aggression received
(a)
Midazolam administêd Midazolam administered
to dominant to subordinate
40
•Saline
[lMidazolam(125 Mg/kg)[
S 30
20
10
I
Aggression given
(b)
40
Í30
ε
520
10 LL
FIG. 4.2. Aggression received (a) and given (b), sexual (c) and social (d) behavior, and visual
monitoring (e) in dominant and subordinate male talapoin monkeys treated with midazolam
(125 Mg/kg, I M ) or vehicle. Each value shows the mean ( ± S D ) of six to eight observations. Sig
nificantly different from vehicle treatment: * Ρ < 0.05; Ρ < 0.001.
acting drugs, but that the presence of the dominant male prevents (or constrains) the
subordinate from exhibiting behaviors such as aggression. As yet, this has not been stud
ied in detail and, thus, the question remains unresolved.
The effects of ß-CCE have previously been studied in chair-restrained baboons by
Ninan et al. (1982) and Crawley et al. (1985), who suggested that the administration of
this drug to monkeys could be a reliable model of human anxiety. The behavioral effects
observed by these authors, which included increased vocalization, abnormal head and
body movements, increased defecation, piloerection, and scratching, could be readily
antagonized by doses of diazepam that were within the clinical therapeutic range. These
overt behavioral effects were not noted with the much lower doses of ß-CCE used by
Vellucci et al. (1986), who noted behavioral effects that would not have been evident if
the monkeys had not been studied within the context of an established social group. It
is possible that the anxiogenic and other behavioral effects of ß-CCE may be dose-related
in a manner analogous to those of pentylenetetrazole (leptazol/metrazol), which is anxio
genic at low doses in man (Rodin, 1958) and animals (Lal and Shearman, 1980), whereas
at high doses, it elicits more dramatic behavioral effects including convulsions. Crowley
et al. (1974) studied the effects of various centrally acting drugs, including ethanol, on
social behavior in a colony of pigtailed macaques (Macaca nemestrina). Single doses of
Sexual behavior
Midazolam administered Midazolam administered

to dominant to subordinate
(c)
• Saline
UMidazolam (125 ug/kg)
C
i 4
Social behavior
(d)
6i
C
ε 4
1
i.
Visual monitoring
Midazolam administered Midazolam administered

(e) to dominant to subordinate
• Saline
I i Midazolam (125 ug/kg)
ε
- 20
i , .
FIG. 4.2. (Continued)
various concentrations of ethanol (from 0.5-2.0 mL/kg) were administered via a naso
gastric tube to the five males that ranked immediately below the dominant male, with
only one subject being treated at any given time. In the absence of any drug treatment,
the dominant male prevented the other males from mounting and copulating with
females (thus, in the absence of drug treatment, 10% of the observed sexual behavior was
heterosexual in nature and 90% was autosexual). Although treatment with ethanol did
not change the absolute amount of sexual behavior, it dramatically altered the relative
96 S . V . VELLUCCI
proportions of these behaviors. Thus, with the highest dose of ethanol, more than 90%
of the sexual behavior shown by the treated subordinate males was heterosexual. Ethanol
did not affect the relative proportions of dominant or submissive behaviors. Winslow
and Miczek (1985) studied the effects of ethanol in established social groups of squirrel
monkeys and found that it produced differential behavioral effects in dominant and sub
ordinate males.
When one considers the nature of the social structure of groups of talapoin monkeys
and the observed differences in behavior and neuroendocrine parameters between dom
inant and subordinate monkeys, it might be expected that, with the type of drug treat
ment used by Vellucci et al. (1986), the most marked effects would have been on the
subordinate animals, as these may be considered to be the most "anxious" within a given
social group. However, that is not to say that dominant monkeys are not anxious, and
indeed, during the period of interaction between males and females, there are increases
in aggressive encounters within the group brought about by the presence of receptive
females, because the dominant animal has to maintain both his rank and the integrity
of the group. This is obviously stressful, but only acutely so; in any event, the dominant
animal is generally in command of the situation in which it finds itself (hence, the lack
of marked increases in plasma Cortisol concentrations), whereas a subordinate is not.
These observations in talapoins are in accord with observations made by Insel et al.
(1988), who investigated two groups of socially housed rhesus monkeys raised under con
ditions differing in the degree of control or "mastery" over appetitive stimuli in the first
year of life. The so-called learned mastery animals (i.e., those having the ability to control
access to food and water by means of lever pressing) were paired with "yoked controls,"
who obtained identical appetitive stimuli, but only at times chosen by the learned mas
tery group. This difference in controllability was associated with significant behavioral
differences at 7 to 10 months of age, with the learned mastery group demonstrating less
fearful behavior and more exploratory behavior when challenged by environmental stres
sors and exposure to novelty (Mineka et al., 1986). In their own environment there was
little behavioral difference between the two groups, with the animals reared in conditions
of uncontroUability (i.e., the yoked group) appearing slightly more passive and less play
ful. The only significant group differences were the higher plasma Cortisol concentrations
in the yoked group. When the animals were approximately 2 years of age, they received
ß'CCE under the assumption that the learned mastery group would be more resistant to
the effects of jS-CCE than the yoked group. However, although there were differences
between the two groups, the most dramatic effects were observed in the learned mastery
group. This group responded with a marked increase in aggressiveness, especially toward
the observer, whereas the yoked controls showed an increase in fearfulness. When the
study was terminated, the animals were placed in new social groups, and it was subse
quently noted that the learned mastery animals became the dominant members of the
group, whereas the yoked controls became the subordinates. These results suggest that
early experience with controllability may have long-term consequences on social behav
ior and social status. This may also have relevance to humans in that individuals brought
up in an environment in which they always lack controllability may be more prone to
developing psychiatric disorders such as anxiety or depression than others placed in a
similar situation. Further studies on this aspect of primate sociobiology would be most
useful in attempting to answer this question.
At this point, it is interesting to note that the degree of support received from others
in adulthood may also help in coping with social stress, thus reducing the Ukelihood of
occurrence of psychiatric problems. The organism's response to a stressful situation
depends not only on the severity and type of stress, but also on past experience and
available options for coping with such a stress. Thus, increases in plasma glucocorticoid
concentrations can be caused by psychological stress, and the animal's abiUty to predict
and control aspects of the aversive or stressful situation can faciUtate coping and, in cer
tain circumstances, reduce the physiological or behavioral manifestations of the stress
(Lazarus, 1968; Gal and Lazarus, 1975; Hennessy and Levine, 1979). This aspect has
been studied in nonhuman primate species such as the squirrel monkey, in which it has
been shown, for example, that the presence of conspecifics can reduce the neuroendo-
crine response to psychological stress (Vogt et al., 1981; Stanton et al., 1985).
Finally, under this heading, the effects of ethanol will be considered. It is well estab-
lished that ethanol administered acutely in low doses has anxiolytic properties in rodents
tested using the active social interaction test (File and Vellucci, 1978) and in nonhuman
primates tested in a conflict paradigm (Glowa and Barett, 1976). Studies relating to the
effects of ethanol on primate social behavior have concentrated mainly on its effects on
aggression (Miczek et al., 1984; Winslow and Miczek, 1985), on the response to social
separation (McKinney et al., 1983), or on the possible reasons for the development of
alcohol dependence (Crowley et al., 1983; Crowley and Andrews, 1987). The observa-
tions of Winslow and Miczek (1985), who studied the behavioral effects of ethanol
administered to squirrel monkeys in established social groups, are relevant here as there
are similarities between their ñndings and the effects that have been noted with mida-
zolam and jS-CCE in talapoin monkeys (Vellucci et al., 1986). Winslow and Miczek
(1985) found that ethanol produced a biphasic, dose-dependent change in the behavior
of squirrel monkeys, with high doses suppressing aggression and low doses increasing
aggression in dominant animals, whereas the subordinates were generally unaffected by
any of the doses used.
2.3.1. Depression
A primate model that is thought to have many features that reflect aspects of human
depression is the separation model, consisting of either mother-infant separation (Suomi
and Harlow, 1977), or peer-peer separation (Suomi, 1970). For example, following the
separation of young rhesus monkeys from their mothers or peers, a severe syndrome
occurs, described as "despair," which may ultimately lead to the development of life-
threatening illness and may involve failure to maintain an adequate intake of food and
water, lethargy, withdrawal, and nonresponsiveness to external stimuli (see also McKin-
ney et al., 1983 for review). Similar behavioral effects have been reported when human
infants have been separated from their mothers (Seay and Harlow, 1965). Not all non-
human primates, however, were found to exhibit the same degree of response to maternal
separation. Rosenblum and Kaufman (1968) separated bonnet macaques from their
mothers and found only a slight protest reaction with no despair, in contrast to the dra-
matic response observed in rhesus or pigtail macaques. These authors, therefore, sug-
gested that the underlying nature of the structure of the social group of a given species
vastly influences the type of response ultimately made to such a situation. Bonnet
macaques have been shown to spend a much larger proportion of their time interacting
with other members of their social group, and the general behavior of the other group
members is more permissive and responsive to the young monkeys than in the case of
rhesus or pigtail macaques, in which the bonds between the infant and mother are didac-
tic in nature. A severe despair response can, in fact, be evoked in the bonnet macaque if
the animal is prevented from interacting with the other members of its social group dur-
ing the time that it is separated from its mother (Kaufman and Stynes, 1978). Mother-
infant relationships are obviously important, but these are of comparatively short dura-
tion. From an early age, a young monkey will form close relationships with other group
members, which will persist, and continue to play a vital role during the adult life of the
animal and include behaviors such as play, sexual behavior, and food-gathering. Domi-
nance hierarchies are developed and the infant learns to take its place within the social
group. The protest and despair responses made following peer separation are similar to
those noted following maternal separation (Suomi, 1970; Bowden and McKinney, 1972).
The peer-peer separation model has the additional advantage that these relationships,
once established, are less likely to change drastically over time compared with the
98 S. V. VELLUCCI
mother-infant relationship and, hence, the precise time at which separation occurs is less
critical. Furthermore, repeated observations can be carried out.
Most of the earlier work concentrated mainly on the behavioral responses to separa
tion. However, studies have also been undertaken in which various physiological param
eters have also been looked at, and these tend to support the contention that mother-
infant or peer-peer separation are indeed good paradigms for the study of some (but not
all) aspects of human depression. For example. Reite and Short (1978) found that the
changes in sleep pattern of young pigtail macaques separated from their mothers were
similar to the altered sleep patterns found in depressed humans.
It is well established that rhesus monkeys experiencing social isolation or long periods
of separation during their first year of life subsequently exhibit disturbances in social
behavior (Hinde and Spencer-Booth, 1971; Suomi et al., 1971; Capitanio and Reite,
1984) and in their response to drug treatment (Kraemer et al., 1984) during adult Ufe.
That maternal separation or peer-peer separation is a valid model of certain types of
human depression, particularly that seen foUowing separation from an affectional object,
is confirmed by the observation that treatment with clinically useful antidepressant drugs
such as imipramine can produce a significant improvement in behavior with a time
course similar to that observed clinically (e.g., Suomi et al., 1978; see later).
In certain types of depressive disorder in man, in particular primary depression (Brown
and Shuey, 1980), there is evidence of Cortisol hypersecretion that normalizes on recov
ery (Sachar et al., 1973; Rubin et al., 1980). Thus, investigation of ΗΡΑ activity in
depressed patients has been an attractive field of research in biological psychiatry. An
abnormality of Cortisol secretion in depressed patients can be detected by resistance to
dexamethasone suppression. This test was first introduced by Carroll et al. (1976) and
Stokes et al. (1975) and is, to date, the most extensively studied test of endocrine function
in psychiatric patients. This steroid nonsuppression is thought to reflect disinhibition of
the limbic HPA system (Carroll et al., 1981). Although initially the dexamethasone sup
pression test (DST) was reported to be highly specific for primary (endogenous) depres
sion and to represent a specific laboratory test for the diagnosis of melancholia (Carroll,
1982), more recent studies have failed to reproduce the diagnostic utility of this test (Ber
ger and Klein, 1984). These authors reviewed the results obtained by various research
groups and revealed a "mean sensitivity" of 40% and a "mean specificity" of 76% for
the diagnosis of endogenous depression. Nevertheless, Cortisol nonsuppression following
dexamethasone administration occurs significantly more frequently in depressed
patients. So far, only a few studies have been carried out in animals using procedures
analogous to the DST. Some authors have investigated the differential sensitivity of some
stimuli to dexamethasone suppression; for example, physostygmine causes escape of Cor
tisol from dexamethasone suppression in normal subjects (Doerr and Berger, 1983) and
in rats (Lurie et al., 1989). Others, on the other hand, have looked at the ability of "help
less" rats to demonstrate dexamethasone suppression (Haracz et al., 1988; Greenberg et
al., 1989) and found a statistically significant difference in the degree of corticosterone
suppression in response to dexamethasone between helpless and nonhelpless, naive ani
mals. We have carried out some preliminary studies in singly housed and group-housed
talapoin monkeys treated with a dose of dexamethasone that was just sufficient to pro
duce suppression of Cortisol (0.1 mg/kg IM, administered at 11 PM). Group-housed ani
mals showed evidence of Cortisol suppression 16 hours later, whereas singly housed ani
mals did not. There was no significant difference in basal plasma Cortisol concentrations
between the two conditions (Fig. 4.3). In this experiment, we used two groups, each con
sisting of three males housed together in large cages, in the absence of females and with
ample food and water. Although each group had a dominant, intermediate, and subor
dinate animal, the groups were long established (several years) with little evidence of
intermale aggression. The singly housed animals that were tested (six in all) were in small
cages in a room containing several other male and female animals, all of which were
similarly housed. Thus, we have demonstrated clear differences in the ability of dexa-
600-
500-
I 400-
f
i 300-
200 Η
2 íooH
GROUP SINGLY
HOUSED HOUSED
FIG. 4.3. Dexamethasone treatment in group and singly housed male talapoin monkeys. The
effects of dexamethasone treatment (0.1 mg/kg, • ) or saline ( • ) administered IM to male talapoin
monkeys at 11 PM on plasma Cortisol (mean (±SE)) sampled 16 hours later. •Significantly dif
ferent from corresponding saline values Ρ < 0.(X)1 (n = 6).
methasone to produce ΗΡΑ suppression in primates, dependent upon the conditions in

which they are housed. At this point, we are making no attempt to analogize between
singly housed talapoin monkeys and some classes of depressed patients with respect to
their lack of Cortisol suppression in the DST and are well aware of the fact that some
authors have questioned the utility of the DST as a diagnostic test in psychiatry (e.g.,
Berger et al., 1984). Nevertheless the consistently different effects of dexamethasone on
adrenocortical function in talapoin monkeys housed under different conditions are inter
esting observations and are worthy of further study.
2.3.2. Antidepressant Drugs

Imipramine, a tricyclic compound, has been used clinically as an effective antidepres
sant. Thus, it is not suφrising that this compound was tested in the primate separation
model in order to ascertain whether it is, indeed, a valid model for certain types of
human depression. Studies by Suomi et al. (1978) involved the use of two groups of
rhesus monkeys that were subjected to repeated peer separation. Halfway through
the procedure, one group of animals was treated with imipramine hydrochloride (10
mg/kg/d), with the other group acting as the control receiving saline treatment. These
authors found that the imipramine-treated animals showed a significant improvement in
their behavior, with a time course similar to that noted when the drug is used cUnically.
Following the cessation of therapy, the behavior resembled that of the saline-treated
group. These observations provide further support for the idea that primate separation
models may be useful in extending our knowledge of the etiology and treatment of cer
tain forms of human depression.
Clinically, amphetamine is not an effective antidepressant when used chronically,
although some data has suggested that it may have short-term antidepressant properties.
Evidence from studies by McKinney et al. (1983) have indicated that, in restricted cir
cumstances, amphetamine reduces despair behavior in socially separated rhesus mon
keys. In this respect, it is similar to imipramine, and it could therefore be suggested that
amphetamine has antidepressant properties. However, these are limited as, unlike imip
ramine, amphetamine was also found to disrupt social behavior at doses that exert anti
depressant efiects, producing dose-related increases in agonistic behavior. Ethanol may
also be considered at this point. The relationships between ethanol and depression in
humans are complex. Humans sometimes drink in an effort to treat their depression;
however, any euphoric effects are temporary and quickly evolve into depressant effects.
100 S. V. VELLUCCI
Studies carried out in rhesus monkeys have demonstrated that, whereas high doses of
ethanol produced effects that were indicative of increased depression, treatment with
lower doses had clear antidepressant effects (Kraemer et al., 1981). In this study, ethanol
was administered to juvenile rhesus monkeys (25% ethanol, in doses of 1,2, or 3 g/kg/d).
The dose levels were chosen so that they produced large, moderate, or minimal acute
effects on behavior. With the higher doses, behavior was altered in the peer-housing con
dition, as well as in the separation condition, by producing a more severe despair
response than that noted with placebo. The lowest dose of ethanol decreased the inci
dence of huddling and passive behaviors during separation, compared with placebo, thus
resulting in a less severe despair response. This dose of ethanol significantly ameliorated
the response to separation without affecting the despair response in the group-housing
condition. Thus, alcohol has a biphasic action in the separation model; at a low dose, it
behaved like an antidepressant, whereas at higher doses it exerted depressant effects.
2.3.3. Drugs Producing Depression

Drugs that interfere with the function of monoaminergic systems have been shown to
produce the symptoms of depression and the following have been investigated in non-
human primates.
2.3.3.1. Drugs Affecting Catecholamine Systems. Redmond et al. (1971) treated two
adult female macaques, who were living in a social group consisting of one adult male
and two other females, with the catecholamine synthesis inhibitor a-methyl-/?-tyrosine
(aMPT). Following control observations, the two subjects then received 160 to 250
mg/kg of aMPT, an amount calculated to produce a 50% to 80% depletion of dopamine
and noradrenaline lasting up to 16 hours. To maintain depletion, the animals received
additional aMPT every 12 hours. Following drug treatment, there were quantifiable
changes in social interaction and appearance. Both subjects initiated fewer social inter
actions, including grooming, threats (despite the fact that one of the animals had been
comparatively dominant), and attacks (although total social responses and sociosexual
presentations remained stable). The animals also showed reduced locomotor activity and
changes in posture and facial expression, suggesting withdrawal and lack of concern with
the environment. Although it is not necessarily appropriate to draw direct analogies
between different animal species (e.g., depression and sadness are best referred in the
context of human behavior), the effects produced by aMPT in nonhuman primates
described by Redmond et al. (1971) bear a striking resemblance to the behavioral
description of depression in humans. Furthermore, it is interesting to note that the
behavioral features described following treatment of macaques with aMPT are similar
to the behavioral patterns exhibited by subordinate male talapoin monkeys. In view of
this, it would be useful to investigate the effects of antidepressant drugs in subordinate
male talapoins, as this has not yet been done.
Finally, reseφine also produces depressive states that are clinically indistinguishable
from the naturally occurring types observed in a small percentage of humans, and it is
used to produce an animal model of depression. When administered to monkeys, reser-
pine produced behavioral features similar to those described by Redmond et al. (1971).
2.3.3.2. Drugs Affecting 5HTSystems. Raleigh and McGuire (1980) carried out extensive
studies of the effects of drugs known to influence central serotoninergic function on
social behavior in vervet monkeys. The drugs used were L-tryptophan and 5-hydroxy-
tryptophan (5-HTP) (which increase brain 5-HT content and turnover), /?-chlorophen-
ylalanine (/?CPA, which reduces brain 5-HT by inhibiting tryptophan hydroxylase non-
competitively), and chlorgyüne (a monoamine oxidase inhibitor, which will thus
enhance the activity of other monoamines as well as 5-HT). These authors reported that
tryptophan-loading led to calmer, more tranquil animals, whereas the administration of
pCP\ produced irritability and aggression. However, the precise response seen depended
upon the social rank of the animal. For example, following the administration of L-tryp-
upon the social rank of the animal. For example, following the administration of L-tryp-
tophan (20 mg/kg/d), the males exhibited an increase in approaching, grooming, resting,
and eating and a decrease in locomotor activity, solitary behavior, vigilance, and avoid
ance. The changes were found to be more rapid and of a greater magnitude in dominant
males. Relative to subordinate males, the dominant males responded to the tryptophan
load with a greater absolute and relative increase in whole blood serotonin concentration
(Brammer, et al., 1983). The 5-HT uptake inhibitor, fluoxetine, and the 5-HT receptor
antagonist, quipazine, evoked status-related behavioral effects similar to those seen with
L-tryptophan (Raleigh et al., 1985).
It is believed that pharmacologically-induced changes in 5-HT systems exert permis
sive effects on behavior. Thus, it is possible that the subordinate males may be con
strained from exhibiting overt changes in behavior by the continued presence of the
dominant male (Fairbanks et al., 1978). As L-tryptophan, fluoxetine, and quipazine act
via different mechanisms, it is considered unlikely that differences in drug metaboUsm
or transport could account for the rank-related differences in behavior. An alternative
explanation is that dominant and subordinate animals differ in their CNS responses to
drugs. Preliminary studies by Brammer et al. (1987) indicated that there were no differ
ences in the properties of the 5-HT2 binding sites of dominant and subordinate animals.
However, this observation does not exclude the possibility that there are differences in
other 5-HT receptor sites. The data of these authors also indicate that there is a relation
ship between the social role of a treated animal and the behavioral changes in nontreated
animals. In the case of established social groups of vervet monkeys, chronic treatment
with pCPA (80 mg/kg daily for 14 days) caused irritability, increased aggression, and
hyperactivity. Changes in a treated animal may alter the behavior of the other group
members, depending on which animal receives the drug; for example, when the domi
nant animal is treated, the degree of social disruption among the other members of the
group is far greater than if a lower-ranking animal had been treated. Even though dom
inant and lower-ranking males may appear equally calm prior to drug treatment, the
low-ranking males may be constrained from exhibiting behavioral alterations once a
drug has been administered. Thus, both metabolic factors and social variables (mediating
their effects through various neuroendocrine/metabolic systems) contribute to these
behavioral differences.
3. SUMMARY
From the data reviewed here, it is evident that social behavior in nonhuman primates
can be used to study both anxiety and depression, depending on the social setting
employed and the way in which the animals are manipulated. In established social
groups of monkeys, the way in which an animal responds to a given situation or manip
ulation is closely dependent on the position or rank the animal occupies within the group
and also on the prior experience(s) of that animal during early life. A study of the neu
roendocrine, neurochemical, and behavioral profiles of dominant and subordinate mon
keys indicates that the behavior of dominant animals is more susceptible to drugs that
are known to manipulate levels of anxiety, whereas subordinate animals appear to be
more susceptible to treatment with antidepressant drugs.
Acknowledgments—I wish to thank the MRC for financial support. I am also grateful to Trevor Humby for
helpful comments.
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PeiSamon Press. Inc. (New York), pp. 107-130
CHAPTER 5
DRUG DISCRIMINATION MODELS IN ANXIOLYTIC

AND ANTIDEPRESSANT RESEARCH
J. S. ANDREWS* AND D . N . S x E P H E N s t
^Scientific Development Group, Organon International BV, Oss. The Netherlands
if Department of Neuropsychopharmacology, Research Laboratories of Schering AG, Berlin, Federal Republic
of Germany
1. INTRODUCTION
The ability of animals to identify distinct interoceptive states has been inferred from
state-dependent learning for many years. Drug discrimination paradigms make use of
this ability to produce a stable behavioral assay that can be used to investigate similarities
and differences within and between classes of psychoactive compounds.
Many species have been trained to identify and respond appropriately to the charac
teristic interoceptive properties of different drugs. Although a wide range of methods has
been used, two procedures with minor variations dominate the ñeld. In the ñrst, animals
(usually rats) are trained to obtain food (or to avoid shock) from one arm or the other
of a T-maze, depending on which drug has been administered. In the other method,
animals are trained to press one lever in a standard operant chamber (again to receive
food or avoid shock) in the presence of one drug and the other lever when administered
a second drug or saline. The training drug is alternated with the placebo (or second train-
ing drug) in a random manner until the animal can, on any test or training day, consis-
tently make the appropriate response.
Once animals are trained, they can be used repeatedly as a behavioral assay for drug
effects. Two basic types of experiment are possible: generalization to the stimulus and
antagonism of the stimulus. In the first method, a test drug is injected in place of the
training drug, and a dose-response curve is generated as to how, and at what dose, the
substance substitutes completely for the training drug. In the second procedure, antag-
onism, the test drug is administered shortly before or after the training drug and a dose-
response curve constructed for reduction of drug-appropriate responding. An as yet unre-
solved question is whether the animals recognize intermediate doses of the training or
test drug in a quantal manner. It has been suggested that recognition of the drug is in an
all-or-none fashion, although different animals may be more sensitive to a particular
drug dose than others, thereby giving rise to a group dose-response curve. On the other
hand, others believe that the discriminative stimulus is graded in response to the drug
dose. For this reason, some researchers use individual trial-based procedures, or individ-
ual percent responding on the appropriate lever (graded effects), and others, a simple
first-lever-chosen principle (quantal effect). In practice, both procedures appear to func-
tion equally well, and therefore, in this paper, all drug discrimination procedures will be
treated as equivalent, the important point being that animals have learned a particular
interoceptive stimulus against which other drugs can be compared.
Drug discrimination using this technique is highly specific: animals trained in a two-
lever discrimination do not learn a simple drug versus no-drug condition. Rather, they
learn a "this-drug'7"not-this-drug" discrimination, or more correctly, "this-class-of-
compounds" versus "all-other-substances." Thus, an animal trained to distinguish
between diazepam and saline will respond on the drug lever only when a similar ben-
zodiazepine-like drug is injected and not when substances such as amphetamine, halo-
peridol, or moφhine are administered. Multiple drug discriminations are also possible
107
108 J. S. ANDREWS AND D . N . STEPHENS
(e.g., White and Hoitzman, 1981; France and Woods, 1987; Overton and Shen, 1988),
even in man (Chait and Johanson, 1988; Preston et al., 1987). Indeed, it has been pos
sible to train animals to distinguish compounds acting at different subtypes of receptor
within the same compound class. For example, animals can learn to distinguish between
drugs acting at subtypes of opiate receptors (Hoitzman, 1985).
This ability of animals to identify specifically the interoceptive stimulus properties of
a substance has led to an increasing interest in defining the limits and uses of the tech
nique. Several postulates have been proposed. For instance, if animals are trained to
identify a particular drug from saline, then all substances that substitute for the training
drug should have similar properties (although not necessarily the identical mechanism
of action). It should be noted that saline-appropriate responding following a test drug
does not mean that the test drug is inactive, only that the interoceptive properties are not
the same as the training drug. The predictions from this approach can thus be simply
stated using one or two examples: all substances thus far identified as heroin-like are
potential drugs of abuse; all substances identified as diazepam-like are anxiolytics. The
idea that stimulus control of behavior by a drug correlates well with a particular psycho
logical construct such as anxiety has been promoted by several workers in recent years.
Thus, drug cues have been described as being based on anxiolytic, sedative, or anxiogenic
properties of the particular compounds involved (Cooper et al., 1987; Sanger, 1988; Lal
and Emmett-Oglesby, 1983). The following sections of this chapter will investigate the
usefulness of such statements. The article will attempt to review the important aspects
of drug discrimination in psychiatric research. Accordingly, an extensive review of every
possible substance used to form a discriminative stimulus will be avoided; instead, we
will concentrate on the salient aspects of a few representatives from, in particular, anxio
lytic research.
2. ANXIOLYTIC CUES
The term "anxiolytic cue" is generally taken to be synonymous with a benzodiazepine
(BZ) agonist cue of one form or another. However, in recent years other substances have
been proposed and/or introduced into the market as novel anxiolytics. The most notable
of these is buspirone, a partial agonist at the 5-hydroxytryptamine 1A (5-HT, A) receptor.
As there are also several good reasons to suspect the involvement of 5-HT in anxiolysis,
data involving such cues will also be reviewed.
2.1. BENZODIAZEPINE CUES
Research into the discriminative stimulus properties of BZs was pioneered by Colpaert
(Colpaert et al., 1976; Colpaert, 1977b), who demonstrated that rats could be trained in
a two-lever task to discriminate an oral dose of 5 mg/kg of chlordiazepoxide (CDP) from
saline. These studies also suggested that the cue was not exclusively attributable to the
sedative or ataxic properties of CDP. Subsequently, the stimulus properties of CDP have
been investigated in several animal species, with different routes of administration, but
in general have all reached similar conclusions.
Further research has shown that all agonistic BZs can substitute for CDP in several
different procedures (e.g., Colpaert, 1977b; Stephens et al., 1987; Sanger, 1988). Accord
ingly, it has also been demonstrated that CDP will itself substitute for other BZs such as
diazepam (Stephens et al., 1984b; Shannon and Herling, 1983; Young and Glennon,
1987). Other investigators have used not only CDP and diazepam as training drugs, but
also many other BZs such as lorazepam (Ator and Griffiths, 1986), oxazepam (Hendry
et al., 1983; de la Garza et al., 1987), and midazolam (Garcha et al., 1985; Rauch and
Stolerman, 1987; Woudenberg and Slangen, 1989). Indeed, it is now clear that BZs sub
stitute readily for one another; that is, BZ-trained animals recognize other active BZs as
being similar, irrespective of which BZ is used as the training drug. The stimulus prop-
Drug discrimination models in anxiolytic and antidepressant research 109
erties of the cue are reported as stable in that there are no marked changes in generaliza
tion curves over time, or between young and old rats (Amrick and Bennett, 1987a). The
discriminative stimulus is specific in that only BZs (or compounds acting at the BZ/
GABA receptor complex, such as barbiturates, e.g., Ator and Griffiths [1983], Nieren
berg and Ator [1990]) generalize; substances from other drug classes, for example, stim
ulants such as amphetamine, do not substitute for the training drug (Colpaert, 1977b;
Sanger, 1988). Although the generalization between BZs and barbiturates has sometimes
been inteφreted as meaning the stimulus properties of these behaviorally depressant
compounds are identical, there are important differences. Benzodiazepine cues can be
antagonized in a dose-dependent manner by BZ receptor antagonists such as Ro 15-
1788, ZK 93 426, or CGS 8216 (Herling and Shannon, 1982; Stephens et al., 1984a;
Bennett et al., 1987; Young et al., 1987), whereas barbiturate cues are typically unaf
fected (Young et al., 1987). In addition, rats can be trained to distinguish BZ and bar
biturates in drug-drug discriminations (e.g., Henteleff and Barry, 1989). Benzodiazepine
cues may also show a stereospecific separation of agonistic stimulus properties (Järbe et
al., 1988).
Potencies for inducing diazepam-like effects in rats and potencies for inhibiting the
binding of radiolabeled diazepam to rat cortex membranes are positively correlated
(Shannon and Herling, 1983). Young and Glennon have therefore suggested that a rela
tionship may exist between relative potencies in a BZ drug discrimination procedure and
therapeutic potency. This suggestion has found some experimental support (Young and
Glennon, 1987). As an animal model for identifying compounds with anxiolytic prop
erties, it appears, then, that the BZ cue has some credibility.
However, in recent years, it has become apparent for several reasons that drug discrim
ination cannot be used to identify anxiolytic drugs in any but the most Umited situations.
The nature of the BZ cue is as yet unclear, but it would be naive to suggest that it is based
exclusively on anxiolytic properties of the drugs. Given the broad range of effects of BZs,
the discriminative stimulus produced is likely to be extremely complex, and as yet it is
unknown which, if any, properties of the BZs dominate in forming a discriminative stim
ulus. For instance, a correlation exists between the anticonvulsant properties of BZs and
their effectiveness as anxiolytics in the clinic (Clody et al., 1983); following Young and
Glennon, a positive correlation is therefore to be expected between anticonvulsant effi
cacy and generalization potencies in BZ cues. Furthermore, convulsant agents such as
pentylenetetrazole will antagonize BZ discriminative properties, and although the inter
pretation is extremely unUkely, the evidence is equally persuasive that the BZ cue is, in
fact, an anticonvulsant cue (see also Colpaert et al., 1976). Despite the obvious complex
ity of the discriminative stimulus, the cue also suffers paradoxically from being too spe
cific. Using such an approach, only BZ receptor ligands with similar properties (apart
perhaps from potency) to those already existing would be (and have been) identified, and
the ability to identify novel anxiolytic compounds with other pharmacological actions is
lost.
Despite this criticism, BZ cues do have an important place in investigating the prop
erties of the BZ receptor complex and in the development of new anxiolytic drugs. Over
the last 12 years, the BZ receptor complex has been characterized, subtypes discovered,
and ligands with unexpected properties identified.
2.2. NONBENZODIAZEPINE BENZODIAZEPINE R E C E P T O R LiGANDS
The discovery of high-affinity central binding sites for BZs (Squires and Braestrup,
1977; Möhler and Okada, 1977) prompted a search for the natural receptor ligand. The
iS-carboline, ie-carboUne-3-carboxylic acid ethyl ester (jS-CCE), was initially thought to be
one candidate (Braestrup et al., 1980). Subsequently, it was shown that this substance
was an artifact of the extraction procedure. Nevertheless, its discovery led to a new appre
ciation of the complexity of the BZ receptor complex (see Fig. 5.1). Beta-CCE binds
, Chloride channel protein
BZ-R protein
agonists barbiturates
antagonists picrotoxinin
inverse agonists Ro 5-3663
GABA agonists
GABA-R protein GABA antagonists
FIG. 5.1. Model of the GABA receptor (GABA-R)—benzodiazepine receptor (BZ-R)-chloride

channel complex including the barbiturate binding site (BARB), illustrating the most important
interactions between the various binding sites. All three types of binding sites shown regulate the
chloride channel. The BZ and barbiturate binding sites can enhance (or inhibit) the binding of
GABA. For discussion of the various discriminative stimulus properties of substances acting at
this site, see Sections 2.1 and 2.2 under Anxiolytic Cues. Adapted from Pole et al., 1982.
specifically to the BZ receptor and is a potent inhibitor of tritiated diazepam or flunitra

zepam binding. However, ß-CCE was the first of a new class of compounds known as
inverse agonists, that is, substances that produce an action opposite to those of the pre
sumed agonist, in this case, a BZ. Depending on their properties, three types of BZ recep
tor ligands have been identified: agonists, neutral antagonists, and inverse agonists.
Moreover, in addition to these three main classes, subclasses of partial agonists and par
tial inverse agonists have been synthesized (e.g., Jensen et al., 1984; Petersen et al., 1984;
Stephens et al., 1987) and tested in the cHnic (Dorow et al., 1987). Subsequently, deriv
atives of other BZ receptor ligands have also been synthesized for each of these ligand
classifications.
Although i8-carboline and BZ structures are distinct, the fact that they bind to the same
receptor suggests that agonist substances should possess similar discriminative properties
with BZs. This has been demonstrated in several studies using CDP or diazepam as the
training drug (Stephens et al., 1984b, 1987); in addition, antagonism of BZ cues with β-
carboline and other non-BZ antagonists and inverse agonists has been reported (Bennett
et al., 1987; Shannon et al., 1988; Stephens et al., 1984a). Many of the newer /8-carboünes
and other non-BZ BZ ligands are partial, as opposed to full, agonists. That is, they do
not show the full range of BZ effects and, in some cases, may antagonize the effects of
full agonists. Typical partial agonists require a higher receptor occupancy to achieve the
same effects as an agonist, and this occupancy rate may differ dramatically from behavior
to behavior.
Given that there now exist BZ receptor Ugands that, in many cases, show only some
of the expected BZ properties (e.g., anticonflict, but no sedative actions), it becomes pos
sible to research the nature of the BZ cue more thoroughly. Nonsedating BZ receptor
ligands such as ZK 91 296 and CGS 9896 generaUze readily to BZs (Stephens et al., 1987;
Sanger, 1988), suggesting that sedative or muscle relaxant properties are not essential for
the establishment of the cue.
A number of non-BZ BZ receptor ligands have been reported recently as producing
discriminative stimuU. However, the effects of BZ ligands in substitution experiments
have been far from simple. ZK 95 962 is a /3-carboline partial agonist at the BZ receptor
(Stephens et al., 1987). It generalizes to CDP and diazepam, and CDP and diazepam
generalize to ZK 95 962 (Andrews and Stephens, 1988). However, further research indi-
cates that the j3-carboline and BZ discriminative stimuli are not as similar as first
thought. Table 5.1 shows the ED50S for generalization and antagonism of the CDP and
ZK 95 962 cues. There are several striking differences, notably the failure of the full
agonist ZK 93 423 to generalize, and Ro 15-1788 to antagonize, the ZK 95 962 cue.
As can also be seen in Table 5.1, Ro 15-1788 generalizes to the ZK 95 962 discrimi
native stimulus. Under certain conditions (i.e., a very low training dose of CDP), Ro 15-
1788 has also been shown to generalize to CDP (E)e Vry and Slangen, 1986). Suφris-
ingly, Ro 15-1788 can also form a discriminative stimulus (Woudenberg and Slangen,
1990). However, the specificity of the cue is poor, and it is assumed that the interoceptive
cue formed is correspondingly weak. Weak, nonspecific cueing properties probably also
account for the fact that a low-dose CDP cue, but not a high-dose CDP cue, will gener
alize to Ro 15-1788. Although compounds with low intrinsic activity at a particular
receptor will probably lead to weak stimulus control (see Shannon and Holtzman, 1979;
Colpaert et al., 1988; Colpaert, 1988 for more detailed discussions), it does not auto
matically follow that a substance displaying only some of the typical BZ properties will
produce weaker stimulus control of behavior. There are increasing reports as to the inho-
mogeneity of BZ receptors in the central nervous system (CNS), and it may be that cer
tain selective effects of partial agonists are, in fact, due to actions at specific receptor
subtypes. Because of the paucity of firm experimental data, this hypothesis must still be
treated with some caution; nevertheless, there are several compounds with intriguing
discriminative stimulus properties.
For example, Zolpidem, an Imidazopyridine that binds to central BZ receptors, has a
profile of action that suggests that, in contrast to other BZ ligands, the dominant effect
is one of sedation (Sanger et al., 1987). In zolpidem-trained rats, substitution by BZs
occurs only at high, extremely sedative doses (Sanger and Zivkovic, 1986; Sanger, 1988).
Moreover, generalization to CDP is usually reported as incomplete and, again, only
occurring at very high sedative doses (Sanger, 1988). The same group has also shown
that Zolpidem interacts with other partial agonists in an atypical manner. The partial
agonists CGS 9896 and ZK 91 296 both substituted for, but did not antagonize, the CDP
TABLE 5.1. Generalization and Antagonism of Cues Provided by

Chlordiazepoxide and ZK 95 962
Drug cue
Chlordiazepoxide ZK 95 962
Training dose (5 mg/kg) (10 mg/kg)
Generalization test drug

Chlordiazepoxide 2.5 1.5
ZK 95 962 1.6 1.5
Diazepam 1.5 3.8
CGS 9896 2 5
Z K 9 1 296 10 40
ZK 93 423 0.1 >1.25
Phenobarbital 22 >40
ZK 93 426 >20 20
Ro 15-1788 >40 2.3
Antagonism test drug
Ro 15-1788 1.4 >10
FG7142 1.8 >5
Z K 9 1 296 0.8 >20
Z K 9 3 426 0.26 10
All values represent EDsos in mg/kg.

> X , Further testing usually not continued as subjects stopped responding.
The ability of various BZ/GABA receptor ligands to substitute for, or to antagonize
the discriminative stimuli induced in rats by the sedative BZ chlordiazepoxide and the
non-sedative /3-carboline ZK 95 962. Data adapted from Andrews and Stephens (1988).
See text under Section 2.2 for further discussion.
discriminative stimulus; in contrast, both substances antagonized the Z o l p i d e m discrim

inative stimulus (Sanger, 1987; Sanger and Zivkovic, 1987). In a recent article, it was
suggested that the anticonvulsant and sedative effects of Z o l p i d e m could not be explained
by a simple receptor-occupancy hypothesis because of Z o l p i d e m ' s unusual interaction
with a selective antagonist for sedative effects (the j8-carboline, 3-[methoxycarbonyl]-
amino-jS-carboline [jS-CMC]; Perrault et al., 1988). However, an equally plausible inter
pretation of this data might be that the sedative effects of BZs require a high receptor
occupancy, so that even a small reduction in the binding of such ligands should result in
a loss of sedative effects. Since jS-CMC binds only weakly to BZ receptors, it is probably
capable of displacing sufficient Z o l p i d e m to block its sedative effects while still leaving
enough attached for anticonvulsant activity, which generally requires much lower doses
of BZs. Nevertheless, it is important to note that distribution of Z o l p i d e m binding sites
in brain shows a different regional selectivity than that found with Ro 15-1788 or fluni
trazepam (Niddam et al., 1987; Benavides et al., 1988; Dennis et al., 1988).
The discriminative stimulus effects of CL 218872 have also been attributed to a site-
specific action at BZ binding site. CL 218872 shows both anxiolytic and sedative effects
(Oakly et al., 1984) and substitutes for both CDP and Z o l p i d e m in appropriately trained
rats (Gardner, 1989; Sanger, 1988; Stephens et al., 1987). However, in rats trained to
identify CL 218872 from vehicle, CDP shows only partial substitution, whereas Z o l p i d e m
generalized more potently to the CL 218872 cue (Gardner, 1989). Although the gener
alization profile of CL 218872 is clearly different from that of CDP, it is not identical to
Z o l p i d e m . Gardner (1989) suggests that this may be due to a lesser activation of the BZ
receptor subtype to which both compounds may preferentially bind (i.e., CL 218872 has
a reduced intrinsic activity at this site in comparison to Z o l p i d e m ) . Zolpidem and CL
218872 are not the only two compounds that show asymmetric generalization with CDP
suggestive of an action at a more selective site. The anxiolytic jS-carboUne abecamil (Ste
phens et al, 1990) substitutes for CDP; however, in abecamil-trained rats, generalization
by CDP, diazepam, or even other jS-carboUnes is not readily elicited, but substitution to
the abecamil stimulus by both Z o l p i d e m and CL 218872 is found (Andrews and Ste
phens, unpublished observations). Zolpidem, abecamil, and CL 218872 discriminative
stimuli are all antagonized by BZ antagonists, but they nevertheless differ from each
other, as well as from BZs, in their pharmacological profiles.
The partial agonist CGS 9896 also has an interesting profile of action in comparison
to the BZs and Z o l p i d e m . This compound is one of a series of pyrazaloquinolines that,
although active in animal models of anxiety (CGS 9896 is also reported active in the
clinic), do not show other effects typical of BZs (Bennett, 1987). Due to the lack of side
effects or interactions with substances from other classes such as ethanol or hexobarbital,
it has been proposed that CGS 9896 should be labeled an anxioselective substance (Ben
nett, 1985; Bennett et al., 1985). Furthermore, Bennett (1985) has suggested that the
discriminative stimulus generated by CGS 9896 is dependent on its anxiolytic activity.
In keeping with the theme of this paper, a specific anxiolytic cue must be regarded with
skepticism. It is certainly tme that the discriminative stimulus properties of CGS 9896
differ somewhat from those of BZs such as diazepam, but this does not mean that the
cue formed is anxiolytic. CGS 9896 displays its activity by an action at the BZ/GABA
receptor complex and has the profile of a classic partial agonist at that complex. CGS
9896 will both generalize and antagonize BZ discriminative stimuU; is itself antagonized
by Ro 15-1788, but not by another type of antagonist (the inverse agonist 6,7-dimethoxy-
4-ethyl-i8-carboline-3-carboxylic acid methyl ester [DMCM]) (Bennett, 1985, 1987; Lei-
denheimer and Schechter, 1988b); and although interactions with barbiturates in other
tests may be minimal, generalization to pentobarbital is readily elicited (Leidenheimer
and Schechter, 1988b). The stimulus properties of this and other BZ receptor ligands are,
however, helpful in identifying behavioral differences between the dmgs and their inter
action with the BZ/GABA receptor complex.
Although the effects of BZ ligands are ultimately achieved by an action at the chloride
ionophore coupled to the GABA receptor, the discriminative stimulus properties of diaz
epam apparently cannot be mimicked by GABA itself; GABA agonists and antagonists
do not usually generalize or antagonize the BZ cue (Haug, 1983; Rauch and Stolerman,
1987). And although GABA agonists also possess muscle relaxant and sedative proper
ties, they are generally not found to possess anxiolytic effects. Again, caution must be
used in interpreting the results; these findings may suggest that the discriminative stim
ulus is not dependent entirely on sedative or muscle-relaxant effects, but it is not proof
that the cue is therefore based on anxiolytic properties. Surprisingly, the GABA agonist
SL 75 102 shows a high degree of stimulus similarity with CGS 9896 (Leidenheimer and
Schechter, 1988b), but not with CDP (Sanger, 1988), and it remains to be seen as to
whether or not this substance also exhibits anxiolytic effects.
In summary, the discriminative stimulus effects of the newer compounds are complex.
There are a range of compounds that both generalize and antagonize BZs and each other;
may interact poorly with barbiturates, but may also generalize to the barbiturate discrim
inative stimuli; are antagonized by some antagonists, but generalize to others. In fact,
these confusing asymmetries can also be observed in the BZs themselves. It appears that
one little-studied phenomena in this substance class is the effect of the training dose on
the discriminative stimulus formed. Ro 15-1788 has been shown to both antagonize and
generalize to the CDP cue, depending on the training dose of CDP used (De Vry and
Slangen, 1986). In addition, the asymmetrical generalization seen between the BZ lor-
azepam and pentobarbital in appropriately trained animals (Ator and Griffiths, 1989a,
1989b) may also be due to the dose used in training and testing, as well as to the devel
opment of tolerance to some of the disruptive effects of the test drugs (although the
authors discuss their work as indicating inhomogeneity in the stimulus effects of depres
sant drugs).
Some of the behavioral data may be inteφreted to suggest that there are several sub
types of BZ receptor and that these subtypes may be selectively responsible for specific
BZ effects. Clearly, drug discrimination studies will play an important part in character
izing these distinct effects. However, it must be noted that although subtypes of the cen
tral BZ receptor (the receptor in the CNS BZ/GABA complex as opposed to the "periph
eral" BZ receptor) have been identified both by biochemical pharmacologists (BZ, and
BZ2 receptors; Klepner et al., 1979) and by molecular biologists (e.g., Levitan et al.,
1988), any possible distinct functional role or specific ligands are as yet relatively unde
fined (but see Langer and Arbilla [1988] for a recent proposal on classification and recent
results with Zolpidem [Sanger, 1988], CL 218872 [Gardner, 1989], and abecamil [Ste
phens et al., 1990b]). The conservative view discussed above, that disparities between
compounds may not be dependent so much on different subtypes as on dosing and train
ing methods, as well as perhaps on pharmacokinetic properties of the compounds, is still
very relevant. It is apparent, however, that there has been a shift in the approach to drug
cues based around BZ receptor ligands from "if-it-substitutes-it's-an-anxiolytic" to the
more pragmatic search for behavioral correlates of BZ receptor function. Indeed, it may
now be of more interest to seek out active BZ ligands that do not generalize to BZ dis
criminative stimuli as one approach to identifying novel BZ anxiolytics.
2.3. SEROTONIN CUES
The introduction of buspirone into the cUnic for the treatment of anxiety has led to a
change in how the search for anxiolytics is approached. Buspirone is a 5-HT,A partial
agonist that, in contrast to the BZs, is report^ to be an anxioselective agent (Taylor et
al., 1984, 1985); that is, buspirone possesses no anticonvulsant or muscle-relaxant effects
in addition to its anxiolytic-like properties. Buspirone does not bind to BZ receptors and
therefore shows no interaction with BZs or with substances thought to exert effects
through the BZ/GABA receptor complex. It has been postulated for many years that the
anxiolytic effects of BZ may be due to actions on the limbic 5-HT system (Robichaud
and Sledge, 1969; Wise et al., 1972; Gray, 1982). However, it was not until the last few
years that intense work on the putative anxiolytic actions of 5-HT ligands began.
Undoubtedly, this was due partly to the relation between 5-HT and hallucinogenic sub
stances such as d-lysergic acid diethylamide (LSD) and partly to be lack of well-defined
ligands for the separate receptor subtypes and their association with particular behavioral
effects.
Classical tests for anxiolytic substances are almost always based on BZ actions. It is
therefore not suφrising that 5-HT anxiolytics are not consistently identified in such tests
(Goldberg et al., 1983; McClosky et al., 1987; Pellow et al., 1987; Taylor et al., 1985) or
that 5-HT agents do not substitute in BZ cues (Rauch and Stolerman, 1987; but see
Spealman, 1985). Several new tests have been introduced to help identify 5-HT-like anxi
olytics (e.g., Jones et al., 1988), although they too may suffer from being tests of drug
action as opposed to animal models of anxiety. In addition, there has been a renewed
interest in the discriminative stimulus properties of the new putative anxiolytic class. In
part, this interest has arisen because of the great proliferation of 5-HT receptor subtypes
and the availability of relatively specific ligands for each subtype. The question, then, is
cleariy what stimulus properties, if any, do the putative 5-HT anxiolytics have in
common?
Since the first 5-HT anxiolytic in the clinic is reported to be selective for the 5-HT,A
subtype, it is not suφrising that drug discrimination studies have concentrated on sub
stances acting at this receptor. The merits of other 5-HT„ S-HTj and 5-HT3 receptor
ligands as anxiolytics will not be discussed, partly through considerations of space, partly
because of a paucity of clinically relevant data, and partly because, in the case of 5-HT3
ligands, no cue has yet been reported.
Buspirone is reported to form a discriminative stimulus in rats (Hendry et al., 1983),
but the discrimination was difficult to train because of strong-response depressant effects.
At the present time, it is unclear whether this difficulty was due to the choice of training
dose or to the discrimination procedure used. Buspirone does, however, form a potent
and specific discriminative stimulus in pigeons (Mansbach and Barrett, 1987). The dif
ference in potency of buspirone in pigeons and rodents has been noted in several studies
(e.g., Barrett et al., 1986), but little is known conceming this difference. Although the
pigeon may offer an interesting species for studying the anxiolytic effects of 5-HT,A
ligands, in this review we will concentrate on studies involving mammalian species. Ip
sapirone, a stmctural analogue of buspirone, but reportedly showing a greater selectivity
for the 5-HT,A receptor, has a similar profile of action to buspirone in animal tests and
is currently undergoing clinical trials for the treatment of anxiety (Glaser, 1988). An
ipsapirone dmg cue was established to investigate the pharmacological nature of the
interoceptive stimulus (Spencer and Traber, 1987; Cunningham, 1989). The cue
appeared to be highly specific. Only substances with a high affinity for the 5-HT,A recep
tor substituted for the training dmg, for example, buspirone, 8-hydroxy-2-(di-n-propyl-
amino)-tetralin (8-OHDPAT), and 5-methoxy-N,N-dimethyltryptamine (5-OMe-DMT).
On the other hand, nonselective 5-HT ligands such as methysergide, 5-HT,Β selective
ligands, or non-5-HT ligands (e.g., diazepam and pentobarbital) failed to generalize.
However, antagonism of the cue by a specific 5-HT,A antagonist has still to be demon
strated. Recently, it was proposed that NAN-190 could serve such a role, and indeed,
NAN-190 antagonized the 8-OHDPAT discriminative stimulus (Glennon et al., 1988,
1989). Unfortunately, the expected antagonistic effects of NAN-190 on ipsapirone, at
least on operant responding, have not been apparent (Deans et al., 1989). Deans et al.
(1989) suggest that NAN-190 may, in fact, also be a partial 5-HT,A agonist, capable of
antagonizing only full 5-HT,A ligands. Tests of the ability of each of the substances iden
tified as 5-HT,A ligands to cross-generalize with one another have produced a bewildering
array of asymmetrical results. Ipsapirone substitutes for the discriminative stimuli pro-
duced by 8-OHDPAT and 5-OMe-DMT (Amt, 1989; Cunningham et al, 1987; Spencer
et al., 1987), but 5-OMe-DMT generally does not substitute successfully for the 8-
OHDPAT discriminative stimulus (Amt, 1989; Cunninghani ét al., 1987; Glennon,
1986), although 8-OHDPAT will substitute for 5-OMe-DMT in trained animals (Spen
cer etal., 1987).
8-OHDPAT has been reported to label both pre- and postsynaptic sites (Hall et al,
1985) though its role at these sites is not completely clear. Although 8-OHDPAT appears
to act as an agonist at both autoreceptor and postsynaptic sites (Bobker and Williams,
1990), reports from electrophysiological studies in CAl hippocampal neurons suggest
either a partial agonist or an antagonist function (Andrade and NicoU, 1987; CoUno and
Halliwell, 1987). Furthermore, the 5-HT,A pre- and postsynaptic receptors may be cou
pled to different second messenger systems, and the postsynaptic mechanisms may
undergo tolerance to repeated stimulation (Larsson et al, 1990).
As far as the discriminative stimulus is concemed, it may be that the autoreceptors are
of lesser importance to the 8-OHDPAT discriminative stimulus (Kalkman, 1990); claims
for such neuronal specificity require detailed confirmation. From the presently available
data, it might be expected that the effects of 8-OHDPAT would be difficult to predict
and may, in fact, show contradictory effects on behavior depending on the dose. Inter
estingly, 8-OHDPAT has been reported as both anxiolytic and anxiogenic in animal tests
(Carli and Samanin, 1988; Critchley and Handley, 1987). Although 8-OHDPAT does
form a discriminative stimulus to which both buspirone and ipsapirone generalize, sev
eral researchers have reported that rats trained in the cue are more aggressive or irritable
than usual (Winter, 1988); however, this effect may be dependent on the training dose
used.
Although the 8 - O H D P A T discriminative generally behaves in a manner consistent
with a 5-HT,A agonist cue, some unusual properties should be noted. Although, in gen
eral, non-5-ΗΤ,Α ligands do not substitute for 8 - O H D P A T , substitution is found with an
a2-antagonist, yohimbine (Winter, 1988; Winter and Rabin, 1989) and with lisuride, an
ergoUne possessing dopamine Dj as well as 5-HT,A properties (Cunningham et al, 1987).
The generalization of Usuride is easy to understand as resulting from its 5-HT,A agonism,
but the generalization of yohimbine is less easy to explain as yohimbine possesses only
micromolar affinity to 8-OHDPAT-labeled receptors (Gozlan et al, 1983). Interestingly,
lisuride, yohimbine, and the partial 5-HT,A agonist ipsapirone all generalize to the LSD
discriminative stimulus (Appel et al, 1982; Colpaert, 1984; Amt, 1989). Although the
LSD discriminative stimulus is generally attributed to LSD's action at 5-HT2 receptors
(Cunningham and Appel, 1987), LSD also possesses 5-HT,A affinity and it seems possible
that this activity provides a commonality of action for all these dmgs. The circle is com
pleted by reports that yohimbine generalizes to the ipsapirone discriminative stimulus
(Winter, 1988; Winter and Rabin, 1989). Nevertheless, in contrast to ipsapirone's anx
iolytic-like action, yohimbine is usually reported to exert anxiogenic-like effects (e.g.,
Davidson and Lucid, 1987). Thus, the usefulness of dmg discrimination to identify phar
macological similarities is clear, but the ability to predict behavioral outcomes is
questionable.
Furthermore, the results with yohimbine would suggest that, unUke other dmg classes,
the 5-HTiA discriminative stimulus is much less specific or that chronic treatment with
5-HT agents produces behaviorally significant interactions with other systems. This fact
is certainly tme for buspirone. Buspirone is known to possess dopaminergic, as well as
serotonergic, properties and can successfully block the discriminative stimulus effects of
apomorphine in monkeys (Kamien and Woolverton, 1990).
These results again illustrate the need to treat dmg discrimination data with care. 5-
H T discriminative stimuli cannot be simply represented as a short cut to identifying
novel anxiolytic agents. It is still uncertain if 5-HT,A activity alone is sufficient for anxio
lytic activity, and the cues generated by current candidates appear to possess several dis
tinct components. Much of the confusion in the present data will be reconciled by the
development of better ligands and further physiological and pharmacological studies,

perhaps characterizing the regional nature of specific responses within the central ner
vous system. As already emphasized, at the present time, anxiolytic cues per se do not
exist, but the discriminative properties of anxiolytic drugs can be used to identify a set
of stimuli associated with the drug to discover what properties are related to, allowable
in, or unnecessary for, an anxiolytic compound.
2.4. EXCITATORY AMINO ACIDS
Recent research has shown that antagonists for excitatory amino acids (EAA) (e.g.,
glutamate and asparate) possess muscle relaxant, anticonvulsant, and anticonflict activ
ity (Bennett and Amrick, 1986; Clineschmidt et al., 1982; Lehmann et al., 1988; Ste
phens et al., 1986; Stephens and Andrews, 1988, Turski et al., 1985) and thus would
appear to have some potential as anxiolytic agents. Of the three main receptor subtypes
identified, A^-methyl-o-aspartate (NMDA) antagonists are the most studied. NMDA can
produce convulsions and would appear to be unsuitable for drug discrimination studies.
However, both pigeons and rats can be trained to discriminate NMDA from saline at
subconvulsant doses (Amrick and Bennett, 1987b; Bennett et al., 1988; Willetts and Bal-
ster, 1989b; Woods et al., 1988). Although this cue has not been fully characterized the
discriminative stimulus is blocked completely by the competitive antagonists CGS
19755, 3-(2-carboxypiperazin-4-yl)propyl-l-phosphonic acid (CPP), and 2-amino-4,5-
(1, 2-cyclohexyl)-7-phosphonoheptanoate (NPC 12626), but only partially by another
NMDA antagonist, 2-amino-7-phosphonoheptanoic acid (AP7) (Amrick and Bennett,
1987b, 1987c; Willetts and Balster, 1989b). Amrick and Bennett (1987b) attribute the
incomplete antagonism by AP7 to the poor penetration of AP7 into the brain. Con
versely noncompetitive NMDA antagonists such as MK 801, as well as pentobarbital
and diazepam, reduced rates of responding, but failed to antagonize the cue (Amrick and
Bennett, 1987b; Willets and Balster, 1989b). However, antagonism of the cue by the
noncompetitive antagonist phencyclidine (PCP) and ketamine, as well as by the kappa
agonists trifluadom and ethylketocyclazocine (Amrick and Bennett, 1987b; Bennett, et
al., 1988; Willetts and Balster, 1989b) indicates that the nature and specificity of the
discriminative stimulus is pharmacologically complex.
In addition, despite reports that NMDA antagonists such as AP7 and CGS 19755 exert
anticonflict activity in animals (Bennett and Amrick, 1986; Bennett et al., 1989; Ste
phens et al., 1986; Stephens and Andrews, 1988), too little is known about the true
nature of EAA anxiolytic effects in animal models or man. Both AP7 and CGS 19755
generalize to the diazepam discriminative stimulus (Bennett and Amrick, 1986; Bennett
et al., 1989), although whether this is due to an as yet unknown interaction with the BZ/
GABA receptor complex or to behavioral similarities such as muscle relaxation or anti-
conflict activity is unclear. A recent study has suggested that another competitive NMDA
antagonist, CPP, may have a greater affinity for benzodiazepine than NMDA receptors
(White et al., 1988). At the present time, excitatory amino acid antagonists do not appear
to be useful anxiolytics (mainly because it is difficult to evaluate substances with such
poor brain penetration). It is therefore too early to consider using EAA antagonist cues
to help identify novel anxiolytics. In addition to compounds like AP7 acting as compet
itive antagonists at NMDA receptors, a second class of noncompetitive antagonists acts
at a site apparently within the ion channel. These substances include the hallucinogen
PCP, the dissociative anesthetic ketamine, and the experimental anticonvulsant
MK 801. MK 801 has been reported to possess anxiolytic properties in animal models
(Clineschmidt et al., 1982). However, in keeping with a common site of action, it shares
a common discriminative stimulus with the hallucinogen PCP (Tricklebank et al., 1987;
Jackson and Sanger, 1988; Koek et al., 1988; Willetts and Balster, 1988a; Sanger and
Zivkovic, 1989), perhaps not the most desirable property for an anxiolytic to possess. In
fairness, the discriminative stimulus properties of noncompetitive NMDA antagonists
such as MK 801 may not be the same as those produced by the competitive NMDA
antagonists (Tricklebank et al., 1989; WUletts and Balster, 1989a, b; Willetts et al., 1990).
Peripheral administration of AP7, CPP, or NPC 12626 does not produce complete gen
eralization to PCP (Jackson and Sanger, 1988; Willetts and Balster, 1988b; Koek and
Woods, 1988). However, centrally administered APT will substitute for PCP in pigeons
and rats (Tricklebank et al., 1987; Willetts and Balster, 1988b), albeit at doses that cause
a marked response suppression in rats; in contrast, CPP only partially generalizes to the
PCP cue (Willetts and Balster, 1988b). Another competitive antagonist, AP5, substitutes
for the PCP discriminative stimulus in pigeons (Koek et al., 1987). It may be that the
inconsistencies in substitution profiles are due to poor brain penetration, selectivity of
the ligands, or the training dose of PCP used (see Koek et al., 1987 and Willetts and
Balster, 1989b for further discussion on the problem of inteφretation).
Studies involving drug cues based on competitive NMDA antagonists are too few to
evaluate realistically. In rats trained to recognize NPC 12626, however, CPP substitutes
for NPC 12626 in a dose-dependent manner, whereas PCP does not (Willetts et al.,
1989). This result is again suggestive of separate discriminative stimulus properties of
competitive and noncompetitive NMDA antagonists. It is also becoming apparent that
there are differences between the various competitive antagonists presently available.
One intriguing possibility is that there are several subtypes to the NMDA receptor. Addi
tionally, the NMDA receptor is regulated by an associated glycine site in a manner sim
ilar to that in which BZs modulate the GABA receptor complex (Monaghan et al., 1988).
Thus, differences in discriminative stimulus properties between ligands at the NMDA
receptor, noncompetitive antagonists acting in the ion channel, and ligands at the glycine
receptor may parallel differences between the discriminative stimulus properties of ben
zodiazepines, barbiturates, and GABA agonists.
Barbiturates are also reported to generalize partially to the PCP discriminative stim
ulus (Willetts and Balster, 1988b, 1989a), and because barbiturates also share some sim
ilar discriminative stimulus properties with BZs, it may be important to characterize fur
ther the discriminative properties of EAA using barbiturate cues. In one recent study in
which rats were trained to discriminate pentobarbital from saline, competitive NMDA
antagonists substituted for the discriminative stimulus, whereas the noncompetitive
antagonists MK 801 and PCP only partially generalized (Willetts and Balster, 1989a).
Differences between the cueing properties of pentobarbital and competitive NMDA
antagonists are also demonstrable. Rats trained to discriminate NPC 12626 from saline
generalize to CPP, but not to pentobarbital or PCP (Willetts et al., 1989). Reasons for
the asymmetrical substitution of NPC 12626 and pentobarbital are not apparent at the
present time.
To date, the interoceptive effects of NMDA competitive antagonists are relatively
undefined, but appear to overlap with BZ, PCP, and possibly barbiturate-Uke stimuU.
Whether this reflects direct actions at the appropriate receptors, due to nonspecificity of
the available ligands, or an indirect effect of NMDA stimulation or antagonism has yet
to be determined. An alternative explanation is that these compounds achieve a common
"psychological" effect through different pharmacological mechanisms. We have recently
proposed a model of interaction between BZ/GABA mechanisms and NMDA receptors
that goes some way both in explaining the pharmacological similarity of BZs and NMDA
competitive antagonists and perhaps also in accounting for the overlapping discrimina
tive stimulus properties of these substances (Stephens et al., 1991). In this regard, it is
interesting to note that Bennett and Amrick (1986) inteφreted the AP7 interoceptive
stimulus as a muscle relaxant cue because of its generalization to diazepam and the sim
ilarity of its effects to other muscle relaxants in conflict tests.
The results from using NMDA itself as a discriminative stimulus have raised some
interesting questions. For example, if NMDA antagonists are indeed anxiolytic in nature,
then is the NMDA cure anxiogenic? (In this respect it is of interest to note preliminary
evidence suggesting generalization of the inverse agonist, and reputedly anxiogenic β-
118 J . S . ANDREWS AND D . N . STEPHENS
carboline, /J-CCE, to the NMDA discriminative stimulus in pigeons [Woods et al.,

1988]). If so, then how should the partial generalization of high doses of competitive
antagonists such as CPP and NPC 12626 to the NMDA discriminative stimulus be inter
preted (Koek et al., 1990; Willetts and Balster, 1989b). By analogy with other substance
classes a partial agonist argument could be advanced to explain both the antagonism and
generalization to a discriminative stimulus by the same drug at different doses. In the
case of NMDA antagonists, such an inteφretation may not be appropriate, and unequiv
ocal data suggesting partial agonist activity are presently lacking. Thus, the behavior of
NMDA ligands in the NMDA cue is difficult to inteφret in terms of anxiolytic or anxio
genic activity. The reported similarities in the discriminative stimuli induced by NMDA
and ß'CCE may reflect similarities in the convulsant properties of the compounds, a
theme that will be pursued in the next section.
3. ANXIOGENIC CUES
Several compounds have been reported to produce anxiogenic effects in man, for
example, the convulsant pentylenetetrazole (PTZ) (Rodin and Calhoun, 1970), the β-
carboline partial inverse agonist FG 7142 (Dorow et al., 1983), and the serotonin recep
tor agonist m-chlorophenyl-piperazine (Chamey et al., 1987). These reports, coupled to
the ever present need for a relevant animal model of anxiety, have led several groups to
investigate the discriminative stimulus properties of putative anxiogenic substances. A
model that is based on a clear and reproducible state of anxiety would clearly be a great
advance over present ''conflict" models in anxiety research, many of which can be easily
inteφreted as models sensitive to the disinhibitory effects of BZs. However, although
models based on chemically induced anxiety possess a certain attraction and some face
validity, they are not without problems. It is worth taking some time to examine thor
oughly one such model and the dangers inherent in inteφreting the data produced.
3.1. PENTYLENETETRAZOLE CUE
Pentylenetetrazole is a well-known convulsant dmg, widely used in both epilepsy and

anxiolytic research. Several groups have reported that subconvulsant doses of PTZ can
serve as a discriminative stimulus (Shearman and Lal, 1980; Stephens et al., 1987), and
it has been suggested that the discriminative stimulus induced by PTZ may be one of
anxiety (Shearman and Lal, 1980). Evidence in support of this proposal has arisen from
several sources. In humans, PTZ has been reported to induce feelings of intense anxiety
and impending doom (Rodin and Calhoun, 1970), and in rats, the autonomic signs fol
lowing PTZ administration are similar to those found in stress or anxiety (Emmett-
Oglesby et al., 1987). The discriminative stimulus itself is antagonized by known anxio
lytics such as diazepam (Shearman and Lal, 1980, 1981; Lal et al., 1980; Lal and Field
ing, 1984; Benjamin et al., 1987), whereas other substances reported to induce anxiety
in humans generalize to the discriminative stimulus, for example, FG 7142 (Stephens et
al., 1984a, 1984b) and cocaine (Shearman and Lal, 1981). Moreover, rats trained to dis
criminate PTZ and then made "dependent" on diazepam or ethanol will select the PTZ-
associated lever during withdrawal in the absence of PTZ, ethanol, or diazepam, a resuh
inteφreted as being due to withdrawal-induced anxiety (Emmett-Oglesby et al., 1987;
Harris et al., 1988; Lal et al., 1988). Interestingly, VeUucci et al. (1988) have demon
strated that rats subjected to conspecific defeat also select the PTZ lever, even when
injected with placebo. Lal and Emmett-Oglesby (1983) have reported that anticonvulsant
potency does not correlate well with activity in the cue, and as several cUnically efiective
antiepileptic dmgs do not appear to antagonize the cue, proposed that the discriminative
stimulus is more related to the anxiogenic than to the convulsant action of PTZ.
However, there are several flaws in this particular argument and in the model in gen
eral. In typical tests for anticonvulsant activity, a high convulsant dose of PTZ is given
and the animals tested only once. Repeated injections of subconvulsant doses of PTZ
eventually lead to seizures in rats, a process known as kindling. The efficacy of anticon-
vulsant drugs in preventing kindled seizures sometimes differs markedly from that
observed in the acute PTZ model (Ito et al., 1977). In our own laboratory, we have
observed that rats trained to discriminate PTZ from saline sometimes exhibit seizures
following administration of the training dose. More often, the rats display wet-dog shakes
or tremors, behaviors similar to those observed by Racine (1972) in his description of
the eariy stages of seizure development in amygdaloid-kindled rats. These symptoms
have also been reported by other groups working with rats receiving chronic PTZ treat-
ment in drug discrimination procedures (Lal and Emmett-Oglesby, 1983; Gauvin et al.,
1989), indicating that kindUng may be occurring in these animals.
Many, if not all, of the substances reported to substitute for PTZ are themselves con-
vulsants (Lal and Emmett-Oglesby, 1983), but perhaps of more interest, many of the
drugs that generalize and are not themselves convulsant on first administration are pro-
convulsant and induce kindled seizures on repeated administration, for example, cocaine
(Stripling and EUinwood, 1977) and FG 7142 (Little et al., 1987; Corda et al., 1987).
Indeed, for some such as cocaine there is cross-sensitization between itself and PTZ in
animals kindled with PTZ (L. Turski, unpubUshed observations). The potent convulsant
strychnine generalizes only partially to the PTZ discriminative stimulus (Shearman and
Lal, 1980) and can be seen as going some way to help answer criticisms of the possible
convulsant nature of the cue. However, not all convulsants have the same mode of action
as PTZ. Strychnine-induced convulsions are primarily spinal in origin and are mediated
by glycinergic rather than by GABAergic mechanisms. Therefore, strychnine might be
expected to produce a rather different internal stimulus from PTZ.
In an experiment designed to investigate the relationship between anticonvulsant
activity and activity in the PTZ cue, groups of rats were trained to discriminate PTZ
from saline or were kindled using a slightly higher, but still subconvulsive, dose of PTZ.
A series of anxiolytic and anticonvulsant drugs were used to antagonize the cue and kin-
dled PTZ seizures, as well as seizures induced by an acute high dose injection of PTZ
(Andrews et al., 1989). In keeping with other reports (Shearman and Lal, 1980; Lal and
Emmett-Oglesby, 1983), the results showed that all benzodiazepine receptor ligands
antagonized the discriminative stimulus regardless of their activity against acutely
induced PTZ seizures. Furthermore, two clinically effective anticonvulsants antagonized
the cue (valproate and ethosuximide), while two others (carbamazepine and Phenytoin)
failed to antagonize the cue. Substances that failed to antagonize the cue failed to antag-
onize PTZ-kindled seizures, and several substances that showed extreme potency differ-
ences in antagonizing acutely induced PTZ seizures and the cue were also strikin¿y more
effective in antagonizing PTZ-kindled seizures (see Table 5.2).
One of the most important criticisms of the PTZ cue as an anxiogenic model is its
reliance for validity on antagonism by BZ-like anxiolytics. Putative non-BZ anxiolytics
such as the serotonergic compounds buspirone, gepirone, and cyproheptadine, as well as
the NMDA antagonists AP7 and CPP, are ineffective in antagonizing the PTZ discrim-
inative stimulus (Table 5.2) (Stephens et al., 1986; Ator et al., 1989; Wilson and Bennett,
1989). In fact, it has been reported that buspirone generalizes to the PTZ discriminative
stimulus in baboons (Ator et al., 1989), an effect that may be dependent on buspirone's
known effects on the dopamine system, but is not consistent with the clinical profile of
buspirone. Interestingly, File (1988) has demonstrated that, in some circumstances such
as in combination with antidepressants, buspirone can induce convulsions in rats. Sev-
eral antidepressants with reported anxiolytic activity are also ineffective in the cue
(Mason et al., 1987). The antidepressants used in this study were reported to be procon-
vulsive in combination with PTZ; moreover, higher doses of the drugs showed some
generalization to PTZ when administered alone. To date, all BZ anxiolytics tested in the
PTZ cue also possess anticonvulsant activity, particularly against kindled seizures. It is
well known that the anticonvulsant activity of BZs is a good predictor of anxiolytic activ-
TABLE 5.2. Antagonism of Pentylenetetrazole (PTZ) Potencies against PTZ Cue. Acute
Clonic Seizures, and Kindled Seizures
Substance PTZ cue Acute PTZ clonic Kindled PTZ seizure
BZ Agonists
Diazepam 0.37 1.86 1.84
Chlordiazepoxide 1.06 3.1 1.87
Lorazepam 0.05 0.36 0.18
Lormetazepam 0.04 0.22 0.24
Triazolam 0.02 0.25 0.17
Midazolam 1.25 2.49 0.82
Clonazepam 0.32 0.6 0.24
Non-BZ BZ agonists
Z K 9 1 296 0.83 10.8 1.86
ZK 93 423 0.08 0.66 0.32
ZK 95 962 0.18 12.87 0.59
ZOLPIDEM 0.96 6.81 1.83
CGS 9896 1.47 >80 1.53
CL 218872 2.16 4.32 4.86
Antiepileptics
Valproate 126.84 293.6 99.99
Phenytoin >60 >100 >I00
Carbamazepine >40 >80 >80
Ethosuximide 107.69 212 176
New/potential anxiolytics
Buspirone >2.5 >30 >30
MK-801 >0.32 >0.5 >0.5
2-APH >80 — >80
All values ED50S (mg/kg, IP) rat.

The relationship between activity in the PTZ cue of full and partial benzodiazepine (BZ) receptor ago
nists and clinically effective antiepileptics and activity against clonic convulsions induced by an acute injec
tion of PTZ and against PTZ kindled seizures (adapted from Andrews et al. 1989). Note that a compound's
ability to antagonize the PTZ discriminative stimulus is predicted by its ability to prevent PTZ-kindled
seizures, but not necessarily by its action against seizures induced following a single, high dose of PTZ. Of
the clinically effective antiepileptics, only those with the ability to antagonize PTZ convulsive effects were
active in the cue. For discussion, see text under Section 3.1 Pentylenetetrazole Cue; for full details of the
experimenul procedures see Andrews et al. 1989.
Adapted from Andrews et al., 1989. Reprinted with permission of the copyright holder, John Wiley and
Sons, New York.
ity in the dinic (Clody et al., 1983), but this does not automatically make an anticon
vulsant an anxiolytic.
These observations raise the question as to whether the PTZ discriminative stimulus
is related to some form of preseizure activity (aura?), and in this context, it is of interest
that electrical kindling can form the basis of state-dependent learning in the rat (Overton,
1984). Neither carbamazepine nor Phenytoin antagonize the PTZ cue (Andrews et al.,
1989; Lal and Emmett-Oglesby, 1983), but neither of these substances are effective
against PTZ-induced seizures. It has been argued that, if the PTZ cue were based on a
preconvulsive aura, then antiepileptic agents should be effective in antagonizing the cue.
Interestingly, although Phenytoin is effective in the clinic in several forms of epilepsy, it
does not prevent the aura many patients report feeling before seizures (Rail and Schleifer,
1985). Since not all antiepileptic drugs are effective in all convulsant models, it might be
expected that agents ineffective in the PTZ model of epilepsy would not be active against
the PTZ cue. In the experiment cited above (Andrews et al., 1989), only substances that
antagonized PTZ-induced seizures antagonized the cue. One of these, valproate, has been
reported to possess anxiolytic-like effects (Lal et al., 1980); however, to date we are
unaware of any clinical data indicating anxiolytic activity of ethosuximide in the clinic.
Indeed, in certain anxious patients, anxiety has been reported as a side effect of ethosux
imide therapy (Rail and Schleifer, 1985).
Studies, showing that, during withdrawal from chronic BZ treatment, animals previ-
ously trained to discriminate PTZ from saline will select the PTZ-associated lever in the
absence of PTZ, have been suggested as a further validation of the model (Harris et al.,
1988). However, although anxiety is one symptom of BZ withdrawal, in some circum
stances, so are convulsions (Breir et al., 1984; Levy, 1984).
This point is of particular relevance to studies purporting to examine withdrawal anx
iety using the PTZ cue. These studies assume the validity of the PTZ discriminative stim
ulus as an anxiety model. Inteφretation is further complicated by the nature of chronic
PTZ and diazepam treatments. Before receiving chronic diazepam, the rats have been
trained on the PTZ discriminative stimulus. The evidence suggests that the rats become
more sensitive to PTZ possibly through kindling. The rats are then treated with diazepam
over several days before withdrawal. Both withdrawal from chronic diazepam treatment
and chronic treatment with PTZ decrease seizure thresholds (in the case of PTZ kindling,
permanently). In particular, it has been demonstrated that discontinuation of chronic
BZ treatment leads to an increased sensitivity (i.e., decreased threshold) to PTZ- and FG
7142-induced convulsions (Schatzki et al., 1989). Accordingly, it is probably not sur
prising that signs of diazepam withdrawal as detected by the PTZ cue are enhanced by
the addition of known convulsants (Idemudia et al., 1987).
A similar case can be made for the inteφretation of PTZ lever choice in ethanol with
drawal (Lal et al., 1988; Harris et al., 1989). Subjective feeUngs of anxiety are certainly
one symptom of alcohol withdrawal, as are convulsions and tremor (Mello and Men
delson, 1977). Recently, Idemudia et al, (1989) reported that PTZ lever choice during
ethanol withdrawal was enhanced by bicucuUine and Picrotoxin. BicucuUine and Picro
toxin alone occasioned 50% PTZ lever responding; ethanol withdrawal, 30%. Assuming
that ethanol withdrawal leads to an increased sensitivity to seizures, the addition of Pic
rotoxin or bicucuUine might be expected to exert an additive effect. The results appear
to bear this out. PTZ lever selection in ethanol-withdrawn animals treated with Picro
toxin or bicucuUine averaged approximately 80%.
Thus, if the PTZ cue is based on a preconvulsant effect, it may not be suφrising that
partial generalization to the PTZ cue is observed in the first few days foUowing with
drawal from diazepam or ethanol. Thus, and unfortunately, this model may not be of
great help in analyzing the psychological state arising from BZ withdrawal in the cUnic,
as was first believed.
3.2. FG 7142 C U E
Recently, a cue based around the /3-carboUne partial inverse agonist FG 7142 has been
established (Leidenheimer and Schechter, 1988a), and although it is not yet fully char
acterized, it has been suggested that the discriminative stimulus may be anxiogenic in
nature. This follows mainly from three Unes of reasoning; first, that FG 7142 has been
reported to induce feeUngs of anxiety in humans (Dorow et al., 1983); second, that FG
7142 generalizes to PTZ, an "anxiogenic" cue; and third, that exposure of rats to a phys
iological stressor elicits FG 7142-appropriate responding. As a model for testing anxio
lytic drugs it is open to criticisms similar to those leveled at the PTZ cue. FG 7142, Uke
PTZ, induces kindled seizures in rats; therefore, substances active in the cue are as Ukely
to be anticonvulsants as they are to be pure anxiolytics. Moreover, FG 7142 is believed
to exert its effects through central BZ binding sites (Stephens et al., 1987; Corda et al.,
1987). Thus, an action at this particular site may be blocked not only by BZ agonists
possessing anxiolytic (and anticonvulsant) properties, but also by neutral BZ receptor
antagonists with no intrinsic action of their own. In fact, recent studies have shown that
Ro 15 1788, as weU as partial agonist and fuU agonist BZ receptor Ugands, will block the
FG 7142 cue (Leidenheimer, personal communication).
These two examples again illustrate the necessity for care in inteφreting the nature of
discriminative stimuU. Undoubtedly, there are many substances that can induce feeUngs
of anxiety when administered acutely to humans. However, many such as PTZ and FG
122 J. S. ANDREWS AND D . N. STEPHENS
7142 are also capable of causing seizures in animals following repeated administration.
Therefore, as suggested above, apparent anxiolytic activity in cues based on such sub
stances is likely to be confounded with anticonvulsant properties. In this respect, it is
interesting to note that rats that have been kindled with FG 7142 show no changes, when
compared with saline-treated animals, in the social interaction, plus-maze, Vogel con
flict, or startle response tests of anxiety (Taylor et al., 1988). In the two examples above,
drug effects are probably more parsimoniously explained by convulsant or anticonvul
sant properties.
4. ANTIDEPRESSANT CUES
The discriminative stimulus properties of antidepressants are as poorly understood as
the exact mode of action of the antidepressants themselves. Several groups have success
fully trained rats to discriminate an antidepressant from saline. Unfortunately, the ques
tion as to whether or not the various antidepressants share a common discriminative
stimulus has not been adequately addressed, as few generalization studies have been car
ried out. This is due partly to the difficulty in training and partly to the high mortality
rates in animals used in such tests (Shearman et al., 1978; Jones et al., 1980).
The prototype class of antidepressants, the tricyclics, present a particular problem.
Using a T-maze, shock-avoidance paradigm, Overton (1982) has successfully trained rats
to discriminate the antidepressant drugs doxepine, imipramine, and amitryptyline.
Although cross-generalization with these tricylics could be demonstrated, the overall suc
cess of the experiments was low (cited in Jones et al., 1980). Training doses were high in
comparison to other behavioral effects observed with such compounds and a low survival
rate following repeated administration was observed. Indeed, it appears that only at near
toxic doses do the tricyclics produce a discriminative stimulus (Shearman et al., 1978;
Overton, 1982; see also Jones et al., 1980). Schechter (1983) compared the effects of
differing stress conditions on the ability of rats to recognize the discriminative stimulus
induced by imipramine. Only the unstressed animals were able to learn the discrimina
tion successfully, and other tricyclics substituted for the training drug.
More recent additions to the antidepressant pharmacy are reported to be less toxic
than the prototype antidepressants such as imipramine. One, the phenylaminoketone
buproprion, although active in the clinic (but see Blackwell and Simon, 1986) has a very
different profile of action to classical antidepressants. Although buproprion forms a spe
cific discriminative stimulus in rats (Jones et al., 1980), other antidepressants, with the
exception of the putative antidepressant viloxazine, do not substitute for the training
drug. In fact, the discriminative stimulus appears to be related to a stimulant effect, since
amphetamine, caffeine, and methylphenidate all substitute for buproprion. Another
novel antidepressant, the phosphodiesterase (PDE) inhibitor rolipram (Davis, 1984;
Wachtel and Schneider, 1986), also forms a discriminative stimulus in rats (Ortmann
and Meisburger, 1986; Yamamoto et al., 1987; Andrews et al., 1988). In these studies,
other specific and nonspecific phosphodiesterase inhibitors generalized to rolipram, but
tricyclic antidepressants did not. This would suggest that the discriminative stimulus is
based on its phosphodiesterase activity. However, it is unlikely to be so simple. Although
rolipram inhibits locomotor activity, lisuride, an antiparkinson agent without PDE
inhibitory properties, but possessing both dopaminergic and serotonergic agonist activ
ity, has been found to generalize or partially generalize to the training drug (Andrews
and Stephens, unpublished observations).
The few results available using antidepressants as discriminative stimuli would appear
to suggest (1) that tricyclics produce a similar discriminative stimulus albeit at near toxic
doses; and (2) that other clinically active antidepressants can form discriminative stimuli,
which are, however, unrelated to other antidepressant drugs. Thus, there is no one com
mon cue for antidepressant drugs, a result in keeping with clinical reports that antide
pressants do not produce common subjective effects in normal human subjects (Leh-
mann and Hopes, 1977). However, it is clear that drug discrimination techniques offer
a unique way of investigating the differing interoceptive effects produced by these
compounds.
5. SUMMARY AND CONCLUSIONS

Drug discrimination techniques are an important method for investigating the intero
ceptive properties of different psychoactive drugs. However, the discriminative stimuU
formed are often complex, and occasionally, unexpectedly related to other behaviorally
active drugs. It appears then that data obtained from such experiments must be treated
cautiously. Because CDP is anxiolytic and forms a discriminative stimulus, it does not
follow that the cue is an anxiolytic cue. In this respect, results with antidepressants are
particularly revealing. Although all antidepressants are more or less equally effective in
the clinic, the interoceptive stimuU produced by the different compounds are unrelated,
and discrimination occurs typically at doses far removed from the therapeutic dose
range. Although it may be disappointing to say that there is no anxiolytic cue, anxiogenic
cue, antidepressant cue, and so on, recognition of this fact is neither a suφrising nor a
particularly discouraging state of affairs.
The specificity of the discriminative stimulus induced by a drug depends on the spec
ificity of the ligand involved, as weU as on the training dose used. Although several sub
types of BZ receptors are thought to exist, the evidence for their existence, as docu
mented by specific agonists and antagonists, is poor at the present time. In areas where
specific ligands are available, there is more certainty in ascribing behavioral effects to a
particular receptor.
It is apparent that many discriminative stimuU formed by apparently specific receptor
ligands are, in fact, extremely complex and possibly composed of effects at several recep
tor subtypes. This is especiaUy notable in the 5-HT cues, where the further characteriza
tion of these cues has revealed unusual generalization profiles, perhaps suggesting that
the specificity of the ligands is not as good as first believed. Separate components of com
plex multiple stimuU can be identified by animals trained on a mixture of substances
(e.g., concurrent nicotine and midazolam; Stolerman et al., 1987) or substances with
effects at multiple receptor sites (e.g., lisuride; Appel et al., 1982; Cunningham and
Appel, 1987). Present findings suggest that different training doses may have an impor
tant role in uncovering a variety of stimulus effects from a particular compound.
One question that has been poorly researched is the stability of the discriminative stim
ulus over time. Discriminative stimuU from a wide range of psychoactive substances
appear to be stable over months or years (see Schechter et al., 1989). Anecdotal evidence
suggests that this is equally true for opiates and for BZs, both of which belong to classes
of drugs in which the phenomenon of tolerance is weU documented. The possibility that
the stimulus properties of BZ receptor ligands are anxiolytic in nature must again be
questioned when tolerance to BZ anxiolytic, as weU as sedative and anticonvulsant,
actions can be readily observed in animals, but not to the BZ discriminative stimulus.
One possible explanation for the apparent lack of tolerance to the discriminative stim
uli associated with BZs might relate to stimulus fading. In conventional (e.g., visual)
discrimination tasks, reducing the intensity of the discriminative stimulus leads to an
initial reduction in discriminative performance. However, providing that the stimulus is
still detectable, further training at the new intensity leads to a reinstatement of perfor
mance to near previous levels. In the case of drug stimuU, the development of tolerance
(reduction of stimulus intensity) would be a gradual process, with the animal receiving
repeated training during stimulus fading. Provided that tolerance is less than complete,
it is to be expected that discriminative performance would remain stable.
Nevertheless, with suitable experimental designs, tolerance to drug-induced discrimi
native StimuU can be readily observed for other substance classes (e.g., caffeine [Holtz
man, 1987], moφhine [Sannerud and Young, 1987; Shannon and Holtzman, 1976]). In
general, the dose response curves for such substances, like those of BZs, remain relatively
stable over time, varying within a Hmited range. However, by removing the animals from
training for a short period of time while continuing the administration of the training
drug, tolerance to the discriminative stimulus, as shown by a right-shift in the general
ization curve, can be seen (Sannerud and Young, 1987; Shannon and Hoitzman, 1976).
Thus, by removing the opportunity to continually releam the stimulus during fading,
tolerance becomes apparent. It is to be expected that a similar procedure would dem
onstrate a shift in the dose response functions for discriminative stimuli based around
BZ ligands.
It has been suggested in the past that discriminative stimuli in animals are analogous
to the subjective effects of the drug in man and, therefore, may be a measure of the abuse
potential of a drug. For the opiates, there may be some truth in this, but for the anxio
lytics, particularly the newer partial-agonist BZ receptor ligands, and 5-HT receptor-
based anxiolytics that do not appear to induce withdrawal symptoms after chronic treat
ment, there is no relationship between ability to support a discriminative stimulus and
abuse potential. Even in other substance groups, many substances that are not active in
self-administration procedures will themselves form discriminative stimuli or generalize
to discriminative stimuli formed by other known drugs of abuse (see Brady et al., 1990
for a discussion).
In conclusion, research into the discriminative stimulus properties of anxiolytics is at
a pivotal stage. New ligands are raising the possibility of an ever finer dissection of drug
effects on behavior and, for drug discrimination research, an ever greater number of
questions, which for reasons of space, have not been adequately discussed. For example,
does partial generalization (or asymmetrical generalization) mean overlapping, but not
identical stimulus properties, or substitution for one component of a complex multiple
stimulus? How important is the training dose and the pharmacokinetics of the drug to
this phenomena? Do nonparallel generalization curves between training and test drugs
mean that generalization is due to a different mechanism of action from that of the train
ing drug? And of increasing interest, how important are contextual cues to the develop
ment and maintenance of a discriminative stimulus? Discussions of some of these topics
can be found in Colpaert (1977a), Holloway and Gauvin (1989), Overton (1984) and
Young (1990). Drug discrimination procedures cannot be viewed as a simple shortcut to
the development of new anxiolytics or antidepressants, but as a tool in a fuller charac
terization of the pharmacological properties of anxiolytic compounds, which may help
shed some light on the reasons for success or failure in the clinic.
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Peisamon Press, Inc. (New York), pp. 131-153
CHAPTER 6
EFFECTS OF DRUGS ON PUNISHED BEHAVIOR:

PRECLINICAL TEST FOR ANXIOLYTICS
JAMES L. HOWARD AND GERALD T. POLLARD
Division of Pharmacology, Burroughs Wellcome Co., Research Triangle Park, NC, USA
1. INTRODUCTION
This is a review of the effects of drugs on punished operant responding and punished
drinking, with emphasis on how these effects predict clinical anxiolytic action. Effects of
anxiogenics are considered only in passing. We do not discuss the application of operant
behavior to identifying antidepressants, because in our opinion no reliable method exists
(see Seiden and O'Donnell, 1985, for a proposal; Howard and Pollard, 1984, and Pollard
and Howard, 1986, for a rebuttal).
The review is based on the following sources: a MEDLINE database search of refer
ences in English to "conflict" or "punishment" in nonhumans since 1966; a Science
Citation Index search for references to three seminal papers. Geller and Seiner (1960),
Davidson and Cook (1969), and Vogel et al. (1971); a MEDLINE search for 1987 and
1988 references to "anxiolytics" or "antianxiety agents" in humans for recent clinical
reports; reference lists of selected reviews and research reports; and our own collection
of reprints. Database searches were done on June 10, 1988; the MEDLINE search on
"conflict" and "punishment" was updated on February 15, 1990. We are interested pri
marily in the validation of screening methods, the demonstration of sensitivity to the
effects of clinically proven anxiolytics and rejection of other classes of drugs. We have
examined a large portion of the literature on the interaction of drugs and punished
behavior in the laboratory; our reference list, while not exhaustive, is intended to identify
the major reviews and fundamentally important research reports and to present a rep
resentative sample of the use of punished responding to identify anxiolytics and to
explore the neural bases of anxiety and anxiolysis.
2. ANXIETY AND ANXIOLYTICS

Anxiety is a heterogeneous condition that is more easily sensed than defined. The most
recent edition of the Diagnostic and Statistical Manual of Mental Disorders of the Amer
ican Psychiatric Association (DSM-III-R, 1987) continues the trend of greater subdivi
sion and elaboration. As a working definition, we characterize cHnical anxiety as a state
of fear in which the object of the fear is not present. This is similar to the category called
generalized anxiety disorder in DSM-III-R. We will not be concemed with other cate
gories, such as panic disorder and post-traumatic stress disorder, for which there are no
accepted animal models, although the fact that some drugs are effective in several cate
gories suggests underlying commonality.
Not only has the definition of anxiety changed with time, but drug therapy has evolved
with experience. Benzodiazepines largely replaced barbiturates and propanediol carba
mates 2 decades ago. Now it seems that tricyclic antidepressants are effective not only in
panic disorder, but in generalized anxiety disorder as well. And the success of the novel
drug buspirone has demonstrated further that anxiety can be relieved specifically, with
out the side effects of the sedative-hypnotics. Table 6.1 is an attempt to categorize drugs
according to the strength of the clinical evidence of anxiolysis; it is the basis for classi-
131
132 J. L. HOWARD AND G. T. POLLARD
TABLE 6.1. Efficacy of Drugs in Generalized Anxiety Disorder*
Clear evidencef Some evidence^ Limited evidence*
Barbiturates Ethanol Antihistamines

Benzodiazepines Neuroleptics (low-dose) /9-blockers
Propanediols Ritanserin Bromides
Chloral hydrate
Clonidine
Buspirone' Monoamine oxidase inhibitors' Opiates
Tricyclic antidepressants(?)' Tricyclic antidepressants' Paraldehyde
•As defined in DSM-III-R. Reviews: Bressa et al., 1987; Dommissc and Hayes, 1987; Uder, 1987; Rick-
els, 1987; Rickels and Schweizer, 1987; Tyrer, 1988.
fMultiple, double-blind, placebo-controlled trials.
tThe weight of the evidence suggests efficacy.
•Old clinical reports or limited scope of efficacy.
'Clinical action occurs only after 1-3 weeks of chronic treatment.
fying drugs as true-positive or false-negative in succeeding tables. This scheme is less than
satisfactory for several reasons: Anxiety is ill defined; many types of drugs have some
anxiolytic activity under some circumstances, although published clinical evidence may
be weak; and it is practically impossible to gather sufficient clinical data to rule out anxio
lytic activity for some drugs, that is, to generate a list of drugs that can be called true-
negatives if they have no positive effect in a model. Given the uncertainties, we built the
tables conservatively.
3. ANIMAL MODELS OF PSYCHOPATHOLOGY

A model is an artificial representation of a disease or some aspects of it. Models are
often evaluated on a continuum of similarity to the disease (Abramson and Seligman,
1977; McKinney and Bunney, 1969; Thompson and Schuster, 1968; Howard and Pol
lard, 1983; Howard et al., 1989). In this review, a model is understood to be a screening
method, validated at the standard drug correlational level. A model of anxiety is a system
that registers positive for clinically proven anxiolytics and negative for drugs that are
thought to be nonanxiolytic in that they usually do nol relieve the symptoms of gener
alized anxiety disorder.
Empirical validation has an element of circular reasoning. This lack of elegance can
be mitigated by viewing the model, not as an intellectual construct or an embodiment
of disease, but as a screening method. The model is validated by its utility. The phar
macologist must wonder whether his model will identify unique compounds or only
compounds similar to the standards. There is some evidence that models validated at the
empirical level can identify new pharmacological entities; for example, the antitetraben-
azine screen for antidepressants registered positive for bupropion (Cooper et al., 1980;
Ferris et al., 1981; Soroko et al., 1977), and punishment procedures registered positive
for zopiclone.
4. OPERANT RESPONDING SUPPRESSED BY PUNISHMENT

4.1. DEFINITIONS
The distinction between operant and consummatory behavior is somewhat arbitrary.

For our puφoses, "operant responding" signifies the emission of an instrumental
response such as a lever-press or key-peck at a stable baseline rate from session to session
under a prescribed set of contingencies by a trained animal. Consummatory behavior,
by contrast, is the emission of an ingestive response from an animal's natural repertoire
such as licking a spout for water. The consummatory behavior of interest here, spout
Effects of drugs on punished behavior 133
Ucking, is measured in naive animals that are tested once and discarded or in habituated
animals that are tested many times and thus yield data conceptually similar to that
yielded by operant subjects. For convenience, we distinguish between "operant behav-
ior" (Section 4) on the one hand and "lick suppression," "conditioned suppression of
drinking," and similar terms (Section 5) on the other.
"Punishment" is likewise problematic. For example, when pressing a lever results in
delivery of electric shock, under ordinary circumstances, the animal will reduce its rate
of lever pressing; however, there is a substantial body of literature on shock-maintained
responding in the monkey, in which, by schedule manipulations, the monkey is induced
to emit the same response at identical rates and in identical patterns for food reinforce-
ment or shock avoidance under one stimulus condition and shock delivery under
another stimulus condition (Barrett, 1981). Conversely, events that are not normally
thought of as punishing, such as the turning on of a small light, have been shown to
suppress responding under some conditions (Dews, 1976). For our purposes, punishment
is deñned operationally as a scheduled event such as electric shock that reduces the rate
of ongoing behaviors such as lever pressing or drinking. For a discussion of these terms
and other behavioral issues, see Morse and Kelleher (1977) and other chapters in the
same volume. Bertsch (1976) reviewed the literature on punishment of instrumental and
consummatory behavior.
"Conflict" is perhaps an unfortunate term, implying a mental condition in which the
urge to do a thing (e.g., press a lever for food) conflicts with an urge to avoid an unpleas-
ant consequence (e.g., receive an electric shock). However, the term has been used in this
sense for 3 decades, and it is used here occasionally for convenience. We prefer the more
precise, less mentalistic terms formed on "punishment." An "anticonflict" or "antipun-
ishment" drug effect is a drug-induced increase in responding that has been suppressed
by punishment, and a "proconflict" or "propunishment" effect is a further decrease in
responding that has been suppressed by punishment.
This review of the operant literature is limited to stationary behaviors such as lever
pressing and key pecking, to the exclusion of wide-ranging behaviors such as maze-run-
ning, because stationary behaviors have been employed in most studies. The review is
organized by species for convenience and because there are important species differences
in the effects of intuitively noxious stimuli upon ongoing behavior and the effects of
drugs upon behavior suppressed by these stimuli.
Most of the operant studies of interest were done with multiple schedules, in which
punishment occurred in one component and not in the other. The unpunished compo-
nent is often thought to register nonspecific drug effects, but in fact unpunished respond-
ing is a complex phenomenon in this context, and the effects of drugs can depend on
many factors. In some cases, conventional anxiolytics increase both punished and
unpunished responding, in other cases, they increase only punished responding, and in
still other cases, they increase punished and decrease unpunished responding. Stimulants
decrease punished responding, but they may concurrently increase or decrease unpun-
ished responding. The unpunished component serves to maintain responding, and it
yields useful information where the effects of a drug can be compared to the effects of
standard anxiolytic and nonanxiolytic drugs under identical experimental conditions
(same schedule, similar baselines, same laboratory, etc.). But the only sound generality
about its use is that if a drug reduces both punished and unpunished responding, then
that drug is probably not a conventional anxiolytic. We will discuss the effects of drugs
on punished responding only.
4 . 2 . EARLY OPERANT MODELS OF ANXIETY
Estes and Skinner ( 1 9 4 1 ) trained rats to press a lever for food and then administered
unavoidable electric shock preceded by a signal. Presentation of the signal acquired the
capacity to suppress lever-pressing, a conditioned emotional response (CER). Although
134 J . L . HOWARD AND G . T . POLLARD
some anxiolytics reverse the CER, this preparation has not shown good drug-class spec
ificity, probably because the punishment is adventitious, not response-dependent (Kel-
leher and Morse, 1964; McMillan and Leander, 1975). The CER is discussed by Davis
in Chapter 8 of this volume.
Masserman and Yum (1946) trained cats to perform a complex operant task for food
and punished approaches to the food goal with shock and air blasts. This manipulation
produced experimental neurosis characterized by aberrant social behavior and somatic
function. Alcohol reversed some symptoms. Yen et al. (1970), using a similar prepara
tion, found that phenobarbital, pentobarbital, chlordiazepoxide, diazepam, and mepro-
bamate produced a qualitative normalization of neurotic behavior, and chloφromazine
and ^/-amphetamine did not; however, the antipsychotic haloperidol also was effective.
4.3. EXPERIMENTS WITH THE R A T
Geller and Seiflter (1960) designed the first operant procedure to be widely applied as
a screening method for putative anxiolytics. Food-deprived rats were trained to press a
lever for sweetened condensed milk on a multiple (mult) schedule of reinforcement in
daily 75-minute sessions. During four 15-minute portions of the session, a drop of milk
was delivered for a lever-press every 2 minutes on average (variable-interval 2-minute
schedule of reinforcement: VI 2 minutes). During four 3-minute portions of the session,
in the presence of a discriminative stimulus, a drop of milk was delivered for every lever-
press (fixed ratio 1: FR 1) and a foot shock was delivered simultaneously. This schedule
may be abbreviated as mult VI 2-minute (food) FR 1 (food -h shock); the FR 1 (food -h
shock) condition, signaled by the tone, is often called the conflict portion. Early in train
ing, before shock was introduced, the rats responded at a high rate for the high payoff
during the tone; shock reduced this rate to near zero. When the rats had achieved stable
baselines, with moderate rates of responding during the VI portion and low rates during
the conflict portion, drugs were tested for their ability to increase the responding sup
pressed by punishment, that is, during the four 3-minute FR 1 (food -h shock) conflict
periods. The VI portion was assumed to register nonspecific drug effects such as sedation.
The anxiolytics meprobamate, phenobarbital, and pentobarbital markedly increased
responding in conflict. They restored responding suppressed by punishment. The anti
psychotic promazine and the stimulant ^/-amphetamine did not have this effect. In sub
sequent studies, in which rats pressed a lever for milk, food pellets, or other reinforcers
under a similar mult schedule, many chnically active anxiolytics increased punished
responding and many nonanxiolytics did not; Table 6.2 gives a representative sample of
these studies. Geller's early work is reviewed in Geller (1962).
Howard and Pollard (1977; PoUard and Howard, 1979; Howard et al., 1982) modified
the Geller-Seifter procedure by having the shock level begin at zero in each conflict
period and increase by 0.05 mA increments with each response. This modification facil
itated training and maintenance of baselines. Drug effects were qualitatively similar to
those found with the original procedures. Results are summarized in Table 6.2.
Davidson and Cook (1969; Cook and Davidson, 1973) modified the Geller-Seifter pro
cedure by shortening the VI requirement to 30 seconds and lengthening the FR require
ment to 10. A daily session of responding for food consisted of seven 5-minute VI seg
ments without punishment and six 2-minute FR segments with punishment
accompanying the pellet. They were able to maintain stable baselines in some rats for 2
years without changing the shock level. In general, drug-class specificity is comparable
to that of the Geller-Seifter procedure. Table 6.3 gives representative results. Howard and
Pollard (unpublished data) replaced the FR 1 component of their modified Geller-Seifter
procedure with FR 10; this change did not improve buspirone's marginal antipunish-
ment effect.
Table 6.4 gives results firom rats responding on schedules that do not fall clearly into
the Geller-Seifter or Davidson-Cook categories.
TABLE 6.2. Effects of Selected Drugs on Punished Responding of the Rat in

the Geller-Seifter Procedure*
Increase No increase
A. Clinical anxiolytic activity demonstrated

(True-positive) (False-negative)
Chlordiazepoxide^'^^'"-^*"'^**
Clobazam*
Diazepam**'*''^'"'P-'*y***
Lorazepam"
Oxazepam*»*^^**
Meprobamate'-'^P''
Pentobarbital*'*''^'^
Phenobarbital»'''
Temazepam*
Buspirone*'*''*'
B. Clinical anxiolytic activity not established
Azepexole** Amphetamine*'''^
Bromolysergic acid^t Atropine''
Cinanserirf'** Chloφromazine*''
Clonidine*''^'^^ Δ.9 TH<?
Emylcamate*" Diphenylhydantoin"'^
Ethanol" Idazoxam**
Gepirone**! Methysergide''
Hedonal" Moφhine'''
Ipsapirone**! Naloxone"
Methysergide** Phenoxybenzamine"
P-chlorophenylalanine"'**t Phentolamine"
Toluene" Promazine*
Tracazolate*'**t Propranolol*'**
Trimethadione*'t Yohimbine**'"
Urethan^t
Valproic acid*'*
Yohimbine*
•Babbini et al., 1975. »»Babbini et al., 1982. *=Bennett et al., 1982. *»Bullock et al., 1978.
Yielding and Hoffmann, 1979. 'Geller, 1964. «Geller and Seiner, 1960. *<3e\\tT and
Seiner, 1962. Oeller et al., 1962. ^Geller et al., 1974b. ''Geller et al., 1976. "Geller and
Hartman, 1982. "Gelleretal., 1983. "Goldbeig and Ciofalo, 1969. *»Handley and Mith-
ani, 1984. »»Hartmann and Geller, 1978. *»Hartmann and Geller, 1981. 'Howaid et
al.,1982. 'Howard and Pollard, 1987. ^Howard and Pollard, 1988. "Koob et al., 1984.
^Kruse et al., 1981. '"Lal et al., 1980. "Myslobodsky et al., 1983. Ôhmori et al., 1980.
^Pollard and Howaid, 1979. "Robichaud and Sledge, 1%9. '*Robichaud et al., 1973.
**Stein et al., 1975. *"Young et al., 1987.
*Rats responded for food or water on a mult schedule consisting of relatively long
segments of unpunished variable-interval (VI) reinforcement and short segments of
punished fixed ratio (FR) 1 reinforcement. Drugs were administered peripherally
(orally, intraperitoneally, subcutaneously). Drugs under A are accepted as anxiolytic or,
for some benzodiazepines and barbiturates, are congeners of accepted anxiolytics; those
separated at the bottom under A are clinically effective only with chronic treatment (all
animal tests cited are acute). Some drugs under Β are probably anxiolytic in some cir
cumstances, but clinical data in generalized anxiety disorder are not strong (for exam
ple, ethanol, some tricyclic antidepressants); other drugs under Β are probably not anx
iolytic, and some may be anxiogenic (for example, amphetamine). See text for other
important caveats.
flndicates that the antipunishment effect was small or inconsistent or the data were
inadequate.
Inspection of Tables 6.2,6.3, and 6.4 reveals several generalities and a few informative
specific points about the effects of drugs on punished operant responding in the rat. (1)
Conventional anxiolytics—benzodiazepines, propanediol carbamates, and barbitu
rates—are almost invariably positive. The two failures, noted in Table 6.4, occurred in
the same study, in which the schedules of reinforcement and punishment were unusual.
In the conflict portion, every response was punished, but only the first response after 100
seconds was reinforced, that is, there were many more punished responses than rein
forced responses, whereas in most other studies, the ratio of punished responses to rein
forced responses was 1:1. Anticonflict effect in the rat identifies conventional anxiolytics
TABLE 6.3. Effects of Selected Drugs on Punished Responding of the Rat in

the Davidson-Cook Procedure*

Amobarbital**-*
Bromazepam*
Chlordiazepoxide**'**''''''"'^
Clonazepam*
Diazepam'''**'*'^^
Flunitrazepam*
Lorazepam*
Meprobamate**
Methaqualone**
Midazolam^
Nitrazepam*
Oxazepam**
Pentobarbital'
Phenobarbital**
Tybamate**t
Buspirone"! Buspirone*-''
B. Clinical anxiolytic activity not firmly established
Benactyzine**t AAOA*
Cinanserine* a-methyl-tryptamine**
Cyproheptadine^t Amoxapine''
8-ΟΗΟΡΑΤ· Amphetamine**
Ethanol**t Caffeine*
7-butyrolactone' Chloφromazine**-»
Gepirone' Cinanserin**
Ketanserin* Diphenhydramine**
Methysergide* *»t DMT**
Mianserin'*! Doxepin''
Moφhine**t Haloperidol**
P-chlorophenylalanine"'t Imipramine**
Phencyclidine**t Iproniazid**
Ritanserin' Maprotiline''
Trifluoperazine***! Mescaline'
Yohimbine't Muscimol^
P-chlorophenylalanine*
Propranolol"*
Theophylline*
THIpf
Trazodone''
•Amrick and Bennett, 1986. '^BenneUand Amrick, 1987. *=BenneUetal., l985.**Cook

and Davidson, 1973. *Cook and Sepinwall, 1975. t o o k and Sepinwall, 1980. »David
son and Cook, 1969. ''Mason et al., 1987. 'Mcintire and Liddell, 1984. JPieri, 1983.
''Sepinwall et al., 1973. 'Sethy and Winter, 1972. "'Shephard et al., 1982. "Sullivan et
al., 1983. <»Wimer, 1972.
•Rats responded for food or water on a mult variable-interval (VI) ñxed-ratio (FR)
schedule. Typically, a session consisted of seven 5-min segments of VI 30-sec (food)
and six 2-min segments of FR 10 (food + shock). Other information is the same as
that for Table 6.2.
inadequate.
very well. (2) Buspirone, the new anxiolytic that has achieved the most clinical success,
is weakly positive or negative. We found no convincing study in which buspirone
released punished responding as much as conventional anxiolytics did; in several exper
iments in our laboratory, with a Geller-Seifter procedure in which every response or
every 10th response was both reinforced and incrementally punished in the conflict por
tion and a sample size of about 10, typically one or two doses produced a small but
significant release. This drug, which is ineffective acutely in the clinic, appears to have
the same action on punished responding in the rat as several nonanxiolytics, which the
test rejects on the basis of magnitude. (3) Drugs that are usually agreed to be ineffective
TABLE 6.4. Effects of Selected Drugs on Punished Responding of the Rat

Under Miscellaneous Schedules of Reinforcement and Punishment*

Bromazepam** Pentobarbital'
Brotizolam*
Chlordiazepoxide'^**'J''*-"''*'*
Diazepam**''^*"'"'*»'
Flurazepam"t
Midazolam**
Nitrazepam'
Pentobarbital*'
Premazepam"
Buspirone'
Atropine*'t ΑΑΟΑ"·Ρ
Chloral hydrate*" Amphetamine'*''"
Chloφromazine't BicucuUine^
Cyclazocine' Caffeine*"
Cyproheptadine*^ Diphenhydramine*"
Diprenoφhine• Ethylketocyclazocine*
g-OHDPAT** 7-vinyl GABA*
Ethanol**t Ketocyclazocine*
Ethchlorvynol** Moφhine"'
Ketocyclazocine't Naloxone*-'
Methysergide*"'** Naltrexone'
Mianserin**t Picrotoxin^
Moφhine't Pirenperone'
MuscimoFt Pyrilamine*"
Phencyclidine^ Ro 15-1788«»
Ritanserinf
Tertiary butanol**
Toluene'
Valproic acid^
Zopiclone**'"
•Mult (VI l-min [food] (conj VI 1-min [food] FR 1 [shock]); DeRossett and Holtz
man, 1985. *»Mult FR 5 (ESSB) FRl (ESSB + shock); Gomita and Ueki, 1981. *<:onc
Π 1-min (water) FR 5 (shock); Graef, 1974. **Mult VI 20-sec (food) VI 20-sec (food +
shock); Hodges et al., 1987. «Mult (FR 4 [food]) (conj FR 4 [food] FR 1 [shock]); Huot
et al., 1981. ^Muh VI 35-sec (food) Π 180-sec (food + shock); Hymowitz and Abram-
son, 1983. 'Mult FR 30 (food) FR 30 (food + shock); Kuríbara and Todokoro, 1980.
''Muh (VI 1-min [food]) (conj VI 1-min [food] VI 1-min [shock]); Lemer et al., 1986.
'Mult (Π 100-sec [food]) (conj Π 100-sec [food] FR 1 [shock]); McMillan and Leander,
1978. JMult Π 1-min (food) FR 1 (food + shock); Porter et al., 1987. ''Mult (VI 32-sec
[food]) (conj VI 32-sec [food] independent VI 32-sec [shock]); Sanger and Blackman,
1978; a similar schedule in Sanger et al., 1985. 'Mult (Π 3-min [food]) (conj Π 3-min
[food] FR 5 [shock]); Snell and Harris, 1982. "Mult VI 30-sec (food) VI 30-sec (food
+ shock); Tye et al., 1979. "Mult FR 20 (food) FR 1 (food + shock); Ueki, 1987; same
schedule in Ueki et al., 1984. "Unpublished observation cited in van Riezen and van
der Buig, 1978. "MuU FR 21 (food) FR 1 (food + shock); Vellucci and Webster, 1984.
*'Mult FR 30 (food) FR 10 (food + shock); Witkin, 1984. 'Mult (Π 5-min [food]) (conj
Π 5-min [food] VR 2 [shock]); Wood et al., 1984. 'Mult Π 30-sec (food) FR 5 (food
+ shock); Gardner, 1986.
•Rats responded for food or water (or ESSB where indicated) on the schedules given
in the footnotes (some schedule designations may be imprecise). Mult = multiple, cone
= concurrent, conj = conjoint; FI = fixed-interval, VI = variable-interval, FR -
fixed-ratio, VR » variable-ratio; ESSB = electrical self-stimulation of brain. Other
information is the same as that for Table 6.2. For discussion of schedules of reinforce
ment and other issues in operant behavior, see Honig and Staddon (1977).
inadequate.
in generalized anxiety, for example, antipsychotics (except perhaps at low doses), stim
ulants, and suspected anxiogenics, are negative. They do not release responding sup
pressed by punishment or they do so to a smaller degree than conventional anxiolytics.
Deciding whether a drug should be categorized as nonanxiolytic is difficult; even the
division between categories A and Β in the tables is somewhat arbitrary. Drugs that
showed any activity were put in the positive column and flagged if the efiect was rela
tively small or questionable; these drugs may belong in the negative column. (4) Several
drugs for which clinical anxiolytic activity has not been determined faU into the positive
column. Some have been reported to be effective in some types of anxiety. Many are
serotonin ligands, which are of interest primarily because buspirone is a serotonin ligand.
The Geller-Seifter and Davidson-Cook procedures seem about equal in predictive ability.
Such practical factors as stability of baseline and magnitude of antipunishment effect
should dictate the choice of schedules for a screening method, with VI and low-value FR,
which generate steady rates of responding, being perhaps preferable to FI, which often
generates a series of scallops.
In summary, release of punished responding in the rat is a good predictor of clinical
activity for conventional anxiolytics. There are a number of problematic positives, but
no known false-negatives except buspirone and the tricyclic antidepressants, which can
be viewed together as a special case because of the delayed onset of therapeutic action.
4.4. EXPERIMENTS WITH THE PIGEON
Table 6.5 gives results from pigeons responding on various multiple and conjoint
schedules of reinforcement with a punishment component. Inspection of the table sug
gests that release of punished responding in the pigeon has about the same degree of drug-
class specificity as in the rat, with one important exception. In the pigeon, the effects of
buspirone are as large and robust as the effects of conventional anxiolytics. To the best
of our knowledge, this effect of buspirone and its congeners has been demonstrated in
only one laboratory; we are attempting to replicate it.
Two other points should be considered: (1) that the positives by amphetamine and
moφhine occurred in only one study, for which the schedule was a 5-minute FI, and the
negatives by chloral hydrate and ethanol occurred also in one study each, also on a 5-
minute FI, in the same laboratory. Although there are instances in which responding on
long FI schedules with a punishment contingency has generated data that support the
contention that anxiolytics, and only anxiolytics, robustly increase punished responding,
it may be that the wide range of response rates maintained by an FI schedule does not
constitute an ideal baseline for registering the effects of anxiolytics specifically; and
(2) that all but one of the drugs in the positive column of category Β of Table 6.5 are
flagged because their effects were not as robust as those of conventional anxiolytics.
4.5. EXPERIMENTS WITH THE MONKEY
Use of the monkey in preclinical screening for anxiolytics is problematic. It is expen

sive, and there is no convincing evidence that monkey models are superior to rat models
in quality of data. The phylogenetic proximity of monkey to man is not demonstrably
advantageous, at least with respect to operant behavior (Howard and Pollard, 1983).
Although the complexity of infrahuman primate behavior presents unique opportunities
for modeling psychopathology, it may be disadvantageous in the practical business of
screening unknown compounds for potential psychotherapeutic action.
Table 6.6 gives results from monkeys responding in several operant procedures with
punishment as an element. Under specified conditions, the effects of anxiolytics and non
anxiolytics are essentially the same as in rats, including the inconsistent effect of buspi
rone. However, the monkey exhibits some peculiar behaviors with respect to experimen
tal history and context (peculiar in that they have not, to our knowledge, been
demonstrated in lower species). The issues are complex, but in general, if the event main
taining behavior is shock presentation (i.e., if the preparation has been manipulated so
the monkey emits a behavior in order to shock itself) or if the subject has a history of
responding to avoid shock, then stimulants increase responding suppressed by punish-
TABLE 6.5. Effects of Selected Drugs on Punished Responding of the

Pigeon*

Amobarbitar**
Chlordiazepoxide*«'»''''"*»''*''"'**'"
Diazepam"''"'*
Meprobamate™
Midazolam'
Nitrazepam"
Oxazepam"
Pentobarbital'*''*'"''"''*'*''"'^''*'*"
Secobarbital"
Buspirone**''**"
Amphetamine" ACTH 4-10·
Atenolol*t a-methyl-tryptamine»
BOLt Amphetamine*'*"''^'*''''*»'^
Chlorimipramine''t Apomoφh¡ne**
Chloφromazine"t Chloral hydrate**
Clozapine't Chloφromazine"'''''''*
8-OHDPAT'''*t Cyproheptadine*'
EthanoP'*'*'t Δ-8 T H C "
Gepirone^'t Δ-9 T H C "
Imipramine'''"'t Doxepin"
Inosine^t Ethanol*
IsopropranoloFf EthchlorvynoP
Ketamine^'**'^t Haloperidol''**
Metergoline'* Imipramine"
Methaqualone** IsopropranoloP
Methysergide*t Mescaline"
Moφhine"' Moφhine"'»''*'^
Phencyclidine*''"t
Propranolol*t Pentazocine"
Propranolol**
Propylene glycol**
Ro 15-1788**'^''
Tetrabenazine"
TRH*"
*Mult (VI 3-min [food]) (conj Π 5-min [food] FR 50 [shock]); Barrett and Witkin,
1976. **Muh FR 30 (food) FR 30 (food + shock); Barrett et al., 1986b. *"Mult ( Π 5-min
[food]) (conj π 5-min [food] FR 1 [shock]); Brandäo et al., 1980. **Muh Fl 5-min (food)
FI 5-min (food + shock); Chait et al., 1981; effects of phencyclidine and ketamine were
as large as effects of pentobarbital. *Mult FR 30 (food) FR 30 (food + shock); Durel et
al., 1986. '^Mult FR 30 (food + shock) Π 5-min (food + shock) and mult (Fl 5-min
[food + shock]) (conj Fl 5-min [food] FR 30 [shock]); Foree et al., 1973. «Conj FI 5-
min (food) FR 30 (shock); Graeff and Schoenfeld, 1970. ''Muh (Fl 3.3-min [food]) (conj
Π 3.3-min [food] FR 2 or 4 [shock]); U m b and McMillan, 1985. *Conj Fl 5-min (food)
FR 1 (shock); Leander et al., 1976. C o n j FI 5-min (food) FR 20 (shock); same reference
as i. ''Muh Fl 5-min (food) Π 5-min (food + shock); Leone et al., 1983. 'Muh FR 30
(food) FR 30 (food + shock); Mansbach et al., 1988. ""Muh Fl 5-min (food) Fl 5-min
(food + shock); McMillan, 1973a. "Muh Π 5-min (food + shock) FR 30 (food +
shock); McMillan, 1973b '»Muh Π 5-min (food + shock) FR 30 (food + shock);
McMillan, 1973c. "Conj Fl 5-min (food) FR 1 (shock); McMillan and Leander, 1975.
*<:onj Π 5-min (food + shock); McMillan, 1976. 'MuU (VI 3-min [food]) (conj VI 3-
min [food] FR 1 [shock]); Morse, 1964. »Muh Fl 130-sec (food) VI 10-sec (food +
shock); Sahgal et al., 1979. »Muh (Fl 3-min [food]) (conj Π 3-min [food] FR 1, 10, or
30 [shock]); Stitzer, 1974; moφhine increased moderately suppressed responding.
"Muh FR 30 (food + shock) VI 160-sec (food + shock); Wenger et al., 1986. ^Mixed
and mult FR 30 (food) FR 30 (food + shock); Wenger, 1980. *Conj Fl 3-min (food)
FR 30 (shock); Witkin et al., 1987. "Muh FR 30 (food) FR 30 (food + shock); same
reference as w. yMuh (Fl 3-min [food]) (conj Fl 3-min [food] FR 30 [shock]); Witkin
and Barrett, 1986a. ' M u h (Fl 3-min [food]) (conj FI 3-min [food] FR 30 [shock]); Wit
kin and Barrett, 1985. **Muh FR 30 (food) FR 30 (food + shock); Witkin and Barrett,
1986b. **Conj Fl 5-min (food) FR 50 (shock); Witkin and Barrett, 1976. **Conj Π 5-
min (food) FR 50 (shock); Witkin et al., 1981. *'*k:onj Fl 3-min (food) FR 30 (shock);
Witkin et al., 1984. «Conj Π 5-min (food) FR 30 (shock); Wuttke and Kelleher, 1970.
•Pigeons responded for food on the schedules given in the footnotes (see Table 6.4
for explanations). Most injections were intramuscular. Other information is the same
as that for Table 6.2.
flndicates that antipunishment effect was small or inconsistent or data were
inadequate.
TABLE 6.6. Effects of Selected Drugs on Punished Responding in the
Monkey

(True-posiiive) (False-negative)
Chlordiazepoxide**"'"""''"'^^-' Pentobarbital'*
Diazepam'"'"''''**
Lorazepam"*'**'**
Meprobamate**'"
Midazolam**
Λ'-desmethyldiazepam''
Pentobarbital*'"*'"'**'''^
Phenobarbital"
Buspirone**'«^ Buspirone*^"-'^
Amphetamine*''*'*^'''*'*'!: Amitriptyline"
Cinanserin^ Amphetamine*'**'''^"'^
Clozapine^'^t ß-CCE^^^*
Cyproheptadine* Buprenoφhine''
Cyproheptadine!: Chlorpromazine*-**"
Ethanor-'t Chloφromazine*t
Ketanserirf Clothiapine"
Metergoline' Cocaine^
Metergoline't Diprenoφhine''
Methysergide^ Ethanol"
Methysergide't Etoφhine'*
Mianserin^ Imipramine"
Mianserin'* Ketanserin'*
Moφhine''* Loxapine"
Periapine" Mecamylamine*"
Pirenperone'* Moφhine"
Suriclone**t Naloxone**
Zopiclone^'**** Pirenperond
Ro 15-1788*·'*·^
Scopolamine**
Spiperone*
Spiperone'*
^ o n j Π 5-min (food) FR 30 (shock); Bacotti and McKeamey, 1979. «»Mult ( Π 10-

min [shock]) (conj Π 10-min [food] FR 30 [shock]); Barrett, 1977a. *<:onj Fl 5-min
(food) FR 30 (shock); Barrett, 1977b. *tonj Fl 3-min (food) FR 30 (shock); Barrett et
al., 1985b. *Conj Π 3-min (food) FR 30 (shock); Barrett et al., 1985a. ^Muh FR 30
(food) FR 30 (food + shock); Barrett et al., 1986a. «Conj Fl 3-min (food) FR 30
(shock); same as f. ''Complex schedules of S-S term and shock presentation; Brady and
Barrett, 1986. "Conj FI 5-min (S-S term) FR 30 (shock); Brady and Barrett, 1985. ^Conj
FI 3-min (food) FR 30 (shock); same as i. '*Mult (VI 1-min [food]) (conj VI 1-min
[food] FR 1 [shock]); DeRossett and Hoitzman, 1984. 'Mult ( H 5-min [food]) (conj Π
5-min [food] FR 30 [shock]); Glowa and Barrett, 1976. ""Muh VI 2-min (food) FR 1
(food + shock); Gluckman and Stein, 1978. "Mult (FR 30 [food]) (conj FR 30 [food]
FR 10 [histamine injection]); Goldberg, 1980. *>Muh (FR 30 [food]) (conj FR 30 [food]
FR 30 [shock]); Goldberg and Spealman, 1983. «»Same as o but with nicotine injection
in place of shock; see also Goldberg and Spealman, 1982, and Goldberg, S. R., et al.,
1983. *'Mult(VI 1-min [food]) (cone FR 10 [food] VR 15[shock]); Hanson etal., 1967.
'Conj FI 5-min (S-S term) FR 30 (shock); McKeamey, 1976. "Conj FI 5-min (food) FR
30 (shock); same as r. kTonj FI 10-min (food) FR 30 (shock); McKeamey and Barrett,
1975. "FR 1 (food + shock) for 3 hr, then FR 1 (food) for 3 hr, Patel and Migler, 1982.
T o n e (VI 6-min [food]) (conj VI 1.5-min [food] VR 24 [shock]); Sepinwall et al., 1978.
*A complex schedule of responding for food and shock reduction; Smith et al., 1978.
"Muh FR 30 (food) FR 30 (food + shock); Spealman, 1985. ^Mult (FR 30 [food]) (conj
FR 30 [food] FR 1 [shock or air pufl]); Spealman, 1979. ^Mult (FR 30 [food]) (conj FR
30 [food] FR 1 [shock]); mult ( Π 3-min [food]) (conj FI 3-min [food] FR 1 [shock or
air pun]); Spealman and Katz, 1980. «"Conc VI 1.5-min (food) VR 24 (shock) VI 6-
min (food) utilizing two levers; Sullivan et al., 1983. **Mult FR 30 (food) FR 30 (food
+ shock) and mult (FI 3-min [food]) (conj FI 3-min [food] FR 30 [shock]); Weissman
et al., 1984. «=Conj Π 3-min (food) FR 30 (shock); Wettstein, 1988. ****Conj Π 3-min
(food) FI 15 or 30 (shock); Wettstein and Spealman, 1987a. **Conj FI 3-min (food) FR
15 or 30 (shock); Wettstein and Spealman, 1987b. "iSame procedure as in Patel and
Migler, 1982; Goldberg, M. E. et al., 1983. »Mult VI 2-min (food) FR 1 (food +
shock); Geller and Hartmann, 1982.
Monkeys (mostly Saimiri sciureus) responded for food, shock avoidance, shock pre
sentation, or dmg injection on the schedules given in the footnotes (see Table 6.4 for
explanations). S-S term = stimulus-shock termination. Most injections were
intramuscular.
inadequate.
Jlndicates a history or behavioral context known to influence dmg effects (e.g.,
amphetamine increases punished responding in monkeys with a history of responding
for S-S term). Other information is the same as that for Table 6.2.
ment and anxiolytics further decrease responding suppressed by punishment (see

McKeamey, 1976, 1979; Barrett, 1981, 1987). Drug history can be critical also (e.g.,
Brady and Barrett, 1986, on moφhine's effect). Results from peculiar situations are
flagged with a double dagger in Table 6.6. The dependence of drug effects on details of
the preparation indicates the peril in approaching models anthropomoφhically or with
unexamined preconceptions.
4.6. EXPERIMENTS WITH OTHER SPECIES
The few studies on the eflfects of drugs upon punished operant responding in the mouse
suggest no qualitative species diflference. Diazepam and chlordiazepoxide were positive
(Prado de Carvalho et al., 1983; Spealman and Katz, 1980). Clozapine was also positive
(Spealman and Katz, 1980). CaflTeine had a small positive eflTect at one dose (Glowa,
1986; Kuribara et al., 1987), as did ethanol (Kuribara et al., 1987). Amphetamine, nic
otine, cocaine, j8-CCM, Ro 15-1788, and chloφromazine were negative (Glowa, 1986;
Kuribara et al., 1987; Prado de Carvalho et al., 1983; Spealman and Katz, 1980).
Pentobarbital was positive and ethanol negative in goldñsh (Geller et al., 1974a), and
diazepam was positive in pig (Dantzer and Roca, 1974) and man (Beer and Migler, 1975;
Carlton et al., 1981). Observational data in the cat (Yen et al., 1970) were mentioned
earlier.
4.7. CONCLUSIONS ABOUT THE U S E OF OPERANT METHODS
When parameters of the preparation are properly speciñed, release of operant respond-
ing suppressed by punishment is a reliable and reasonably specific predictor of clinical
activity by conventional anxiolytics. All standard anxiolytics are positive, as is the novel
putative anxiolytic zopiclone; buspirone is clearly positive only in the pigeon. Most stan-
dard drugs that are generally thought to be nonanxiolytic are negative; in almost every
case of an increase in punished responding by a nonanxiolytic, the drug can be excluded
because the eflfect is small.
Of the larger number of drugs that we were unwilling to classify as definitely anxiolytic
or nonanxiolytic because of insufläcient clinical data, about half were positive to some
degree and half were negative; a few were both. The positives tended to fall into two
groups, those with some sedative-hypnotic or muscle-relaxant properties (e.g., chloral
hydrate) and those that bind to serotonin receptor subtypes (e.g., ritanserin, which shows
some promise in the clinic). The model errs a bit on the side of inclusiveness.
The cases of Clonidine and yohimbine are intriguing. The first is an «2 adrenergic ago-
nist, the second an «2 adrenergic antagonist, yet both released punished responding (in
four studies in the case of Clonidine). Clonidine has been prescribed experimentally for
several psychiatric conditions, including opiate withdrawal; it may find its way into a
category of specialized anxiolytics (Hoehn-Saric et al., 1981), along with monoamine
oxidase (MAO) inhibitors for panic, /8-blockers for stage fright, and the like. Yohimbine's
antipunishment eflfect, on the other hand, is puzzling. The drug is anxiogenic in man
(Holmberg and Gershon, 1961), and its discriminative stimulus properties in rat are
blocked by anxiolytics (Browne, 1981).
5. DRINKING SUPPRESSED BY PUNISHMENT

5.1. DEFINITIONS
The distinction between unconditioned and conditioned suppression is a matter of

emphasis. Some aspects of the experimental situation may come to serve as cues (for
example, elapsed time when drinking is punished on a fixed-interval or fixed-ratio basis)
in the absence of overt discriminative stimuli. On the other hand, the subject may ignore
an intended warning cue such as a light or tone. We shall use "unconditioned" to denote
a preparation (usually acute, with only one drug test trial) in which there is no overt
discriminative stimulus. We shall use "conditioned" to denote a chronic preparation in
which an overt discriminative stimulus signals that responses will be punished, and there
are many drug test trials with several days intervening.
5.2. UNCONDITIONED SUPPRESSION
Naess and Rasmussen (1958) restricted cats' access to water and electrified the water
source, which suppressed approach. The anxiolytics amobarbital and meprobamate
overcame the suppression; benactyzine and chloφromazine did not. Leaf and Muller
(1965) applied this procedure with the rat; moφhine increased punished drinking, but
the session was long (60 minutes) and only one dose was tested.
Vogel et al. (1971) designed a procedure that, with many variations, has been widely
employed as a screening method for anxiolytics and a tool for exploration of the neural
basis of anxiety. They deprived naive rats of water for 48 hours, placed them individually
into an experimental chamber with a water spout, allowed them to drink briefly, and
then applied a 2-second shock to the spout for every 20th lick during a 3-minute trial.
The shock suppressed drinking. Five anxiolytics reversed this suppression, and three
nonanxiolytics did not. Other experimenters have used several daily sessions of habitu
ation and longer test sessions. Table 6.7 gives representative results.
5.3. CONDITIONED SUPPRESSION
In a procedure that may be seen as a hybrid of the Geller-Seifter and Vogel methods,
rats were water deprived and given daily sessions on a multiple schedule in which drink
ing was unpunished in one component and punished with mouth shock or foot shock in
the other (signaled) component. Stable baselines were established, and the effects of drugs
were determined with each subject serving as its own control. Table 6.8 gives represen
tative results. To judge by the number of references we found, conditioned suppression
of drinking is used about half as often as unconditioned suppression.
5.4. CONCLUSIONS ABOUT THE U S E OF PUNISHED DRINKING
Inspection of Tables 6.7 and 6.8 suggests that the punished drinking method produces
essentially the same results as the punished lever-pressing method in the rat. Although
punished drinking has received less attention from behavioral scientists and relatively
few nonanxiolytics have been tested, existing data show good drug-class specificity. Con
ventional anxiolytics are positive, ethanol is equivocally positive, and the few standard
nonanxiolytics are negative (in some cases, excludable, because the positive efiect is
weak). Systematic investigation of procedure (e.g., direct comparison of mouth and foot
shock or signaled and unsignaled shock, variation of the ratio of unpunished to punished
drinking, temporal placement of shock periods) would facilitate evaluation of the sensi
tivity of punished drinking.
Buspirone is strongly positive in three studies, weakly positive (i.e., excludable, if
weakly positive nonanxiolytics are excludable) in three studies, and negative in two stud
ies. In some studies, methodology was not reported in detail. In our laboratory, we exam
ined several variables (e.g., strain of rat, time of day, pretreatment time) in an unsignaled
procedure; chloridazepoxide was positive, but the effects of buspirone were weak and
variable.
Recently, Commissaris and co-workers found that chronic treatment with the tricyclic
antidepressants imipramine, desmethylimipramine, and amitriptyline, and the MAO
inhibitor phenelzine reversed conditioned suppression of drinking in rats (Fontana and
Commissaris, 1988; Fontana et al., 1989). The onset of effect was temporally similar to
the onset of clinical therapeutic effect in panic disorder and generalized anxiety disorder.
TABLE 6.7. Ejgfects of Selected Drugs on Drinking Suppressed by
Unsignaled Shock (Naive Rats)*

Bromazepam''
Chlorazepate''
Chlordiazepoxide^''J'''™«P'*''''^
Chlormethyldiazepam"
Clobazam*
Clonazepam'*'*
Cloxazolam"
Desmethyldiazepam"
Oesmethylfludiazepam"
Diazepam*'*^**''*'''"*^'*'^''
Fludiazepam"
Flunitrazepam'**-''
Flurazepam''*
Halazepam**
Lorazepam'**
Lormetazepam*
Medazepam'*'"
Meprobamate*»*^
Midazolam**
Nimetazepam"
Nitrazepam'-'*'"
Oxazepam'-'*-"-'
Oxazolam"
Pentobarbital'-*-'
Phenobarbital**-*
Ripazepam'*
Triazolam**
Buspirone^'^-y-"**-**'***''* (somef) Buspirone^*
a-PDAC Amphetamine'
Apomoφhine^t jö-carbolines"
Cyproheptadine" Chlorimipramine"
8-OHDPAT*'t ChloφΓomazine·
Erythrosine (Red No. 3y Cinanserin'
Ethanol"t Clonidine'
Fenobam** Cyproheptadine*
Gepirone*^ Δ-9 T H C
LSD" DMT"
Mescaline" Ethanol*
Morphine''! Mg pemoline'
/'-chlorophenylalanine***f Methyseigide*
Tertiary butanol*^ Muscimol*
Tofisopam' Neostigmine""
Tracazolate**-**-*» Nicotinamide*
Valproic acid*-* Paroxetine*
Zolpidem** Physostigmine""
Zopiclone**-* Propranolol*
Scopolamine'"-'
Strychnine*
Theophylline*
ΤΗΙΡ
•Corda et al., 1983. "»Depoortere et al., 1986, *tison et al., 1986. fEngel et al., 1984.
"Gardner and Piper, 1982. ^Hjorth et al., 1986. «Jones et al., 1988. "Leaf and Muller,
1965. 'Lippa et al., 1978. ^Mailman et al., 1980. ''Malick and Enna, 1979. 'Mendelson
et al., 1983. '"Chronic subjects; Miczek and U u , 1975. "Nakatsuka et al., 1985. **Patel
et al., 1983. PPatel et al., 1985. *»Patel and Malick, 1982. TPellow and File, 1986.
«Chronic subjects: Petersen and Lassen, 1981. »Sänger et al., 1985. "Schoenfeld, 1976.
^Shimizu et al., 1987. ''Stephens et al., 1987. *Sullivan et al., 1983. ^Taylor et al., 1985.
'Vogel et a L 1971. ••Vogel et al., 1980; also ethanol and chloidiazepoxide were positive
in mouse. '***Weissman, et al., 1984. «Oakley and Jones, 1983. *"Heym et al., 1987.
«File, 1985.
•Procedures differed between studies; following is a general description. Water-
deprived naive rats (except as indicated in footnotes) were habituated to drink in the
apparatus. In a single test session, typically 3 or 10 minutes long, drinking was sup
pressed by electric shock applied through the spout (or in some cases the grid), and
drugs were tested. Other information is the same as that for Table 6.2.
tindicates that the antipunishment effect was small or inconsistent or the data were
inadequate.
144 J . L . HOWARD AND G. T . POLLARD
TABLE 6.8. Effects of Selected Drugs on Drinking Suppressed by Signaled

Shock (Trained Rats)*

Amobarbital*
Barbital*
Chlordiazepoxide''""
Clobazam"
Diazepam*-'***''*;^'''
Flunitrazepam**'
Methaqualone**-*
yV-desmethyldiazepam*
Oxazepam*""
Pentobarbital**'^**'^
Phenobarbital^-*
Premazepam"
Secobarbital*^
Buspirone''t Buspirone""
Cinanserin^'t Amitriptyline*-''
DOM***t Amphetamine''^
Ethanort Caffeine'''*
Haloperidol't Carbidopa**
LSD**'*t Chloφromazine*·''^
Ρ-chlorophenylalanine''t Cinanserin*
Sulpiride'! Cyproheptadine''
Tracazolate" Diphenhydramine'
8-OHDPAr
Ethanol*
Fenfluramine''
S-HTP"
Imipramine**^
Isoproterenol'*
LSD"
M-CPP"
Metergoline*-''
Mescaline"
Methysergide*-"
Moφhine''^
Quipazine*
"Caccia et al., 1980. 'ârli and Samanin, 1982. '^Carli and Samanin, 1988. **Com-
missaris et al., 1981. ^Commissaris and Rech, 1982. t o m m i s s a r i s e t al., 1988. *Kiltset
al., 1981. "Kilts et al., 1982. 'McQoskey et al., 1987. ^McCown et al., 1983. ''Mokier
and Rech, 1985. 'Pich and Samanin, 1986; buspirone, haloperidol, and sulpiride pro
duced the same effect; diazepam's effect was much larger. "Gardner, 1986. "Budhram
etal., 1986.
•Procedures were the same as for Table 6.7 except that (1) subjects were trained to
stable baselines of drinking and were tested repeatedly, and (2) a discriminative stim
ulus during parts of the session signaled that drinking would produce shock.
inadequate.
These researchers also found that acute buspirone had no effect on its own, but reversed
conditioned suppression of drinking in rats that had been treated chronically with bu
spirone (Schefke et al., 1989). These results lend some face validity to the antipunish
ment effect as a model of anxiolysis.
6. NEUROCHEMISTRY OF ANXIOLYSIS
The neurochemical mechanisms of anxiety and anxiolysis are not known. However,
the literature is large, and there is good concordance between certain neurochemical
manipulations and consequent behavioral phenomena.
The benzodiazepine-gamma-aminobutyric acid (GABA)-chloride ionophore complex

has received much attention. Following are recent reviews on the benzodiazepine recep-
tor: Boast et al. (1983), Gardner (1988), Sepinwall and Cook (1980a,b). Sepinwall and
Cook (1980b) and Sanger (1985) (üscuss GABA. Following are reviews focused on anxio-
genic action at this complex: File and Baldwin (1987), Pellow and File (1984), Skolnick
and Paul (1983), Stephens et al. (1986), Thiébot et al. (1988).
Serotonin is currently of interest because buspirone and some other putative anxio-
lytics such as ritanserin bind to serotonin receptor subtypes. The evidence is suggestive,
but drugs that act on different subtypes have similar behavioral effects and drugs that
appear to act the same neurochemically have different effects. Following are some recent
reviews: Gardner (1986), Kahn et al. (1988), Riblet et al. (1984), Thierbot (1986). Bu-
spirone's therapeutic effect is often attributed to its action on serotonin, but it also binds
to dopamine receptors (Taylor et al., 1982).
7. GENERAL CONCLUSIONS
Tables 6.1 through 6.8 contain no category for nonanxiolytics, yet in the text, we have
characterized several drugs as nonanxiolytic. This reflects the ambiguity of any exhaus-
tive classificatory scheme for anxiolytics and the necessity to be arbitrary—to practice a
seat-of-the-pants taxonomy—in order to make a review coherent. Although the tables
do not reflect it, we feel that the foUowing scheme is worth entertaining. Benzodiaze-
pines, barbiturates, propanediol carbamates, and many sedative-relaxants such as meth-
aqualone and chloral hydrate have anxiolytic activity acutely. Buspirone and probably
the tricyclic antidepressants have anxiolytic activity in many patients after chronic treat-
ment. Ritanserin and some other drugs that work on the serotonin system show promise
as the next generation of anxiospeciñc drugs. Antipsychotics, opiates, and phencyclidine-
like drugs are not anxiolytic, or not so in the same way that those mentioned immedi-
ately above are, and, in any case, should register negative in an ideal model because of
their untoward effects. Amphetamine, strychnine, and many jS-carbolines are anxiogenic
and should also be rejected by an ideal model. After much agonizing, we present this
scheme, the tables, and the following remarks as a sort of informative failure—the best
we could do given the temporal restraints and the ambiguities in the subject matter.
An increase in responding suppressed by punishment is the most reliable model for
predicting anxiolytic action. AU anxiolytics in widespread clinical use have this effect in
one or more behavioral preparations. There are some unexplained results, primarily
false-positives, but in most cases, they can be dealt with: The effects are small or subject
to exclusion on the basis of secondary tests. The increase in punished responding by
stimulants seems to be conñned to the monkey, which suggests the need for care in spec-
ifying behavioral history and context, as weU as the possible inappropriateness of this
expensive species for primary drug development.
Buspirone is problematic. Its effects are small or inconsistent in all preparations except
the pigeon. However, this does not invalidate punished responding as a screen for acutely
active anxiolytics, because buspirone and the tricyclic antidepressants are inactive
acutely in the cUnic. The case of buspirone does point up the lack of a viable screen for
anxiolytics that work only after chronic treatment and the possibUity that existing screens
register not pure anxiolysis but some combination of anxiolysis and side effects such as
euphoria. On the other hand, there may be neurochemically distinct types of anxiety.
Tests using suppression of operant behavior are more highly developed than tests using
suppression of drinking, probably because operant behavior has a tradition and a strong
advocacy group in the experimental analysis of behavior. Basic behavioral processes are
subject to continual investigation and elaboration, a rigorous process that functions not
only to improve sensitivity, but to reveal inadequacy and puzzling phenomena.
Suppression of drinking has been more employed than developed. The literature sug-
gests that experimenters adapt the basic method to suit the immediate purpose. There
146 J. L . HOWARD AND G . T . POLLARD
are few papers in which many anxiolytic and nonanxiolytic drugs have been tested or
several parameter values varied. On balance, suppression of drinking appears to be no
more specific and reliable than operant tests, and it has the liabilities, at least with naive
subjects, of intersubject variability and the interaction of this factor with environmental
variables that, in chronic operant subjects, may be canceled by habituation.
To the use of punished responding as a screen for anxiolytics in the industrial labo
ratory there is one unresolved objection. It has not produced a successful unique
anxiolytic. However, when used with adequate consideration for the limitations noted,
the release of punished behavior is a reasonably precise and economical predictor of clin
ical anxiolytic activity and is perhaps the most specific behavioral screening method for
psychotherapeutic drugs.
Following are some articles in which the effects of drugs upon punished behavior are
reviewed: Bamett, 1986; Beer et al., 1972 (suppression of drinking); Cook, 1982;
Dantzer, 1977; File and Pellow, 1987; Iversen, 1980; Lippa et al., 1979; Patel and Malick,
1983.
Acknowledgments—The authors extend appreciation to the several persons at Burroughs Wellcome Co. who
rendered valuable service in the preparation of this chapter, especially the following: Robert Kilgore, Technical
Information Division, for on-line database searches and procurement of reprints; Jerry D. Thomas, Division
of Pharmacology, for assistance in organizing the material; the staff of the Word Processing Center for turning
a Hydra-headed bundle into a typescript; and the keen-eyed E. Allen Jones, Technical Information Division,
for editing.
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CHAPTER 7
ETHOLOGICALLY BASED ANIMAL MODELS OF

ANXIETY DISORDERS
RICHARD G. LISTER
Laboratory of Clinical Studies, NIAAA, Bethesda, Md, USA
1. INTRODUCTION
Several problems confront the behavioral scientist interested in developing animal
models of anxiety. An issue that has frequently been ignored in the animal literature
concerns classification. Is anxiety a unitary phenomenon or are there many different
forms, each with a separate underlying neurobiology? Clinicians have been discussing
this issue for some time (see, e.g., Tyrer, 1986; Marks, 1987; Levin and Liebowitz, 1988;
Barlow, 1988) and in the revised version of the Diagnostic and Statistical Manual of
Mental Disorders ([DSM III-R] American Psychiatric Association, 1987), generalized
anxiety disorder (GAD), panic disorder (with and without agoraphobia), simple phobias,
obsessive compulsive disorder (OCD), social phobia, and post-traumatic stress disorder
are all considered separately in the section on anxiety disorders. In addition, it is recog
nized that anxiety can be associated with other clinical diagnoses such as depression, or
can have an organic origin, resulting from the intake of psychotropic drugs such as caf
feine. It must be emphasized that the present division of anxiety disorders into these
separate categories is still highly controversial, but the apparent heterogeneity of anxiety
disorders certainly needs to be considered in discussing animal models of anxiety. If anx
iety is being modeled by a particular paradigm, just what is the nature of that anxiety?
On September 3, 1931, Pavlov stated
T o make analogies between the neurotic state o f our dogs and the various neuroses o f man
is to me, a physiologist not thoroughly acquainted with human neuropathology, a problem
hardly attainable. But I a m convinced that the decision, or the conditions favourable to a
decision, of many important questions of etiology, the natural systematisation, the mecha
nism and finally the treatment o f neuroses in the human being lies in the hands of the a n i m i l
experimenter (see Pavlov, 1941, p. 74).
Today, it remains difficult for the behavioral pharmacologist working with animals
and with minimal patient contact to integrate animal and human data. It still seems
likely, however, that studies in laboratory animals will indeed shed light on the appro
priate classification and treatment of anxiety disorders.
A second issue concerns what we understand by the term "model". At one level, the
ideal model would reproduce all the features of the phenomenon under investigation
and, as such, would be an exact reproduction rather than a model. An example of such
a "substitutive" model might be the use of the visual system of chimpanzees as a model
of human vision (Suomi, 1987, pp. 7-8). Whether it is possible to reproduce, as opposed
to model, in animals any form of anxiety that is observed in humans is questionable. For
example, cultural factors appear to play a role in the human response to anxiety-provok
ing situations (Kanfer, 1985; Good and Kleinman, 1985; Tan, 1988). It is tempting to
speculate that cultural factors play an important role in the sex differences observed in
the incidence of anxiety disorders (e.g., Raguram and Bhide, 1985; Showalter, 1985). In
rodents, females appear less anxious than males in a number of models of anxiety (see
Gray, 1979; FaraboUini et al., 1987), whereas in humans, females with anxiety disorders
155
156 R. C L I S T E R
are encountered somewhat more frequently in the clinic than males. Another issue that
is difficult to address is whether the cognitive differences between humans and animals
leads to qualitative differences in behavior associated with anxiety.
Lorenz (1974) made the important distinction between analogy and homology in the
discussion of animal behavior. Homology was defined as "any resemblance between two
species that can be explained by their common descent from an ancestor possessing the
same character in which they are similar to each other." Analogous behaviors are those
that have evolved independently. Lorenz (1974) argued that there is no such thing as a
false analogy. "An analogy can be more or less detailed and hence more or less infor
mative." He felt comfortable in using the term "jealousy" to describe certain behavior
patterns of geese and humans, although a number of psychologists strongly disapproved
of such anthropomoφhism. These two forms of jealousy are considered analogous rather
than homologous, since the last common ancestor of humans and geese was incapable
of complicated social behavior. In discussing anxiety, it is not always easy to distinguish
between analogy and homology in human and animal behaviors. In view of this diffi
culty, the term "anxiety" will be used in this paper in the discussion of animal behavior
without an a priori assumption that the animal anxiety is homologous with that seen in
humans.
Finally, it is important to consider the function of animal models. The ρυφθ8€ that a
model is intended to serve determines the criteria by which it is appropriately evaluated
(Overmier and Patterson, 1988). Do we wish to use the model to examine signs or symp
toms of anxiety in humans or to investigate the effects of potential anxiolytic (or anxi
ogenic) treatments? Is the model to allow us to examine a mechanism involved in anxiety
and, if so, is the mechanism under investigation a cognitive, neurochemical, environ
mental or genetic one? Clearly, the assessment of a model is also critically dependent on
our knowledge of the phenomenon that is being modeled. Unfortunately, our knowledge
of anxiety in humans is still rather limited, and many of the problems confronting the
animal models stem from this incomplete understanding.
Before discussing animal models of anxiety individually, a few general points will be
made that should be considered in reading the following sections. First, an assumption
implicit in many animal models is that it is possible to investigate anxiety in an unse-
lected general population of animals. Such populations are used in many studies exam
ining the efficacy of potential anxiolytic treatments (but see Soubrie et al., 1974). There
is general agreement that a random sample of the human population will have varying
levels of anxiety, but only a significant minority are sufficiently disturbed by their anxiety
to seek clinical help. Whether the anxiety that these individuals experience differs only
quantitatively, or also qualitatively, from that of the rest of the population, is at present
unclear. It should be noted that, in humans, a drug's efficacy as an anxiolytic is often not
apparent in populations of normal volunteers, but is only evident in clinically anxious
populations.
Second, it is important to distinguish between trait and state anxiety. State anxiety is
the anxiety a subject experiences at a particular moment in time and is increased by the
presence of an anxiogenic stimulus. Trait anxiety, in contrast, does not vary from
moment to moment and is considered to be an enduring feature of an individual. (For
a discussion of the stability of personality traits, see Brody, 1988.) A subject with arach-
naphobia may have a trait anxiety in the normal range and, under most circumstances,
a low state anxiety level. However, the introduction of a spider into the individual's envi
ronment will produce a marked increase in state anxiety. Most research in behavioral
pharmacology has focused on drug-induced changes in state anxiety, where an animal is
confronted with an anxiety-provoking situation and the effect of the drug of interest is
examined. While this approach is convenient, because it is generally rapid, it does have
some important limitations. Most notably, it ignores factors that contribute to high trait
anxiety, which presumably underlie the development of GAD. It would be predicted,
therefore, that treatments that were effective in the state models may reduce state anxiety
Ethologically based animal models of anxiety disorders 157
in humans in situations in which this had been increased by some anxiety-provoking

situation. Such treatments, however, might not be of great long-term benefit in the
chronically anxious patient. In this context, it should be noted that in DSM-III-R to
qualify for a diagnosis of GAD, a subject must have had more or less continuous signs
of anxiety for at least 6 months.
There are currently a number of paradigms that have been used to assess drug effects
on anxiety. Some are more generally accepted as useful models of anxiety than others,
and some paradigms that have not been proposed as models of anxiety (e.g., the hole-
board test), have often provided results that have been loosely discussed with reference
to anxiety. This paper focuses on ethologically based paradigms that focus primarily on
what may be loosely described as spontaneous unconditioned behaviors. Tests based on
operant conditioning, classical conditioning, and drug discrimination will not be consid
ered here, although some conditioning paradigms are discussed in the sections dealing
with phobias and obsessive compulsive disorder.
In the following sections, each animal model will be discussed from several perspec
tives. First, the validity of the test will be considered from a behavioral standpoint, and
the important features of the test will be discussed from a psychological perspective. Sec
ond, the pharmacological characteristics of the test will be considered where data are
available. The test should be able to successfully discriminate drugs that are clinically
effective from those that are not, without making errors of omission or commission. The
task of discussing the animal tests from each of these perspectives is made more difficuh
by the fact that neither psychological nor pharmacological theories of anxiety in humans
are definitive. For example, the pharmacological validation of a test presupposes that we
have standard anxiolytic and anxiogenic drugs. As will become apparent below, there is
even controversy surrounding which drugs should be used as standards. This review will
focus on the effects of drugs whose effects have been studied in humans. The extensive
literature on compounds that have only been tested in animals will be mentioned only
briefly. Certainly many new and interesting compounds have tested positive in a number
of animal models of anxiety. Some of these drugs alter gamma-amino (GABA)ergic neu
rotransmission, and there has also been considerable recent interest in drugs that selec
tively interact with subpopulations of 5-hydroxytryptamine (5-HT) receptors. However,
only after the effects of these agents have been evaluated in humans will it be possible to
assess their impact on the validity of the various animal models.
The reader should note that pharmacological validity alone does not make a test a
model of anxiety. For example, many anxiolytics cause other behavioral effects such as
sedation, anterograde amnesia, ataxia, and myorelaxation and also induce hypothermia.
If an anxiolytic is active in one particular paradigm, this does not necessarily mean the
paradigm is assessing anxiolysis. It might be sensitive to another effect of the drug. This
reasoning is elementary, but is often not taken into consideration in the validation of
animal models. Sometimes it is difficult to dissociate two different behavioral effects. For
example, anxiolytic agents are often anticonvulsant (see Pellow, 1985). The ability of
drugs to antagonize the convulsant action of pentylenetetrazole has been extensively
used as a screen for potential anxiolytic drugs (Zbinden and Randall, 1967; Clody et al.,
1982). This procedure, however, must be classified as a correlational model (see Treit,
1985) and is no more a model of anxiety than are the various in vitro neuronal prepa
rations sensitive to drugs that enhance the effects of GABA (e.g., Pole and Haefely, 1976;
Haefely, 1980). No further reference will be made to correlational models in this paper.
In evaluating a particular behavioral measure as a potential index of anxiety, a ques
tion that the reader should bear in mind is, "If two groups of animals differ from one
another on the measure in question, does this necessarily reflect a group difference in
anxiety?" It will be seen that this can rarely be answered in the affirmative. A theme that
will run throughout this review is that, in order to be able to inteφret the results from
any given paradigm unambiguously, a multivariate approach is required so that the spec
ificity of the effects of any given manipulation can be adequately assessed.
The models of anxiety will be discussed in two sections. The first considers generalized
anxiety and panic disorder and the second discusses other anxiety disorders.
2. GENERALIZED ANXIETY AND PANIC DISORDERS

While GAD and panic disorder are considered separately in DSM-III-R, the evidence
in favor of their dissociation is controversial. Neither the genetic nor the pharmacological
evidence supporting the dissociation is compelling, and it is possible that panic may just
be a more severe form of GAD. It is no longer clear that drugs effective in treating panic
are ineffective in treating GAD, and the importance of genetic factors in panic disorder
and the apparent lack of such factors in GAD (Torgersen, 1988) might be explained by
a difference in severity of a single disorder. In view of the importance of genetic factors
in personality traits, including neuroticism (see Cattell, 1982), it would be somewhat
$υφπ$ίη§ if anxiety had no genetic component. Further, the selection of the Maudsley
reactive and nonreactive lines of rat considered below suggests a genetic involvement in
individual differences in anxiety.
At present, there are no animal models that can be said to relate specifically to panic
disorder and, therefore, this is not considered separately in this review (but see Johnston
and File, 1988a). It is, however, difficult to know what would characterize a distinct ani
mal model of panic disorder. A distinction based purely on pharmacological grounds
would currently be questionable in view of the present controversy as to whether panic
and GAD can really be distinguished by their different responses to drug treatment in
humans. Further, the development of an animal model of panic would seem difficult if
catastrophic inteφretation of bodily sensations is a crucial aspect of the disorder, as sug
gested by Clark (1986, 1988).
Before discussing the pharmacological validation of models of GAD, there is a need
for determining just which drugs should be considered standard anxiolytics and which
drugs, anxiogenic. It is important to appreciate that just because a drug exerts an anx
iolytic effect in one context, does not mean that it will exert a similar effect in other
contexts. For example, alcohol, which is anxiolytic in some situations, may increase the
anxiety of a subject performing a task requiring skill by virtue of its psychomotor impair
ing effect (Logue et al., 1978). The behavioral effects of a drug are thus not immutable
properties (Hughes et al., 1988).
Table 7.1 lists a number of drugs that might be considered the best candidates for
standard anxiolytic or anxiogenic agents, together with their putative mechanism of
action. The assessment of the effects of these agents is based primarily on their effects in
humans, so as to avoid circularity in the following discussion of animal models. How
ever, it must be noted that there are problems inherent in a number of the human stud
ies, particularly concerning expectancy (see Schacter and Singer, 1962), which make an
unequivocal inteφretation of some of the data impossible. For example, the anxiogenic
effects of drugs infused into patients with panic disorder may to some extent result from
an interaction of drug with expectancy, since the patients are told for ethical reasons that
anxiety and/or panic is a possible outcome of the treatment.
There is general agreement that benzodiazepines (BZs) and barbiturates reduce anxi
ety. Although ethanol is widely accepted as an anxiolytic, this view is not held by all (see
Wilson, 1988). As noted above, context probably plays an important role, and alcohol's
effects may differ in different subject populations. Cloninger (1987), for example, has
suggested that a subtype of (type I) alcoholics may drink alcohol for its anxiolytic effects.
Buspirone is a novel anxiolytic drug that interacts with 5-HT,Λ receptors (Dompert et
al., 1985; Traber and Glaser, 1987), but which also affects dopaminergic pathways (Stan
ton et al., 1981; Wood et al., 1983). Its onset of action, however, is much slower than
that of the BZs, and its anxiolytic action may not be apparent until several weeks into
treatment (e.g., Jacobson et al., 1985; Tyrer et al., 1985; Goa and Ward, 1986). The
TABLE 7.1. Drugs That Have Been Reported to Alter Anxiety in Humans
Drug Efficacy Site/mechanism of action
Anxiolytics
Benzodiazepines + + -f BZ receptors
Barbiturates + + Barbiturate site
Ethanol ??
Buspirone 5 - H T i A receptors
Clonidine +/- a2-adrenoceptors
Imipramine -f+• Monoamine reuptake inhibition
Nicotine -f Nicotinic receptors
Propranolol + -h iS-adrenoceptors
MAOIs + +* MAO inhibition
Anxiogenics
FG7142 BZ receptors
Pentylenetetrazole Picrotoxin site
Caffeine + + Adenosine receptors
Yohimbine -f a2-adrenoceptors?
Benzodiazepine withdrawal -}- + 77
Ethanol withdrawal -f-h 77
* = Only active following chronic treatment.
relative lack of success of various animal models in detecting an anxiolytic effect of this
drug may in part be due to the fact that most investigators have focused on the effects of
acute, rather than chronic, treatment. Tricyclic antidepressants (in particular, Imipra
mine) have proven successful in the management of panic disorder and may also be
efficacious in GAD (Kahn et al., 1986; Thase and Shipley, 1988). Monoamine oxidase
inhibitors (MAOIs) are effective in the treatment of panic disorder (e.g., Sheehan et al.,
1980), although their potential in the treatment of GAD is at present unclear. The effects
of the tricyclics and of MAOIs are only clearly observed in humans following chronic
treatment, and this should be noted in assessing their effects in animal models (see e.g.,
Bodnoffet al., 1989; Fontana et al., 1989). Clonidine (Hoehn-Saric et al., 1981) and ß-
blockers such as propranolol (see Noyes, 1988) are also effective in the management of
anxiety disorders, although it is still unclear where their greatest utility lies. While nico
tine is not used in the treatment of anxiety disorders in the clinic, there is some evidence
that it can act as an anxiolytic (Warburton et al., 1987), which may contribute to the
reasons why people smoke. The relevant animal literature has recently been reviewed by
Clarke (1987).
Just as there is general agreement about the anxiolytic effects of BZs, most would use
BZ-receptor inverse agonists such as the jS-carboline FG 7142 as examples of anxiogenic
agents. However, this assumption is not beyond question (e.g., see Thiebot et al., 1988).
Certainly, there are only limited data on the eflFects of these drugs in humans (Dorow et
al., 1983). Pentylenetetrazole is a drug that reduces the effects of GABA (like benzodi
azepine receptor inverse agonists), although via a different site on the BZ/GABA receptor
complex (Ticku and Maksay, 1983). It was used in a number of human studies earlier
this century and appeared to have an anxiogenic action (Rodin, 1958). The anxiogenic
effects of caffeine have been documented in both normal volunteers and patients with
anxiety disorders (Chamey et al., 1984; Veleber and Templer, 1984; Chamey et al.,
1985). Yohimbine is also widely considered to exert anxiogenic effects in humans,
although the evidence here is not convincing (see Wamboldt and Insel, 1988). Even if
yohimbine is anxiogenic it is not clear that this effect is mediated by its «z-adrenoceptor
antagonist properties, since preUminary studies suggest that other more specific «j-recep-
tor antagonists (e.g., idazoxan and atipamezole) do not produce marked anxiogenic
effects. Sodium lactate and carbon dioxide have also received much recent attention as
agents that increase anxiety (see Wamboldt and Insel, 1988), although the mechanisms
underlying these effects remain unclear. Finally, withdrawal from various dmgs such as
alcohol (Naranjo and Sellers, 1986; George et al., 1988) and BZs (Tyrer et al., 1983;
Rickels et al., 1988) can cause significant increases in anxiety in humans.
Having considered some of the agents that might serve as standards, it is now possible
to discuss the various behavioral paradigms. These paradigms can be loosely divided into
three categories. The first involves tests based on exploratory behavior, the second com
prises tests involving social behavior, and the third involves other paradigms.
2.1. TESTS OF EXPLORATORY BEHAVIOR
Since many of the currently used models of anxiety use animals' exploratory behavior,
either implicitly or explicitly, as an index of anxiety, it is important to consider some of
the factors that play a crucial role in exploration. In so doing, two points will emerge.
First, it is often difficult to assess an animal's exploratory behavior independently of
other behaviors such as locomotion. Second, factors that do not appear to be directly
related to anxiety are capable of producing marked changes in exploration.
During the 1950s and early 1960s, there was considerable interest in psychological
explanations of exploratory behavior (e.g., Berlyne, 1960; Fowler, 1965), because they
were closely linked to the contemporary theories of motivation. Since that time, how
ever, the psychology of exploration has received much less attention (but see Russell,
1983; Archer and Birke, 1983). Some workers (Montgomery, 1955) proposed that the
behavior of animals exposed to a novel situation results from a competition between an
exploratory tendency (motivated by curiosity or boredom) and a withdrawal tendency
(motivated by fear). At one level, this might be considered a form of approach-avoidance
conflict. Many factors can alter the relative strengths of these drives; for example, the
complexity of the situation, the degree of novelty, and the internal state of the animal
(Berlyne, 1955; File and Day, 1972; Russell, 1973; Taylor, 1974; Corey, 1978). In some
circumstances, for example, the initial reaction of an animal to a novel stimulus is pri
marily avoidance. As time passes, the animal will eventually approach the stimulus and
explore it. With further exposure, exploratory behavior decreases as the novelty disap
pears. In this case, the response to novelty is an inverted U function. Whenever a behav
ioral parameter varies in a nonmonotic manner, considerable caution must be exercised
when inteφreting the results of a single treatment (see, e.g., Weiskrantz, 1968). For
example, a treatment might reduce exploration via either an increase or a decrease in the
fear drive. It may be impossible to determine which (if either) of these two opposite
explanations applies without further experimental data.
2.1.1. The Open Field

This test has been extremely popular in behavioral research, since it requires minimal
apparatus and is quick to perform. The open field consists of a large, usually circular
arena under a high level of illumination. It must be emphasized that the term "open
field" should not be applied loosely to any test arena assessing an animal's activity under
an arbitrary level of illumination. The behavior of an animal may be quite different and
its response to a drug markedly altered by changes in the size of the arena or the ambient
lighting (Walsh and Cummins, 1976). The floor of the arena is marked to allow a quan
tification of an animal's activity. An animal is placed in the arena for a fixed period of
time, and its behavior is noted. Hall (1934) used the term "emotionality" to describe
what he believed to be assessed by the test. The argument is essentially that an animal
will typically freeze when exposed to a strange or noxious stimulus. An activation of the
autonomic nervous system will also occur, one consequence of which is that the animal
defecates. An "emotional" animal is thus taken as one that has little ambulatory activity
but defecates in the open field. This basic hypothesis has been supported by a number
of workers (e.g., Broadhurst, 1960a; Denenberg, 1969; Royce, 1977; Gray, 1979), but has
been questioned by others (e.g.. Archer, 1973). Just what is understood by the term emo-
tional, however, is not altogether clear, and the reader may note that Dews (1976) argued
that "emotion is a useless concept in current behavioral pharmacology!"
If the open field is to provide us with a useful model of anxiety, the first question we
must consider is what measures should be used as indices of anxiety. From the above
analysis, the suggestions that defecation and locomotion are useful indices will be
examined.
The use of locomotor activity as an index of emotionality or anxiety is highly ques
tionable. There appears to be a consistent but low negative correlation between loco
motion and defecation in undrugged animals, but in reviewing this hterature. Gray
(1979) notes that no one has suggested that ambulation in the open field is related only
to emotionality. Treatments can be expected to cause changes in locomotor activity by
mechanisms that do not involve changes in anxiety. A number of investigators have been
critical of locomotor activity measures in the open field for their inability to dissociate
locomotion from exploration (Sheldon, 1968; Kumar et al., 1970; Robbins, 1977; File,
1985a). If motor activity were to function as an index of anxiety, the argument would
presumably involve the suggestion that increased locomotion reflects decreased anxiety.
Certainly, BZs at low doses will increase the locomotion of animals unfamiliar with the
test arena (e.g., Christmas and Maxwell, 1970). However, motor stimulants also increase
activity in the open field, even though they are not anxiolytics (e.g., Cunha and Masur,
1978). It should also be noted that the effects of a drug can be qualitatively altered if
animals have previous experience in the apparatus (Christmas and Maxwell, 1970). In
short the contaminated nature of the locomotor activity measure and its alteration by
drugs that are not anxiolytics make it an unsuitable index of anxiety in psychopharma-
cological research.
On balance, the evidence from undrugged animals suggests that the incidence of def
ecation in the open field is related to an animal's emotionality. However, it can also be
altered by factors that may be unrelated to this construct (e.g., food intake). In particular,
many different drug treatments alter gut motility by a direct action on the gut rather than
via changes in emotionality. Drug-induced alterations in defecation in the open field,
therefore, must be inteφreted with great caution (e.g., Silva and Calil, 1975).
Hall (1934) also suggested that the approach to the consumption of food in an open
field might be sensitive to individual differences in emotionality. More recently, Britton
and Britton (1981) examined the effects of a number of anxiolytics in food-deprived rats
on the consumption of a food pellet placed in a pedestal in the center of an open field.
They found that diazepam, chlordiazepoxide, sodium pentobarbital, and ethanol all
increased the amount of food eaten each time the food pedestal was approached at doses
that failed to alter rearing, grooming, or the number of approaches to the pedestal. Mor
phine and haloperidol did not produce this pattem of results. It was argued that the
effects of the benzodiazepines was not due to a direct effect on appetite, since food con
sumption in the home cage was not altered by these dmgs. It should be noted, however,
that BZs do increase food consumption in a number of other testing situations (Cooper,
1980; Cooper and Estall, 1985). Increased food consumption in the open field has also
been reported in BZ-treated animals that have been habituated to the open field, sug
gesting that the dmg-induced increase in food intake may not be mediated via a reduc
tion in anxiety (Boissier et al., 1976). Anxiogenic effects of BZ antagonists and inverse
agonists have also been reported using this paradigm (Hoffman and Britton, 1983),
although this may reflect dmg-induced decreases in food consumption independent of
anxiety.
Bodnoff and co-workers (1988; 1989) have recently examined the effects of a number
of antidepressants in this paradigm. They found that, whereas acute treatment with diaz
epam decreased the latency to begin eating in a novel environment, rats treated acutely
with desipramine, amitriptyline, mianserin, fluoxetine, buspirone, or nomifensine failed
to show a similar decrease. In fact, desipramine, amitriptyline, fluoxetine, and nomifen
sine all increased the latency to eat. However, if rats were chronically treated with these
162 R . G . LISTER
drugs for 21 days, all agents except nomifensine reduced the latency to eat. This effect
did not appear to be related to drug effects on eating in a familiar environment (Bodnoff
et al., 1988) and, therefore, it was argued, reflects anxiolytic activity.
2.1.1.1. The Maudsley Reactive and Nonreactive Rat Strains. Hall (1938) performed
genetic selection studies based on open-field defecation. The Maudsley reactive and non
reactive rat strains provided essentially a replication of this early work and were selec
tively bred in London in the 1950s following a restandardization of the open-field test
(Broadhurst, 1960a, 1962, 1975). There have been problems with the British stocks of
these lines, and most of the recent research has been performed on colonies established
in the United States in the 1960s at NIH in Bethesda and at the University of Northern
Iowa. Both these stocks exhibit open-field behavior similar to that observed by the British
stocks early during the selection (Blizard, 1981). The results of a large number of studies
examining differences between the reactive and nonreactive lines are reviewed elsewhere
(Eysenck and Broadhurst, 1964; Broadhurst, 1975; Blizard, 1981). On balance, the evi
dence supports the suggestion that the strains differ in emotional reactivity. Unfortu
nately, only a limited number of studies have looked for differences between the strains
in animal models of anxiety. This is in part because many of the animal models were
only developed after problems had reduced the availability of the two lines. Recent
reports in the North American strains suggest that the Maudsley reactive rats differ from
the nonreactive ones in a way consistent with their being more "anxious." In a condi
tioned suppression of drinking paradigm, the Maudsley reactive rats accepted signifi
cantly fewer shocks than the nonreactive rats (Commissaris et al., 1986, 1990).
In the infant separation model discussed in more detail below, the reactive line emitted
more isolation calls (Insel and Hill, 1987). This latter finding is interesting in that it sug
gests that the strains differ from one another early in their development. While an
increase in BZ binding has been reported in the Maudsley nonreactive line (Robertson
et al., 1978), a more recent study failed to find differences in either peripheral or central
BZ receptors in the Maudsley rats (Tamborska et al., 1986).
Continued experimentation with the Maudsley strains is likely to add to our under
standing of the neurobiology of anxiety and will also be of value in characterizing behav
ioral tests used to model anxiety.
2.1.1.2. The Roman High- and Low-Avoidance Rat Strains. The behavioral differences
between two other lines of rats have also been discussed in terms of emotionality. These
lines, known as the Roman High Avoiders (RHA) and Roman Low Avoiders (RLA),
were selected on the basis of their avoidance conditioning in a shuttle box (Bignami,
1965). The suggestion is that the low avoiders are more emotional than the high avoiders
(DriscoU and Battig, 1982). The evidence to support this is somewhat sketchy, and these
lines have not been widely tested in animal models of anxiety. Their defecation in the
open field, which has been used as one of the arguments for that proposal, is rather incon
sistent. Initial studies failed to find differences in defecation between the lines (Broad
hurst and Bignami, 1965). A later series of studies found the RLA rats defecated more
than the RHA rats (Imada, 1972; Gentsch et al., 1982), but a more recent study found
an effect in the opposite direction (Durcan et al., 1984). The RLA, however, seem to
have consistently lower activities than the RHA (Broadhurst and Bignami, 1965;
Gentsch et al., 1982; Durcan et al., 1984). At present, it seems that further studies are
needed before a conclusion can be drawn that the low avoiders are, in fact, more anxious
than the high avoiders. It should also be noted that a significant amount of inbreeding
was present during the selection of these lines (and also of the Maudsley lines). Therefore,
differences in their behavior may not be due to the selection pressure, but rather to ran
dom gene fixation. More recent experiments partially overcome this problem of inter
pretation by using replicate Hues and control lines (see, e.g., Crabbe et al., 1990).
2.1.2. The Holeboard Test

The holeboard is an apparatus with a number of holes (frequently four) in the floor,
through which an animal can poke its head. The test assesses an animal's directed explo-
ration (head dipping) and its locomotor activity (Boissier and Simon, 1964; Boissier et
al., 1964; File and Wardill, 1975). Head dipping has been validated as a measure of
directed exploration (File and Wardill, 1975). A number of studies have shown that head
dipping and locomotion can vary independently (e.g., File, 1977; Lister 1987b; Durcan
and Lister, 1989). It is important to appreciate that the effects of a drug on the behavior
of an animal in the holeboard test (as in the open-ñeld test) can be critically dependent
on the familiarity of the animal with the test environment (File, 1973; Nolan and Parkes,
1973). For example, in mice naive to the testing apparatus, BZs exert a biphasic effect
on exploratory head dipping, low doses increasing and high doses decreasing this mea-
sure (Nolan and Parkes, 1973). If the mice have previously been familiarized with the
apparatus in an undrugged state, then doses of BZs that increase exploration in naive
mice fail to alter or decrease exploratory head dipping (Nolan and Parkes, 1973). A sim-
ilar pattem of results has been obtained with ethanol (Lister, 1987b). Note also that the
increase in exploration seen following benzodiazepine treatment is most easily observed
during the ñrst few minutes of exposure to the apparatus (Nolan and Parkes, 1973; File,
1976), and decreases in exploration may be found as the duration of the test increases.
2.1.2.1. Head Dipping and Anxiety. An important question in the context of this article
is ''Can a dmg-induced alteration in exploratory head dipping in the holeboard be inter-
preted in terms of a change in anxiety?" In considering this question, the ñrst issue that
must be addressed is the one of behavioral speciñcity. An increase (or decrease) in explo-
ration may reflect a general stimulant (or depressant) action. The concomitant assess-
ment of locomotor activity provides one method of examining the speciñcity of any
changes in directed exploration. In rats, decreases in both locomotion and exploration
are frequently observed following the administration of even low doses of BZs acutely
(File, 1981). This decrease in both measures has been interpreted in terms of the dmg's
sedative action (see File, 1985a for a discussion) to which tolerance rapidly develops
(File, 1981; Lister and File, 1983). Increased exploration in rats is more easily seen fol-
lowing chronic dmg administration, when tolerance to the sedative effects has occurred
(File, 1984a). As in the social interaction paradigm discussed below, therefore, the acute
sedative effect of BZs in rats makes an assessment of other effects difficult.
Mice appear less sensitive to the sedative action of low doses of BZs, and increases in
exploration are more easily observed in this species following acute treatment (Nolan
and Parkes, 1973; File and Pellow, 1985; File et al., 1985b). Mice, however, also show a
locomotor stimulant response to BZs (e.g., Marriott and Smith, 1972). Although signif-
icant increases in exploration have been observed at doses that fail to signiñcantly
increase locomotor activity, it is not clear whether this reflects a difference in the sensi-
tivity of the two measures (e.g.. File et al., 1985b). In mice, low doses of BZs, ethanol,
or barbiturates will frequently increase both measures (e.g.. Lister, 1987e). However,
studies with stimulants such as amphetamine and caffeine have found increases in loco-
motor activity with no increase, and often a decrease, in exploratory head dipping (e.g.,
Pellow et al., 1985; File et al., 1988).
While it can be argued that if a dmg alters locomotor activity and directed exploration
in the same direction the dmg effect is nonspeciñc, another argument is that locomotor
activity and directed exploration are competing behaviors, and therefore, an increase in
one might lead to a decrease in the other. The absence of holes in the floor of the hole-
board does indeed cause increased locomotor activity, although it appears not to produce
qualitative changes in the response to dmgs (Durcan and Lister, 1989).
A number of anxiogenic dmgs have been tested in the holeboard. Benzodiazepine-
receptor inverse agonists appear to reduce exploratory head dipping in both rats (File
164 R. G. LISTER
and Lister, 1982) and mice (Lister, 1987d) without exerting much influence on loco
motor activity. A dissociation between the effects of FG 7142 on exploration and loco
motion is more easily observed in mice than in rats (Lister, 1987d; File et al., 1985a).
Yohimbine reduced both exploration and locomotion in rats (Chopin et al., 1986). Caf
feine reduces exploration in rats more clearly than in mice (Pellow et al., 1985; File et
al., 1988; Durcan and Lister, 1989), but increases motor activity. Drugs acting at the
Picrotoxin site reduce exploration and locomotion in both rats and mice (File, 1984c;
Pellow etal., 1985; Lister, 1987a).
Despite a large number of experimental studies, it seems premature to accept the hole-
board test as a valid model of anxiety. An increase in exploratory head dipping is sug
gestive of an anxiolytic action in animals naive to the test apparatus. However, certain
drugs that appear to lack anxiolytic effects such as the BZ-receptor antagonists flumazenil
and ZK 93426 also increase directed exploration (File et al., 1982b; File and Pellow,
1986; Lister, 1988b). There seems to be a relatively consistent trend for anxiogenic drugs
to cause a specific decrease in exploratory head dipping. However, decreased exploration
can also be observed following treatment with sedatives, although this is accompanied
by decreases in motor activity. In mice, decreases in exploration can also be observed
with concomitant increases in locomotor activity following moderate doses of BZs or
ethanol (Lister, 1987b, 1987e). Whether this reflects a biphasic effect of anxiety on
directed exploration is at present unclear. In conclusion, drug-induced changes in explor
atory head dipping in the holeboard test may be caused by drug-induced changes in anx
iety. Whether alterations in anxiety do mediate such changes in exploration should be
further investigated using other, more selective tests.
2.1.3. The Plus-Maze Test

The plus-maze is a test that was developed from the work of Montgomery (1955).
Handley and Mithani (1984) used it to investigate the effects of a variety of α-adreno
ceptor agonists and antagonists. Pellow and co-workers (1985) performed an extensive
series of studies validating the procedure as an animal model of anxiety in rats, and more
recently, Lister (1987a) has validated the test in mice. This test has proved to be very
popular in the last few years, in part, because it is rapid and appears to be sensitive to
the effects of both anxiolytic and anxiogenic agents.
The apparatus consists of a plus-shaped maze with two open and two enclosed arms.
The test involves placing the rat (or mouse) in the center of the apparatus and allowing
it to explore for a short period (usually 5 minutes). Two indices of anxiety are used: the
proportion of entries that are made on to the open arms of the maze and the time spent
on the open arms of the maze expressed as a percentage of the total time spent on both
the open and closed arms. These two measures are highly correlated (Pellow et al., 1985;
Lister 1987a). The total number of arm entries is also recorded.
The test has some face validity, in that the reluctance of animals to explore the open
arms of the maze probably results from a combination of rodents' aversion to open
spaces and the elevation of the maze. At present, however, it is unclear whether it is the
open space or the elevation of the maze that provides the predominant anxiogenic stim
ulus. Plasma corticosterone concentrations are higher in animals confined to the open,
as opposed to the closed, arms of the maze (Pellow et al., 1985). While Copland and
Balfour (1987) failed to find similar results, they suggested that this may have been due
to a ceiling effect.
If a treatment increases an animal's preference for the open arms without altering the
total number of arm entries, this is taken to reflect an anxiolytic action. Similarly, if a
treatment decreases an animal's preference for the open arms without altering the total
number of arm entries, this is taken to reflect an anxiogenic action. A problem occurs if
a treatment alters both an animal's preference for the open arms and the total number
of arm entries in a similar way. Does a reduction in both measures reflect a sedative or
an anxiogenic action? Attempts have been made to answer this question using an anal
ysis of covariance.
2.1.3.1. The Use of Analysis of Covariance. Pellow and co-workers (1985) used the total
number of arm entries as a covariate and examined whether drugs still affected the
adjusted preference for the closed arms. This procedure can certainly be legitimately per
formed, however, caution must be exercised in inteφreting the results.
A change in the total number of arm entries may not merely reflect changes in activity
but is also probably sensitive to changes in anxiety. Therefore, if a treatment no longer
alters preference for the open arms following an analysis of covariance, this does not
necessarily imply that the treatment is not changing anxiety. A small, but signiñcant
correlation between the total number of arm entries and each index of anxiety was found
in a study examining the behavior of 91 undrugged mice (Lister, 1987a). It is not, per-
haps, suφrising that there is a relationship between preference for the closed arms and
the total number of arm entries. An animal that has a marked aversion to the open arms
is restricted to exploring only two (closed) arms, in contrast to the animal that does ven
ture onto the open arms. It may, therefore,.habituate more rapidly to the two closed
arms, thereby making fewer total entries than the animal that explores all four arms.
To make an independent assessment of activity, Pellow et al. (1985) and Lister (1987a)
test the animals in a holeboard apparatus immediately before the plus-maze test. Loco
motor activity in this test does not correlate with either index of anxiety (Lister, 1987a).
If a treatment increases an animal's preference for the open arms without altering loco
motor activity in the holeboard, this provides strong evidence for an anxiolytic action.
Similarly, if a treatment reduces an animal's preference for the open arms without reduc
ing activity in the holeboard, this provides strong evidence for an anxiogenic action. If
both measures are changed then an analysis of covariance using activity in the holeboard
as the covariate is a useful procedure. Again, it is only able to address the issue of whether
the change in preference for the closed arms is due to a stimulant (or sedative) effect,
insofar as activity in the holeboard is a reliable index of stimulation (or sedation). If
activity in the holeboard is altered as a result of a change in anxiety (and in some cir
cumstances this may happen), then a failure to find an effect on the adjusted preference
for the closed arms does not indicate that the treatment failed to alter anxiety. The cor
relational study of Lister (1987a) in undrugged animals suggests that using activity in the
holeboard as the covariate is probably preferable to using the total number of arm
entries.
2.1.3.2. Repeated Testing and Drug Effects. The behavior of undrugged animals on the
plus-maze does not appear to change with repeated testing (Pellow et al., 1985; Lister
1987a). Halliday (1967) repeatedly tested rats on a Y-maze with either three open arms,
or three enclosed arms and noted a day-to-day decline in the activity of rats tested on
the enclosed maze, but no such decline in rats tested on the open maze. He suggested
that the enclosed maze differed from the elevated maze in that it loses its capacity to
evoke fear as it becomes more familiar. Perhaps this reflects habituation to the anxi
ogenic effect of novelty, but a lack of habituation to the fear of an open space. The results
of Copland and Balfour (1987), however, do not support this hypothesis. They found
habituation of the corticosterone response in rats tested on an open elevated X-maze,
but no habituation if the rats were tested on an enclosed maze. They used a large number
(25) of relatively long (15-minute) exposures and, unlike Halliday (1967), found that the
activity of both groups habituated over this period.
Interestingly, the response of mice to an anxiolytic dose of a BZ was markedly reduced
if the animals had previously been tested on the plus-maze apparatus (Lister, 1987a).
This phenomenon is not easily explained in view of the lack of change in baseline behav
ior. However, the results suggest that great caution should be used in inteφreting data
obtained from animals that have previous experience of the test.
Anxiolytic profiles have been observed in both rats and mice treated with benzodiaz-
166 R . G . LISTER
epines, barbiturates, and ethanol (Pellow et al., 1985; Balfour et al., 1986; Lister, 1987a),
and anxiogenic profiles have been obtained with BZ receptor inverse agonists, caffeine,
and yohimbine (Handley and Mithani, 1984; Pellow et al., 1985; File and Johnston,
1987; Lister, 1987a; Lister, 1988a; Ferrari et al., 1989). The effect of yohimbine may not,
however, be mediated through aj-adrenoceptors since other more specific «z-^ceptor
antagonists do not produce such clear effects (File, 1987; Durcan et al., 1989). Cloni-
dine's effects vary with the dose (Handley and Mithani, 1984), and other aj-adrenoceptor
agonists fail to produce anxiolytic effects (Johnston et al., 1988). Neither acute nor
chronic treatment with nicotine altered the preference of rats for the closed arms, but the
7-day chronic treatment regimen increased the total number of arm entries (Balfour et
al., 1986). Both ethanol and BZ withdrawal produce anxiogenic effects (File et al., 1987;
Baldwin et al., 1989; File et al., 1990).
Chronic treatment with the tricyclic antidepressant imipramine failed to produce an
anxiolytic effect in rats and failed to reverse the effects of the anxiogenic drugs yohim
bine, FG 7142, or PTZ (File and Johnston, 1987).
The effects of chronic treatment with the MAOI phenelzine are still unclear. A 9 mg/kg
dose increased the preference of rats for the open arms of the maze, but this effect failed
to reach statistical significance (Johnston and File, 1988b). The size of phenelzine's effect,
however, appeared equivalent to that of the other anxiolytics tested, and the lack of sta
tistical significance was probably a result of the small number of animals in the phenel
zine test groups.
A number of investigators have examined the effects of manipulating 5-HT function.
The results have not all been consistent. For example, Critchley and Handley (1987)
found the 5-HT2 antagonist ritanserin to exert an anxiolytic effect, whereas Pellow et al.
(1987) reported it to be anxiogenic. Critchley and Handley (1987) also noted that the 5-
ΗΤ,Α agonist 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OHDPAT) was anxiogenic,
while Pellow et al. (1987) reported no significant effect. Acute treatment with buspirone
has failed to produce an anxiolytic effect (Pellow et al., 1987), although such an action
may have been obscured by the drug's sedative effect. As noted above, however, a failure
to find an acute anxiolytic effect of buspirone should not be considered a major problem,
since the clinical benefits of buspirone only manifest themselves after chronic treatment.
Moser (1989) recently performed an extensive series of studies examining the effects
of both acute and chronic treatment with buspirone in the plus-maze. An acute anxi
ogenic action of the drug did not disappear with 16 days of treatment.
The inability of the plus-maze to detect an anxiolytic action of chronic treatment with
buspirone and imipramine calls into question the general ability of the test to detect
novel anxiolytics.
2.1.4. Light-Dark Transitions

Crawley and co-workers have used the number of transitions made by mice between
two experimental chambers as an index of anxiety. One of the chambers is large and
brightly lit and the other is smaller and maintained under a low level of illumination.
The specificity of a drug effect is assessed by measuring the locomotor activity of inde
pendent groups of animals in an activity box. This test posseses a certain degree of face
validity in that light serves as an anxiogenic stimulus, and there is an apparent conflict
between the desire to explore and the desire to avoid the brightly lit part of the apparatus.
Drugs that increase the number of light-dark transitions at doses that do not increase
locomotor activity are said to be anxiolytic (Crawley and Goodwin, 1980; Crawley,
1981). A number of BZs and meprobamate produced anxiolytic profiles. Pentobarbitol,
however, failed to alter the number of transitions at doses that did not increase activity.
Acute administration of clorgyline (a MAOI) or butriptyline (a tricyclic antidepressant)
did not produce anxiolytic profiles (Crawley, 1981). Chronic treatment with these drugs
was not investigated. The neuroleptic chloφromazine also did not produce an anxiolytic
profile. Initial studies with BZ-receptor inverse agonists (e.g., FG 7142) in this test failed
to demonstrate intrinsic anxiogenic actions of these drugs, although they were capable
of reducing the anxiolytic effect of BZs (Crawley et al., 1984). Although methyl-/J-car-
boline-3-carboxylate (jS-CCM), another BZ-receptor inverse agonist, reduced the number
of light-dark transitions, it also reduced locomotor activity precluding an anxiogenic
interpretation of the data. Ro 5-4864, which binds to peripheral rather than central BZ
receptors, but is believed to exert an anxiogenic action via the Picrotoxin site (see Pellow
and File, 1984), caused marked reductions in both the number of light-dark transitions
and locomotor activity (Crawley, 1981).
More recently, Belzung et al. (1987a) modified the test, by making the size of the light
and dark compartments equal, and reported that ß-CCM did possess anxiogenic prop
erties, that is, it reduced the number of light-dark transitions at a dose that failed to alter
motor activity. They have also reported anxiogenic actions of other BZ-receptor inverse
agonists (Misslin et al., 1988) and anxiolytic effects of ethanol (Belzung et al., 1987b,
1988).
In the version of the test used by Belzung et al. (1987a), the time spent in the light part
of the apparatus was significantly altered by anxiolytic (chlorazepate) and anxiogenic (jS-
CCM) treatments. In contrast, Crawley and Goodwin (1980) found that BZs did not alter
the preference for the dark compartment.
Costall and co-workers (1987) have also modified the original test and measured motor
activity and rearing separately, in both the brightly lit and dark portions of their appa
ratus. An anxiolytic profile was defined (based upon the effects of the BZs diazepam and
triazolam) as an increase in rearing and motor activity in the light compartment with a
concomitant decrease in these measures in the dark compartment. Amphetamine and
haloperidol failed to produce anxiolytic profiles. Ethanol appears anxiolytic, and ethanol
withdrawal produces an anxiogenic profile (Costall et al., 1988). Currently, it is difficult
to appropriately evaluate the claims of Costall et al. (1987) as to what constitutes an
anxiolytic profile in view of the limited data available. When a test is modified, caution
should always be exercised in interpreting data. This is particularly true when the mod
ification includes the use of completely different behavioral measures to define an anx
iolytic profile. Further work is clearly needed before the validity of this variation can be
assessed.
2.1.4.1. Repeated Testing, Blumstein and Crawley (1983) reported that a diazepam-
induced increase in the number of light-dark transitions was significant over the first
three uses of their mice and concluded that animals could, therefore, be tested repeatedly
without invalidating the test. However, their experimental design was incomplete, and
their conclusion premature. Only two groups of animals were used, one that received
vehicle on all 3 test days, and the other that received diazepam on all 3 days. It was
impossible, therefore, to assess whether the response to diazepam is affected by previous
exposure to the test in an undrugged state. Studies with various pieces of apparatus,
including the plus-maze (Lister, 1987a), have shown that previous test experience in an
undrugged state can markedly alter an animal's subsequent response to a drug (Rushton
et al., 1968; Nolan and Parkes, 1973; Dantzer, 1977; Lister, 1987c). The reuse of animals
should, therefore, be avoided until a more comprehensive investigation is undertaken.
2A.5.TheStaircase Test
The staircase test was adapted by Simiand et al. (1984) from the work of Thiebot et
al. (1973) as a screen for anxiolytic drugs. A naive mouse is placed in a chamber con
taining a five-step staircase and the number of rearings and steps climbed are recorded
for 3 minutes. It was suggested that anxiolytics reduce rearings at doses that do not
reduce the number of steps climbed. It is difiicult to evaluate this procedure from a
behavioral perspective. Rearing is, to some extent, an exploratory behavior, although it
does not correlate with exploratory head dipping in the holeboard test (File, 1985a), but
168 R . G . LISTER
does correlate with locomotor activity (Soubrie, 1971; File 1985a). It is likely that rearing
would be reduced by agents that induce ataxia independently of any drug-induced
change in anxiety. Of course a drug-induced ataxia may also reduce the number of steps
climbed. In short, the validity of the test at present must rely primarily on the pharma
cological data. Whether its value is greater than that of correlational models mentioned
in the introduction remains to be determined.
The effects of a number of different classes of drug have been examined, and BZs,
meprobamate, barbiturates, and sodium valproate all tested positive. Chloφromazine,
haloperidol, amphetamine, imipramine, and amitriptyline all tested negative (Simiand
et al., 1984). More recently. Pollard and Howard (1986) found that ethanol, nicotine,
and moφhine also tested positive, but failed to find an anxiolytic effect of buspirone,
ketanserin, CGS 9896, and tracazolate. Interestingly, pentylenetetrazole and FG 7142
failed to produce effects opposite to those of BZs, so the test does not appear suitable for
screening potentially anxiogenic drugs.
2.2. TESTS OF SOCIAL BEHAVIOR
This section focuses on tests of social behavior in which aggression is infrequently

observed. The effects of drugs on aggressive behavior have been recently reviewed else
where (Miczek, 1987), and here it will only be noted that the relationship between anx
iety and aggression is not a simple one. While drug-induced alterations in anxiety may
modulate aggressive behavior, great caution must be exercised in attributing changes in
aggression to changes in anxiety.
2.2.1. The Social Interaction Test

The social interaction test developed by File and co-workers uses novelty and high
lighting conditions as anxiogenic stimuli. It has been the subject of a number of reviews
(File, 1980, 1985b, 1988). The time spent by pairs of male rats in active social interaction
is measured in a test arena under high or low lighting conditions and when either familiar
or unfamiliar with the testing apparatus. Four testing conditions are used corresponding
to high-light unfamiliar, low-light unfamiliar, high-light familiar, and low-light familiar
conditions. Undrugged rats spend a greater time interacting in the low-light familiar con
dition than in the high-light unfamiliar condition, and exhibit intermediate periods of
interaction in the other two conditions (see File, 1980). Locomotor activity is also mea
sured, and this assists in determining whether any change in social interaction is due to
a general stimulant or sedative effect. As in the plus-maze test, when both the time spent
in social interaction and locomotor activity are affected by a treatment, analyses of
covariance have been performed with locomotor activity as the covariate. As was noted
in discussing the use of the procedure to analyze data from the plus-maze test, the anal
ysis is only valid insofar as locomotor activity is sensitive to changes along the stimulant/
sedative dimension and is unaffected by changes in anxiety. For example, a treatment
might reduce activity by increasing freezing (perhaps as a result of a large increase in
activity) and also decrease social interaction. An analysis of covariance might find no
treatment effect on the adjusted social interaction, even though the treatment was anxi
ogenic. Providing the appropriate caution is used in inteφreting negative results, an anal
ysis of covariance can be useful in discussing data from the test.
An anxiolytic profile of a BZ is observed following chronic treatment after tolerance
has developed to the drug's sedative effects. It is characterized by a drug χ test condition
interaction. The BZ increases the amount of time of interaction under conditions of high
lighting or unfamiliarity with the test arena, but exerts a minimal effect in the low-light
familar condition, which is the least anxiety-provoking test condition. It might be argued
that the effect of BZs can be attributed to a rate-dependency phenomenon, with BZs
selectively increasing low rates of responding. However, reducing the baseline scores by
varying the weight of the animals, or the time of day, failed to produce similar changes
in sensitivity to BZs (File and Hyde, 1978).
Attributing the reductions in social interaction caused by increasing the ambient light
level and by decreasing familiarity with the test arena to increased "anxiety" appears to
have some face validity. Ethological studies suggest that rats find high light aversive, and
the anxiogenic effects of novelty were discussed in the section on exploration. File and
Peet (1980) have shown that plasma corticosterone levels are higher under the high light
ing conditions that under the low lighting conditions, and are higher in unfamiliar than
in familiar testing conditions.
Other anxiolytics such as phenobarbitone have been found to increase social interac
tion across all test conditions (File, 1980). The significance of the difference between the
profiles of BZs and barbiturates is not clear. Propranolol did not exhibit anxiolytic effects
(File, 1980) and Clonidine was anxiogenic (Pellow and File, 1987). Neither chronic treat
ment with imipramine nor with the MAOI phenelzine produced anxiolytic profiles in
the test (Pellow and File, 1987; Johnston and File, 1988b). In fact, chronic phenelzine
appeared to be anxiogenic (Johnston and File, 1988b). Chronic (5-day) treatment with
buspirone also failed to produce a clear anxiolytic profile (File, 1984b).
Anxiogenic agents reduce the time spent in social interaction without altering loco
motor activity. Interestingly, the effects of some anxiogenic agents do not appear to vary
with the testing condition (e.g.. File and Vellucci, 1978; File et al., 1984), that is, there
does not appear to be a drug χ test condition interaction. The effect of caffeine, however,
did appear greater in the low light familiar test condition than when the environment
was familiar or brightly illuminated (File and Hyde, 1979). The finding that some anxi
ogenic agents decrease social interaction across all test conditions, and some anxiolytics
increase social interaction independently of the test conditions makes it difficult to deter
mine the importance of a drug χ test condition interaction.
There have been a number of attempts to establish the social interaction test in mice
(De Angelis and File, 1979; Lister and Hilakivi, 1988). Pairs of male mice have a ten
dency to be much more aggressive in the test than pairs of male rats, and attacks with
bites, which are not seen in rats, are commonly observed in mice. The effect of manip
ulating familiarity with the test arena also does not appear to be have as marked an effect
on the social behavior of mice as it does rats (De Angelis and File, 1979; Lister and
Hilakivi, 1988). Ethanol, which acts as an anxiolytic in the rat test (File, 1980), reduced
rather than increased social interaction between mice (Lister and Hilakivi, 1988). The
anxiogenic drugs caffeine and BZ-receptor partial inverse agonists failed to produce a
specific reduction in social interaction in mice (Hilakivi and Lister, 1988; Hilakivi et al.,
1989), although such drugs are anxiogenic in the rat test (File and Hyde, 1979; File et
al., 1982a). In rats, both BZ withdrawal (Baldwin and File, 1989) and ethanol withdrawal
(File et al., 1989) are anxiogenic.
Krsiak and co-workers (1984) performed an extensive series of studies examining the
effects of a variety of drugs on the social interaction between pairs of male mice, one of
which had been isolated for 3 to 6 weeks, and the other of which was a group housed
animal. Drug treatment was only given to the isolated mice. They found that, in general,
most of the drugs tested that possessed clinical anxiolytic activity increased social inves
tigation and reduced the incidence of active escape and defensive behavior and that most
of the drugs that lacked anxiolytic activity did not. However, in a number of cases, the
effect was only observed in subpopulations of mice previously characterized as being
"timid" or "aggressive." Antichohnergics such as scopolamine, whose clinical anxiolytic
effects are unclear, also increased social behavior. More recently, Sulcova and Krsiak
(1989) compared the effects of nine BZs in this paradigm. They reported that alprazolam,
oxazepam, and diazepam reduced defensive behavior at doses that failed to reduce social
sniffing or ambulation, while triazolam and lorazepam reduced defensive behavior only
at doses that suppressed other activities as well. The data were inteφreted in terms of
differences between the various BZs' sedative effects.
170 R . G . LISTER
A disadvantage of the social interaction test is that the test involves the use of pairs of
animals. It is, therefore, difficuh to examine individual differences in behavior with this
test, since the behavior of any one animal is critically dependent on the behavior of its
test partner.
2.2.2. Separation Vocalization

Several groups have used the ultrasonic vocalization emitted by rat pups following
separation from their mother as an animal model of anxiety (Gardner, 1985a, 1985b;
Insel et al., 1986). If a drug reduces vocalization without causing sedation, then this is
taken to reflect an anxiolytic action. To determine whether a drug is exerting a sedative
effect, Insel et al. (1986) made an independent assessment of locomotor activity. The
method has some face validity since the ultrasonic cries are considered to be "distress"
calls (Noirot, 1972). Ultrasonic calls are emitted in response to cold and to tactile stim
ulation resulting, for example, from retrieval by the mother or contact with unfamiliar
surfaces (Okon, 1970, 1972; Noirot, 1972; Hofer and Shair, 1987). The nature of the call
appears to vary with the motivating condition (Smith, 1972).
In view of the effect of changes in temperature on vocalizations, it is important to
examine whether a drug-induced change in ultrasonic calls is secondary to some altera
tion in body temperature. Insel et al. (1986) found that diazepam decreased the number
of calls without altering activity or body temperature, and pentylenetetrazole specifically
increased the number of calls. The effects of BZ-receptor inverse agonists were a little
less clear, but a tendency toward increased vocalization was noted (Insel et al., 1986;
Gardner and Budhram, 1987). Gardner (1985a) found decreases in vocalization follow
ing chlordiazepoxide and diazepam at doses that did not appear sedative. He also argued
that a myorelaxant action did not underly these effects, since mephensin was relatively
inactive. Clonidine increased vocalization, though this was probably due to its hypo
thermic action. Yohimbine decreased vocalization, but also induced marked tremors
(Gardner, 1985b). Herman and Panksepp (1978) reported that moφhine reduced the
vocalization of infant guinea pigs at doses that did not appear sedative and, conversely,
naloxone increased separation distress vocalizations. More recently, Kehoe and Blass
(1986) obtained similar results in rat pups.
While the pharmacological data are at present limited, the results so far suggest that
this test may prove useful in assessing potential anxiolytic or anxiogenic drugs. Clearly,
in this paradigm attention needs to be paid to the ontogeny of the receptors where the
drugs act (e.g., Insel and Harbaugh, 1989; Insel et al., 1988). The paradigm is probably
limited in its ability to predict the anxiolytic or anxiogenic effects of chronic drug treat
ments in view of likely drug interactions with development.
It is at present difficult to determine the relationship between the separation-induced
vocalizations observed in rodent pups and those observed in other species (e.g., Panksepp
et al., 1980). A recent prehminary report by Lehr (1989) found that several BZs, but also
caffeine and yohimbine, inhibited distress call activity in isolated chicks. No data were
presented to assess whether sedation or changes in body temperature might mediate these
effects.
2.3. OTHER TESTS
2.3.1. Observer-Rated Anxiety in Primates

A number of investigators have used observer rating scales to assess drug effects on
anxiety in primates. Examples include studies on the anxiogenic actions of the BZ-recep
tor partial inverse agonist ¡8-CCE in rhesus monkeys (Ninan et al., 1982; Skolnick et al.,
1984) and the anxiolytic effect of the novel 5-HT3 receptor antagonist GR38032F in
marmosets and cynomolgus monkeys (Jones et al., 1988). While the physiological effects
of ß'CCE in the former studies were certainly consistent with an anxiogenic drug effect.
the behavioral validation of these procedures is not entirely satisfactory. The observation
of struggling in a restraining chair, head and body turning, periods of immobility,
scratching, vocalization, urination, and penile erection may indeed be behavioral man
ifestations of anxiety. However, in order to assert a direct relationship, these behaviors
should be selectively eUcited by other anxiety-provoking situations and not be observed
in nonanxious animals. Squirrel monkeys will react to a mirror or a conspecific with a
display that includes penile erection, vocalization, urination, and scratching (MacLean,
1964). This pattern of behavior, which shares a number of features with the pattern
observed in the /8-CCE-treated rhesus monkeys of Ninan et al. (1982), does not appear
to be so clearly associated with a lai^ge increase in anxiety. That the same monkeys will
work in order to gain access to the mirror also suggests that the mirror is not a very
anxiogenic stimulus. In short, great care must be taken in inteφreting behavioral changes
produced by a given treatment, and measures must be validated whenever possible in
any species without recourse to anthropomoφhism. There is often an unwritten assump
tion that behavioral studies in nonhuman primates do not require the same degree of
validation as those using other species. While the phylogenetic proximity of humans to
nonhuman primates certainly makes studies in the latter extremely valuable, it in no way
implies that such studies should be less rigorously controlled than those using other
species.
A number of studies have examined the response of nonhuman primates to anxiogenic
stimuli in settings in which they were not restrained. The pattern of behavioral responses
appeared to vary considerably with the age of the subjects (e.g., Suomi et al., 1981). A
problem with a number of the early studies was that behavioral changes were the only
indices of anxiety, and physiological validation was not undertaken. More recent studies
have addressed tiiis issue, and there are reports of activation of the hypothalamic-pitu
itary-adrenal (HPA) axis following the separation of monkeys from their social groups
(e.g., Mendoza et al., 1978; Coe and Levine, 1981). It is interesting that the effects of
separation on the HPA do not seem to vary with age (Scanlan, 1984 cited in Suomi,
1987), despite changes in the behavioral reaction to separation. There is still a clear need
for further studies in primates examining both the behavioral and physiological changes
accompanying exposure to anxiety-provoking situations.
2.3.2. The Use of Defensive Behaviors to Study Anxiety in Rats

Blanchard and co-workers (Blanchard and Blanchard, 1989; Blanchard et al., 1990a,
b, c) have recently examined the defensive behaviors of rats in a visible burrow system
following the presentation of a cat. The defensive behaviors were examined in great detail
as a function of time after the removal of the cat. Withdrawal, immobility, increased risk
assessment, and a suppression of nondefensive behaviors such as eating, drinking,
grooming, and mounting were observed. Blanchard and Blanchard (1989) contrast the
defensive behaviors observed in the absence of the cat from the defensive behaviors
exhibited by rats in the presence of a threat stimulus (Blanchard and Blanchard, 1987).
They suggest that the former behaviors may reflect anxiety and the latter, fear (see also
Blanchard and Blanchard, 1990). Two test batteries have been developed based on this
analysis: a fear/defense test battery (Blanchard et al., 1986) and an anxiety/defense test
battery (Blanchard and Blanchard, 1990). In the former, defensive reactions such as
freezing, vocalizations and defensive jump attacks to a human experimenter, brush stim
ulation of the mustacial vibrissae, and an anesthetized conspecific are assessed. In the
latter, defensive behaviors are assessed in situations where threat has previously been
encountered, but is no longer present. Preliminary pharmacological analyses of these
paradigms have been performed (Blanchard et al., 1990c). In the fear/defense test bat
tery, BZs reduced vocalizations, but failed to alter freezing or flight. In the anxiety/
defense test battery, the effect of diazepam on risk assessment behaviors varied with the
paradigm. In situations of high-level threat, where control animals tended to show avoid-
172 R . G . LISTER
anee and freezing behaviors, diazepam increased risk assessment. However, in other sit
uations in which controls showed higher levels of risk assessment, diazepam decreased
these behaviors (Blanchard et al., 1990a).
These paradigms certainly have a high degree of face validity as methods of assessing
fear and anxiety. Their further characterization, followed by a comparison with the other
paradigms discussed in this paper, should considerably enhance our understanding of
the neurobiology of fear and anxiety.
2.4. SUMMARY
The effects of drugs in five of the models discussed above are summarized in Tables
7.2 through 7.6. In each case, the criteria used to determine whether a drug is anxiolytic
or anxiogenic are stated at the top. Only the effects of chronic treatment with buspirone,
tricyclics or MAOIs are reported (when known). Although the effects of acute treatment
with these drugs have been examined in a number of the paradigms (in each case, with
out finding an anxiolytic effect), these studies are not considered directly relevant since
the clinical efficacy of these drugs is only seen after chronic treatment. Not all studies
have produced the same results, and so in some cases, the author has taken the liberty
of making a personal overall assessment of the available data.
2.5. FUTURE DIRECTIONS
None of the "models" so far discussed, with the possible exception of the Maudsley
rat strains, has focused on etiology. Unfortunately, very little is known of the etiology of
GAD in humans. A number of theories have been proposed based upon either classical
conditioning (Wölpe, 1958), instrumental conditioning (Kimmel, 1975), or unpredicta
bility and lack of control (see Seligman, 1975, Chapter 6). Many of the best examples of
symptoms resembling generalized anxiety states come from the animal laboratories of
Pavlov, Gantt, Liddell, Masserman, and Wölpe (see Beach, 1953; Broadhurst, 1960b).
Unfortunately, many of their experiments lacked appropriate controls and statistical
analysis. Little is known of the response of these experimentally induced neuroses, which
TABLE 7.2 Plus-Maze

Anxiolytic profile: Increase in preference for the open
arms independent of change in activity.
Anxiogenic profile: Decrease in preference for the open
arms independent of change in activity.
Drug Profile
Anxiolytics
Benzodiazepines -h
Barbiturates -f-
Ethanol +
Buspirone (c) -
Clonidine +/-
Imipramine (c) 0
Nicotine 0
Propranolol 0
MAOIs (c) -f ?
Anxiogenics
BZ inverse agonists —
Pentylenetetrazole —
Caffeine —
Yohimbine -
Benzodiazepine withdrawal
Ethanol withdrawal
c = c h r o n i c treatment; -h = a n x i o l y t i c ; — = anxiogenic; 0
no effect.
TABLE 7.3. Light-Dark Transitions

Anxiolytic profile: Increase in number of transitions with
no change in locomotor activity.
Anxiogenic profile: I>ecrease in number of transitions
with no change in locomotor activity.
Drug Profile
Anxiolytics
Benzodiazepines +
Barbiturates 0?
Ethanol +
Buspirone (c) ?
Clonidine ?
Imipramine (c) ?
Nicotine ?
Propranolol ?
MAOIs (c) 7
Anxiogenics
BZ inverse agonists 0/-
Pentylenetetrazole 7
Caffeine 7
Yohimbine 7
Benzodiazepine withdrawal ?
Ethanol withdrawal —
c - chronic treatment; + - anxiolytic; - = anxiogenic; 0 =

no effect.
were often long lasting, to different pharmacological agents. Some of Pavlov's neurotic
dogs responded favorably to bromides (Pavlov, 1941). Masserman and colleagues (Wik
ler and Masserman, 1943; Masserman and Yum, 1946) reported that alcohol and mor
phine were effective in ameliorating the reactions to conflict.
Mineka and Kihlstrom (1978) have discussed the work of these investigators at some
length and emphasized the importance of exposure to controllable, as opposed to uncon
trollable, aversive events. Mineka (1985) presents an overview of recent work in this area.
TABLE 7.4. The Staircase Test

Anxiolytic profile: Decrease in the number of rears with
no change in the number of steps climbed.
Anxiogenic profile: Increase in the number of rears with
no change in locomotor activity.
Drug Profile
Anxiolytics
Benzodiazepines
Barbiturates -1-
Ethanol +
Buspirone (c) ?
Clonidine ?
Imipramine (c) 7
Nicotine +
Propranolol 7
MAOIS 7
Anxiogenics
BZ inverse agonists 0
Pentylenetetrazole 0
Caffeine 7
Yohimbine 7
Benzodiazepine withdrawal 7
Ethanol withdrawal ?
c = chronic treatment; + = anxiolytic; - = anxiogenic; 0 =

no effect.
174 R . G . LISTER
TABLE 7.5. Social Interaction

Anxiolytic profile: Increase in social interaction
independent of change in locomotor activity.
Anxiogenic profile: Decrease in social interaction
independent of change in locomotor activity.
Drug Profile
Anxiolytics
Benzodiazepines (c) -I-
Barbiturates +
Ethanol -h
Buspirone (c) 0
Clonidine -
Imipramine (c) 0
Nicotine ?
Propranolol 0
MAOIs (c)
Anxiogenics
BZ inverse agonists —
Caffeine -
Yohimbine —
Benzodiazepine withdrawal —
Ethanol withdrawal —
c = chronic treatment; -I- = anxiolytic; - ~ anxiogenic; 0

no effect.
While much of the research has been conducted in nonhuman primates, there have also
been studies in rodents. Joffe et al. (1973), for example, found that rats who controlled
the lighting conditions and the delivery of food and water were less "emotional" in the
open field test than yoked controls. Unfortunately, no standard rearing group was
included and the open field was the only behavioral test reported. More recent work in
primates (with appropriate controls) has considerably extended these findings (Mineka
et al., 1986). Animals who had access to manipulanda that allowed them to control the
delivery of food, water, and treats during their development were more exploratory in a
playroom, and displayed less fear to a mechanical toy in front of their cage than yoked
or standard-reared controls.
It is likely that pharmacological research would greatly benefit from animal models
TABLE 7.6. Rat Pup Vocalization

Anxiolytic profile: Decrease in vocalization with no
change in activity or temperature.
Anxiogenic profile: Increase in vocalization with no
change in activity or temperature.
Drug Profile
Anxiolytics
Benzodiazepines +
Barbiturates ?
Ethanol ?
Clonidine -?
Nicotine ?
Propranolol ?
Anxiogenics
BZ inverse agonists -
Caffeine ?
Yohimbine
-I- = anxiolytic; - « anxiogenic; 0 « no effect.

derived from an etiological perspective of anxiety disorders. In particular, experiments

in which manipulations of environmental control and predictability are used should pro
vide a fruitful area for future study. Qearly, such research requires a greater investment
of time and resources than is needed for most of the procedures described in the preced
ing sections. However, what is lost in time may be gained in validity.
3. OTHER ANXIETY DISORDERS

3.1. PHOBIC DISORDERS
Although there has been considerable interest in phobias from a psychological per
spective, there is little pharmacological data from animal studies that can be considered
directly pertinent to the treatment of phobic disorders. CUnically, BZs alone do not
appear helpful in alleviating phobias other than agoraphobia. However, benzodiazepines
can be beneficial when used in conjunction with behavioral therapies such as flooding
(Marks et al., 1972; Johnston and Gath, 1973; Hafner and Marks, 1976). That diazepam
should enhance rather than impair the effects of the behavioral therapies is of interest in
view of the well-documented amnestic properties of BZs (see Lister, 1985; Lister et al.,
1988).
Despite the paucity of pharmacological data, there are many behavioral studies inves
tigating phobias in animals. Such studies should provide a firm basis for future phar
macological research. It is not the intention to review these studies in detail here and the
interested reader is referred to a number of excellent reviews elsewhere on the etiology
of phobias in both animals and humans (e.g., Mineka, 1985; Marks, 1987). However, a
few selective points will be made.
First, it appears that phobias develop more readily to some stimuli (such as spiders and
snakes) than to other salient and dangerous objects (e.g., guns and electric outlets) that
today account for many more deaths. Whether this selectivity reflects a tendency for
some phobias to be prepared via some evolutionary mechanism (Seligman, 1971;
Ohman et al., 1985; Ohman, 1986; McNally 1987) or is a consequence of other (e.g.,
ontogenetic) factors (LoLordo, 1979; Mackintosh, 1974; Delprato, 1980) is still subject
to debate.
Second, phobias can be transmitted by observation. For example, rhesus monkeys
raised in captivity do not have a fear of snakes (unlike those reared in the wild), but
rapidly acquire such a fear by watching an adult monkey behaving fearfully in the pres
ence of a snake (Cook et al., 1985). Interestingly, the fear of a flower cannot be trans
mitted in a similar manner (Cook et al., 1985).
Finally, some phobias in animals appear to be innate. For example, specific fears of
poisonous coral snakes are found in some snake-eating birds who have never seen a
snake before (Smith, 1975, 1977). Whether these reflect true phobias is perhaps ques
tionable, since a phobia is "a fear of a situation that is out of proportion to its danger,
can neither be explained or reasoned away, is largely beyond voluntary control, and leads
to avoidance of the feared situation" (Marks, 1987, p. 5). Many of the fears seen in the
animal kingdom are not clearly out of proportion to their danger. In this context, a
human fear of snakes in Ireland constitutes a phobia, but in the Australian outback is,
perhaps, realistic.
To conclude this section, there has been much animal research into phobias from both
ethological and classic experimental psychological perspectives. Such research provides
a firm basis from which to develop models for assessing the effects of pharmacological
treatments. In humans, all the most efficacious treatment programs involve exposing
patients to their phobic stimuli (see Marks, 1987; Marks and O'Sullivan, 1988). It seems
likely that the greatest contribution that can be made from animal pharmacological
research may be the suggestion of treatments that benefit the patient when used in con
junction with the behavior therapies.
176 R . G . LISTER
3.2. OBSESSIVE COMPULSIVE DISORDER
As with the phobic disorders, there is hmited pharmacological data on the successful
treatment of OCD. More data are available on the effects of clomipramine than on the
effects of any other antidepressant, although it is not yet clear whether this reflects clom
ipramine's superiority over other tricyclics (see Marks, 1987; Marks and O'SuUivan,
1988; Leonard etal., 1990).
At present, there is no adequate animal model of OCD. However, a number of behav
ioral phenomena observed in animals that might be of some relevance to OCD have been
discussed by several authors (Mineka, 1985; Insel, 1988). From the ethology literature,
displacement behaviors and stereotypes share some features with OCD. Displacement
activities occur in a variety of situations in which there is a conflict between different
drives (e.g., fight and flight). It has been suggested that repetitive grooming behavior has
some parallels with compulsive hand washing. However, a number of displacement
activities are observed in humans such as adjusting nonexistent disorders of the coiffure
or scratching behind one's ear (Tinbergen, 1951) that have no clear obsessional dimen
sion. In humans, these displacement activities often lack a clear, conscious cognitive
component and may be executed with little apparent awareness.
Some of the stereotyped behavior exhibited by zoo animals can give the impression
that the animals are engaging in compulsive acts (e.g., Meyer-Holzapfel, 1968). Interest
ingly, bears seem to be particularly susceptible to such behavior. Similar stereotypes can
be observed in nonhuman primates, particularly those that have been reared in isolation.
Solomon et al. (1953) noted that, as an avoidance response became well leamed, it
became more stereotyped and persisted over hundreds of trials even without further
exposures to the unconditioned stimulus. The role of avoidance in OCD has been dis
cussed by Teasdale (1974) and by Rachman and Hodgson (1980), and the interested
reader is referred to an excellent recent review of the relevant animal literature (Mineka,
1985). The reader should also appreciate that avoidance also plays an important role in
the maintenance of phobias discussed in the previous section.
Interestingly, following local media coverage of the effects of clomipramine in tricho
tillomania (human hair pulling) (Swedo et al., 1989) and OCD, owners of two pets with
acral lick dermatitis suggested that clomipramine might be useful in treating this disor
der. Acral lick dermatitis is a common self-inflicted skin disorder in dogs caused by con
tinued licking, biting, or scratching (Veith, 1986). Its etiology is unknown, although it is
commonly considered to be of psychogenic origin (Muller et al., 1983). Following the
suggestion of the pet owners, Goldberger and Rapoport (1990) recently reported that
clomipramine, but not desipramine, significantly improved the dogs' licking behavior.
The possibility that there is a link between the abnormal grooming behavior of the dogs
and OCD is intriguing and certainly seems worthy of further study.
It will probably be some time before there is an adequate animal model of OCD.
Although a number of phenomena in animals bear some similarity to certain aspects of
the human disorder, it is premature to suggest that any of the above paradigms provide
a firm basis for screening potential pharmacological treatments.
3.3. POST-TRAUMATIC STRESS DISORDER
Most of the pharmacological studies in humans on post-traumatic stress disorder

(PTSD) have been open and have focused on the efiects of tricyclic antidepressants and
MAOIs (Hogben and Comfield, 1981; Bleich et al., 1986). Both classes of dmg appeared
to be effective. A recent placebo-controlled, double-blind clinical trial also found imip
ramine and phenelzine to be superior to placebo in alleviating PTSD (Frank et al., 1988).
To my knowledge, no animal model has been proposed specifically for PTSD,
although several Hues of investigation may be of use in the search for such a model. The
behavioral changes following exposure to inescapable shock have been the subject of
much research (e.g., Maier and Jackson, 1979) and alterations in behavior have also been
found following a single exposure to a pharmacological stressor (Drugan et al., 1985).
While much of this work has been discussed in terms of depression, it may have some
relevance to PTSD. There is clearly a need for further work examining the long-term
behavioral changes produced by severe stressors in laboratory animals and the effects of
drugs on these changes.
4. CONCLUDING REMARKS
There may be more animal models of anxiety disorders than of any other psychiatric
condition. Many of the tests used are rapid and have been successful in discriminating
the effects of drug acting at the BZ/GABA receptor complex from other psychotropic
agents. This success has led, perhaps, to a certain degree of complacency among behav
ioral pharmacologists. The validity of many of the tests, however, is now being ques
tioned by a growing literature on the cUnical efficacy of agents that reduce anxiety by
non-GABAergic mechanisms. These new generation anxiolytics are not showing clear
anxiolytic profiles in all the animals models.
Most of the animal models currently in use assess the effects of drugs in an unselected
population of animals in which no attempt has been made to induce a chronically anx
ious state. A drug effect is investigated by placing animals in an environment designed
to induce some form of anxiety or conflict. As such, it seems likely that drugs that pro
duce anxiolytic profiles in these tests would alleviate short-term increases in state anxiety
that occur in humans. If the mechanisms that mediate these short-term increases in anx
iety are similar to those underlying conditions of chronic anxiety, like those found in
GAD, then that would account for the relative success of these tests in predicting treat
ments for GAD.
In screening for agents that may be useful in treating a particular anxiety disorder, it
would currently be unwise to rely too heavily on any one model. Our present knowledge
requires that the effects of any putative anxiolytic be examined in several paradigms
before its behavioral effects can be appropriately evaluated.
The success of any model is limited by the characterization of the phenomenon that
is being modeled. The present review has used DSM-III-R classifications as a basis for
discussing animal models of anxiety disorders. These classifications will no doubt change
in the coming years. Further, the pharmacological validation of the models is made dif
ficult by some current controversies in the literature concerning the sensitivity of the
various disorders to certain classes of drugs. Future animal research will benefit from
attending to the apparent heterogeneity of anxiety disorders and placing a somewhat
greater emphasis on models with an etiological basis. Indeed, a reciprocal relationship
between clinicians and investigators studying anxiety in animals is likely to be invaluable
in helping to determine the most appropriate classification of anxiety disorders. It is not
the intention to argue that etiological models are always the most useful (see Overmier
and Patterson, 1988), but they are underrepresented in current pharmacological research
on anxiety. While an etiological approach may require a greater investment of time for
each animal tested, the results are likely to enhance considerably our understanding of
the anxiety disorders and their treatment.
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CHAPTER 8
ANIMAL MODELS OF ANXIETY BASED ON CLASSICAL

CONDMONING: THE CONDITIONED EMOTIONAL RESPONSE
A N D THE FEAR-FOTENTIATED STARTLE EFFECT
MICHAEL DAVIS
Yale University School of Medicine and Ribicoff Research Facilities of the Connecticut Mental Health Center,
New Haven, Conn., USA
1. INTRODUCTION
When a stimulus such as a Ught, which does not produce a very profound reaction
before pairing, is paired with an aversive stimulus such as a foot shock, the hght can now
elicit a constellation of behaviors that are typically used to define a state of fear in ani
mals. To explain these findings, it is generally assumed that during light-shock pairing
(training session) the shock elicits a variety of behaviors that can be used to infer a central
state of fear (unconditioned responses) (Fig. 8.1). After pairing, the light can now pro
duce the same central fear state and thus the same set of behaviors formerly produced
by the shock. Moreover, the behavioral effects in animals that are produced by this for
merly neutral stimulus, now called a conditioned stimulus (CS), are similar in many
respects to the constellation of behaviors that are used to diagnose generalized anxiety in
humans (Table 8.1). Viewed in this way, a pharmacological and anatomical analysis of
conditioned fear should produce information that would be important and relevant to
the study of human anxiety.
The puφOse of this article is to review work on two measures of conditioned fear, the
conditioned emotional response (CER) and fear-potentiated startle, which have been
investigated at both pharmacological and anatomical levels of analysis.
2. THE CONDITIONED EMOTIONAL RESPONSE

In 1941, Estes and Skinner published a highly influential paper that suggested a tech
nique for the objective measurement of fear in animals. In the prototypic experiment,
food-deprived rats are first trained to press a lever for food using intermittent reinforce
ment. After giving a sufficient number of training sessions to estabUsh stable rates of bar
pressing, some neutral stimulus such as a light is paired with a shock. After a small num
ber of pairings, the light now produces a suppression of the lever-press response. A vari
ant of this procedure is to measure the disruptive effects of the conditioned stimulus on
some consummatory response such as a thirsty rat licking for water on a water tube. Both
phenomena have been termed conditioned suppression and are now widely used as a
measure of classical conditioning where the amount of suppression of ongoing operant
or consummatory behavior is used as an index of the strength of fear.
2.1. W H A T DOES CONDITIONED SUPPRESSION MEASURE?
Although the conclusion generally prevails that conditioned suppression reflects a cen
tral state of fear, this inteφretation has been questioned on a number of levels. First, it
is clear that a suppression of lever pressing per se is not sufficient to infer a central state
of fear. Thus, salient or novel stimuli by themselves can suppress bar pressing, probably
because they elicit an unconditioned orienting response. In fact, in many experiments,
187
188 Μ. DAVIS
Training
Increased heart rate
Decreased salivation
Stomach ulcers
Light Respiration change
Tone Shock S c a n n i n g and vigilance
Puff Increased startle
Urination
Defecation
Grooming
Freezing
Testing
Increased heart rate

Decreased salivation
Stomach ulcers
Respiration change
S c a n n i n g and vigilance
Increased startle
Urination
Defecation
Grooming
Freezing
FIG. 8.1. General scheme believed to occur during classical conditioning using an aversive con
ditioned stimulus. During training, the aversive stimulus (e.g., shock) activates a central fear sys
tem that produces a constellation of behaviors generally associated with aversive stimuli (uncon
ditioned responses). After consistent pairings of some neutral stimulus such as a light or tone or
puff of air with shock during the training phase, the neutral stimulus is now capable of producing
a similar fear state and hence the same set of behaviors (conditioned responses) formeriy pro
duced only by the shock.
the unconditioned suppressant effects of the conditioned stimulus have to be habituated

before pairing, to be sure its suppression of bar pressing resulted from its association with
shock and not from its ability to produce a disruptive orienting response.
Second, it has been shown that stimuli paired with the receipt of food can also suppress
bar pressing (Azrin and Hake, 1969; Miczek and Grossman, 1971). Because this type of
suppression would be difficult to ascribe to conditioned fear, it indicates once again that
stimulus-induced suppression of bar pressing by itself is not sufficient to infer a state of
conditioned fear.
Third, it has been argued that response suppression by the conditioned stimulus may
simply reflect response competition caused by conditioned freezing. If one only views
freezing as an unconditioned reaction to foot shock in the rat, which might have nothing
to do with fear, then it would not be necessary to appeal to a central fear state to explain
conditioned suppression. Instead, it would simply be an instance where one conditioned
TABLE 8.1. Comparison of Measures in Animals Typically Used To Index Fear and Those in the DSM-III-R
Manual (APA, 1987) To Index Generalized Anxiety in People
Measures of fear in animal models DSM-III-R criteria-generalized anxiety
Increased heart rate Heart pounding

Decreased salivation Dry mouth
Stomach ulcers Upset stomach
Respiration change Increased respiration
Scanning and vigilance Scanning and vigilance
Increased startle Jumpiness, easy startle
Urination Frequent urination
Defecation Diarrhea
Grooming Fidgeting
Freezing Apprehensive expectation—something bad is going to happen
CER and fear-potentiated startle 189
response competes with another. However, in many cases, conditioned suppression is

associated with defecation, urination, piloerection, and fast, shallow breathing; reactions
normally used to define a state of fear. While freezing is also a prominent response (and
one incidentally that classically is associated with fear), there are special cases in which
the same training procedures that allow a conditioned stimulus to suppress bar pressing
will actually facilitate an avoidance response (Hermstein and Sidman, 1958; Scobie,
1972). Because both of these effects cannot be ascribed to freezing, it seems more prudent
to conclude that the conditioned stimulus evokes a central fear state, which has different
behavioral consequences depending on the operant behavior being used to index fear.
Fourth, in the typical paradigm, presentation of the conditioned stimulus and the
shock occurs when the animal is engaged in the operant task. Fear conditioning done in
this way (conditioning on the baseUne) provides opportunities for a bar-press to be fol
lowed closely by a shock. Hence, suppression could result from "adventitious punish
ment" rather than a central state of fear. In fact, it has been argued that the ability of
benzodiazepines to attenuate conditioned suppression only occurs following condition
ing on the baseUne, reflecting the well-known ability of benzodiazepines to block pun
ished responding, rather than reflecting a blockade of fear (Gray, 1977). However, it is
unlikely that the rather profound levels of suppression after such training result from a
few punished responses. Moreover, it is well established that conditioned suppression can
certainly occur when conditioning occurs off the baseline (e.g., with the manipulandum
retracted or covered), and more recent papers show that benzodiazepines can attenuate
conditioned suppression using this method of training (see Table 8.2).
Thus, while acknowledging the possibility that conditioned suppression to a stimulus
paired with foot shock does not reflect only fear, on balance, this still seems to be a fair
conclusion. If so, then drugs that would be expected to reduce fear or anxiety should also
attenuate the CER.
2.2. EFFECTS OF DIFFERENT DRUGS ON THE CONDITIONED EMOTIONAL RESPONSE
Table 8.1 shows a sampHng of representative experiments done over the last 30 years,
which have evaluated the effects of different classes of drugs on conditioned suppression,
when suppression has been measured by a change in some operant or consummatory
response. Drug effects on freezing itself (e.g., Babbini et al., 1973; Blanchard et al., 1988;
Fanselow and Bolles, 1979; Kameyama and Nagasaka, 1982), where decreases in loco
motor activity are used as an index of fear, are not included. While a significant per
centage of drug studies on the CER are represented, only results that used rats or pigeons
have been included in Table 8.2, and the Usting does not claim to be exhaustive. Instead,
it represents a range of studies across a long period of time to give the flavor of the lit
erature on how different drugs alter the CER. Importantly, Table 8.2 only includes stud
ies in which the effects of drugs were measured on CER performance (i.e., drugs given
after the conditioned stimulus was paired with the foot shock). A very interesting liter
ature on the acquisition of CER fear conditioning, where drugs are administered before
CS-US (conditioned stimulus-unconditioned stimulus) pairings, is not included, since
this would be more in the purview of an article on the effects of drugs on the leaming
process.
22Λ. Effects of Ethanol

Ethanol has not been reported to be effective in blocking the CER in rats (Cicala and
Hartley, 1967; Lauener, 1963) or cats (Goldman and Docter, 1966), although it appar
ently did in one study using dogs (Tamura, 1973, in Edwards and Eckerman, 1979). A
very modest effect was reported in pigeons (Edwards and Eckerman, 1979), but only one
animal showed a robust blockade of the CER at the highest dose (2000 mg/kg). While
diazepam also attenuated the CER in this study, a combination of diazepam and ethanol
TABLE 8.2. Effects of Various Drugs on the Conditioned Emotional Response
Method
Response ofCER Chronic Effect on Effect on
measure CS US training Drug Dose(mg/kg) or acute baseline CER Reference
LP-F Ν Shock Off Alcohol 600-1200 A NM No effect Cicala and Hartley, 1967
LP-W L + N 40-50 volts On Alcohol 200-1600 A Down No effect Uuener, 1963
KP-F CC Shock On Alcohol 500-2000 A Down Attenuate Edwards and Eckerman, 1979
LP-F Ν Shock Off Chlordiazepoxide 5-10 A NM Attenuate Cicala and Hartley, 1967
LP-W L -f- Ν 40-50 volts On Chlordiazepoxide 5-40 A Up Attenuate Lauener, 1963
LP-F L 900 volts-47 k On Chlordiazepoxide 5-33.3 A None Attenuate Miczek, 1973
LI-M L-l-N 1.0 mA Off Chlordiazepoxide 12 A None No effect Scobie and Garske, 1970
190
LP-F Ν 1.0 mA Off Chlordiazepoxide 12 A None No effect Scobie and Garske, 1970
LI-M CL 1.0 mA Off Chlordiazepoxide 15-25 A NB No effect Stein and Berger, 1969
LI-S L + T 1.5 mA On Chlordiazepoxide 10,31.6,75 A NB Fast recovery Tenen, 1967
LP-F L + Ν 1.0 mA Off Diazepam 2.5-5 A NM Attenuate Lane et al., 1982
LP-F Τ 3.0 mA On Diazepam 0.37-2.0 A Up-down Attenuate Haug and Gotestam, 1981
KP-F CC Shock On Diazepam 0.3-10 A Down Attenuate Edwards and Eckerman, 1979
LI-W Τ 1.6 mA Off Diazepam 2.5 A o r C NB Fast recovery Methot and Deutsch, 1984
LI-S L-HT 1.5 mA On Diazepam 17.8 A NB Fast recovery Tenen, 1967
LI-M CL 1.0 mA Off Diazepam 4 C NB No effect Stein and Berger, 1969
LP-F Ν Off 0.4-1.3 mA On Diazepam 1-10 A Up No effect Hymowitz, 1981
KP-F CC Shock On Diazepam + ethanol A Down No effect Edwards and Eckerman, 1979
LI-S L + T 1.5 mA On Nitrazepam 10 A NB Fast recovery Tenen, 1967
LP-W L 0.7 mA On Oxazepam 10 C None Attenuate Maser and Hammond, 1972
LI-M CL 1.0 mA Off Oxazepam 20 C NB No effect Stein and Berger, 1969
LI-M CL 1.0 mA Off WY 4036 0.5,2 A or C NB Enhance Stein and Beider, 1969
LP-W L-fN 40-50 volts On Hexobarbital 50-75 A None No effect Uuener, 1963
LP-W L + N 40-50 volts On Methoxyphenobarbital 45-70 A None Attenuate Uuener, 1963
LP-F Τ 60 volts On Pentobarbital 4,6,10 A Down No effect Hill et al., 1967
LP-F TorL 0.5 mA On Pentobarbital 4,8,12,16 A Down Attenuate Miczek and Luttinger, 1978
LP-W L + Ν 40-50 volts On Pentobarbital 45-70 A None Attenuate Uuener, 1963
LP-W L + N 40-50 volts On Sodium amytal 45-100 A None Attenuate Uuener, 1963
LI-M CL 1.0 mA Off Sodium amytal 30 AorC NB Enhance Stein and Beiger, 1969
LI-S L + T 1.5 mA On Sodium amytal 23.7 A NB Fast recovery Tenen, 1967
LP-W L-HN 40-50 volts On Meprobamate 50-200 A Up No effect Uuener, 1963
LP-W Ν 1.0 mA On Meprobamate 30,160,240 A None No effect Hunt, 1957
LP-W Τ 1.0mA On Meprobamate 80-180 A Down No effect Ray, 1964
LP-W Ν 1.0 mA On Meprobamate 240 C None Attenuate Hunt, 1957
LP-F Τ 40-80 volts On Meperidine 10-30 A Down Attenuate Hill et al., 1966
LP-F Τ 40-80 volts On Methadone 0.75-4.5 A Down Attenuate Hill et al., 1966
LP-F Τ 40-80 volts On Moφhine 1-9 A Down Attenuate Hill et al., 1966
LP-W L + Ν 40-50 volts On Moφhine 2-8 A Down No effect Uuener, 1963
LP-F Τ 60 volts On Morphine 5,7,9 A Down Attenuate Hill et al., 1967
LP-F Τ 60 volts On Naloφhrine 7,17,25 A Down No effect Hill et al., 1967
LP-W L-l-N 40-50 volts On Chloral hydrate 200 A None No effect Uuener, 1963
LP-F Τ 60 volts On Chloφromazine 1,2,3 A Down No effect Hill et al., 1967
LP-M L-fN 0.8 or 1.0 mA On Chloφromazine 1.5 5 days IDown Attenuate Appel, 1963
LP-W L + N 40-50 volts On Chloφromazine 1.5 A Down No effect Uuener, 1963
LP-F Ν Shock Off Chloφromazine 1-2 A NM No effect Cicala and Hartley, 1967
LP-W Τ 0.5-1.0 mA On Chloφromazine 0.5-3.0 A NM No effect Kinnard et al., 1962
LP-W Τ 1.0 mA On Chloφromazine 1.0-3.5 A Down No effect Ray, 1964
LI-M CL 1.0 mA Off Chloφromazine 3 A NB No effect Stein and Berger, 1969
LI-S L + T 1.5 mA On Chloφromazine 3.16 A NB Slow recovery Tenen, 1967
LP-M L + N 0.8 or 1.0 mA On Rescφine 0.2 C Down Attenuate Appel, 1963
LP-W Τ 0.5-1.0 mA On Reseφine 0.25-0.60 A NM No effect Kinnard et al., 1962
LP-W Ν 1.5 mA On Reseφine 0.2 C Down Attenuate Brady, 1956
LP-W Τ 1.0 mA On Reseφine 1.0 C Down Attenuate Ray, 1964
LP-W L-l-N 40-50 volts On Amphetamine 1-5 A Up No effect Uuener, 1963
LP-F L 900volts-47k On Amphetamine 1-2.0 A Down No effect Miczek, 1973
LP-F TorL 0.5 mA On Amphetamine 0.25-3.0 A None Enhance Miczek and Luttinger, 1978
191
LP-M Τ 0.5 or 1.5 mA On Amphetamine 0.5,1.2 A Down Attenuate Cappell et al., 1972
LP-W Ν 1.5 mA On Amphetamine 2 A Up Enhance Brady, 1956
LP-F Τ 60 volts On Amphetamine 1,2,3 A Up No effect Hill et al., 1967
LI-S L-HT 1.5 mA On Amphetamine 1 A NB No effect Tenen, 1967
LP-F Τ 60 volts On Cocaine 5,10,15 A Up No effect Hill et al., 1967
LP-F Τ 60 volts On LSD 0.10,0.2,3,4 A Down Attenuate Hill et al., 1967
LP-F L 900volts-47k On Scopolamine 0.1-1.0 A Down No effect Miczek, 1973
LI-M CL 1.0 mA Off Scopolamine 1 C NB No effect Stein and Berger, 1969
A = acute; C = chronic; CC = color change; CER » conditioned emotional response; CL = clicker, CS = conditioned stimulus; F = food; KP « key peck; L « light; LI « lick; LP « lever press; Μ • milk; Ν «
noise; NB = no baseline in this paradigm; NM » not mentioned; OFF » off the baseline training; ON » on the baseline training; S » sucrose; Τ » tone; US » unconditioned stimulus; W « water.
192 M.DAVIS
failed to block the CER in the same animals in which each drug by itself tended to do
so. The authors contend that this was not due to narcosis produced by the two drugs
given together, because the drug combination did not suppress baseline key pecking (i.e.,
in the absence of the CS) any more than either drug did by itself Instead, they conclude
that alcohol and diazepam antagonized each other's actions. However, this somewhat
suφrising result is not assimilated with other clinical or animal literature.
2.2.2. Effects of Benzodiazepines

Several studies have shown that benzodiazepines (chlordiazepoxide, diazepam, oxaz
epam) attenuate the CER using either an operant or consummatory measure, ahhough
the data have not been consistent. Thus, chlordiazepoxide has been reported to attenuate
the CER using a lever press for food (Cicala and Hartley, 1967) or for water (Lauener,
1963), although administration of chlordiazepoxide at a similar dose, route of adminis
tration, and injection-test interval did not block the CER using a lever press for food
(Scobie and Garske, 1970). Because conditioning was done off the baseline in the two
studies that reported different results, the source of the discrepancy does not seem to be
attributable to this variable.
More recently, Miczek (1973) showed that chlordiazepoxide produced a robust, dose-
related attenuation of the CER using a bar-press response for food. Baseline rates of
responding also tended to be elevated by chlordiazepoxide at the lower doses (5-25 mg/
kg). However, a dose of 33.3 mg/kg completely blocked suppression without significantly
affecting the baseline response rate. Moreover, neither ^/-amphetamine nor scopolamine
had any effect on conditioned suppression, although the peripheral actions of scopol
amine markedly depressed baseline levels of responding, which might have masked its
effects on suppressed behavior. Finally, in a very important control experiment, Miczek
found that chlordiazepoxide or diazepam did not block conditioned suppression when
the conditioned stimulus had previously been paired with sweetened milk, even though
the baseline and suppressed levels of the operant response were very similar to those
obtained when chlordiazepoxide blocked suppression to a conditioned stimulus paired
with foot shock. While acknowledging the possibility that the slight parametric differ
ences between the two studies might account for the different drug effects on the two
types of conditioned suppression, Miczek concluded that the ability of chlordiazepoxide
to attenuate conditioned suppression following aversive conditioning could not be attrib
uted to general disinhibitory effects or appetite stimulatory effects. Moreover, the lack of
effect of chlordiazepoxide on positive conditioned suppression has been replicated by
Poling et al. (1977).
Using a potentially powerful approach, Umemoto and Olds (1975) tested the CER
while concomitantly measuring single- or multiple-unit activity in the hippocampus,
hypothalamus, geniculate nuclei, and amygdala. In this case, the aversive stimulus paired
with a visual CS was electrical stimulation of the central gray, instead of foot shock.
Pairing the light with central gray stimulation resulted in robust suppression of a lever-
press response for food, as well as a change in unit firing in each of the recorded brain
areas. In about half the animals, the benzodiazepines (chlordiazepoxide or diazepam)
attenuated the CER, whereas chloφromazine had no effect in any animal. Most inter
esting, in several cases in which benzodiazepines attenuated the CER, they also blocked
the change in unit activity produced by the conditioned stimulus in each of the recorded
brain areas. In contrast, chloφromazine had no disinhibitory effect on the CER in spite
of consistently antagonizing conditioned unit activity in the hippocampus. Although the
correlations between the CER and unit activity were not tight, these and other data
showed that the most consistent pattern of drug effects on the two measures occurred in
the amygdala rather than in the hippocampus, geniculate nuclei, or hypothalamus.
In another interesting experiment, Maser and Hammond (1972) paired a relatively
long, 5-minute light-tone CS with shock, using a large number of training trials. Consis-
tent with previous work (e.g., Millenson and Hendry, 1967), suppression at CS onset (a
time that actually signals that no shock would occur for 5 minutes) was minimum, but
then grew steadily over the duration of the 5-minute CS. Under such conditions, oxaz
epam only altered suppression at the end of the CS (i.e., when anticipatory fear should
be maximal). While this result is clearly consistent with an anxiolytic effect of benzodi
azepines in the CER, this type of finding has been used to support a rate-dependent
interpretation of benzodiazepine disruption, along with an effect of the drug on acquired
timing behavior (cf., Millenson and Leslie, 1974). However, as mentioned eariier, rate-
dependent effects cannot universally explain the effects of benzodiazepines on the CER
(e.g., Miczek, 1973), and drugs like scopolamine, which alter acquired timing behavior
(cf.. Meek and Church, 1987), do not necessarily alter the CER (Miczek, 1973), although
admittedly this may not be germane because the duration of the light was only 20 sec
onds in the Miczek study.
Goldman (1977) also found that chlordiazepoxide attenuated the CER when testing
during extinction was used. Interestingly, when a second extinction test day was given,
in which all animals were tested in the absence of drug administration, rats given chlor
diazepoxide the previous day had much greater suppression than other rats given saline
the previous day. Goldman concluded, therefore, that chlordiazepoxide decreased
extinction of the CER that normally occurs in this paradigm using a repeated extinction
procedure. Further studies will be required, however, to evaluate whether these results
could be attributed to a chlordiazepoxide-induced amnesia for the prior extinction
session.
In another very thorough study, Tenen (1967) showed that recovery time (the time it
takes a rat to resume licking after presentation of a short 10-second conditioned stimu
lus) was directly related to the shock intensity paired with the CS, as well as the number
of training trials, and inversely related to the deprivation level of the animal (i.e., a very
thirsty rat will resume drinking faster than a moderately thirsty one). Using recovery time
as a measure, Tenen found that chlordiazepoxide, diazepam, and nitrazepam caused a
marked reduction in recovery time, even though high, sedating doses were used. In con
trast, amphetamine had no effect and chlorpromazine actually increased recovery time.
Finally, Tenen showed that the effects of chlordiazepoxide could not easily be attributed
to state-dependent generalization decrement from training to testing, because groups
injected with chlordiazepoxide during training, but tested under sahne (i.e., a change in
drug state from training to testing), had a nonsignificant attenuation of conditioning.
Groups injected with the drug on both occasions (i.e., the same drug state in training
and testing) still had reduced recovery times. More recently, very similar effects of a lower
dose of diazepam on a recovery time measure of CS-induced lick suppression have been
obtained by Methot and Deutsch (1984) when training was carried out off the baseline.
Moreover, there was no tolerance to this effect, since diazepam produced even shorter
recovery time (i.e., it was more effective in blocking conditioned suppression) in rats
injected with diazepam (5 mg/kg) for 22 days before testing.
Other studies have also found diazepam to block the CER (Lane et al., 1982; Haug
and Gotestam, 1981; Edwards and Eckerman, 1979), although Hymowitz (1981)
reported positive effects of diazepam on punished responding but not on responding sup
pressed by a conditioned stimulus. However, this was a very complex schedule in which
the same animals were exposed to a punishment phase and a CER phase in the same test
session. A more glaring exception to the generally positive effects of benzodiazepines on
the CER was a widely quoted study by Stein and Berger (1969). In this study, diazepam
and oxazepam had no effect, whereas sodium amytal and a novel benzodiazepine (WY
4036) actually enhanced suppression, as if they were producing anxiogenic effects.
Because this did not occur with a sedative dose of chlordiazepoxide and still occurred
after repeated injections of WY 4036 or oxazepam, when the sedative effects should have
shown tolerance, it was concluded that the longer latencies could not be ascribed to drug-
induced sedation. However, in another phase of the study, it was found that WY 4036
194 Μ . DAVIS
did slightly reduce drink latency when a weak foot shock was used, even though it once
again increased drink latency when a strong foot shock had been used. To explain the
main results, the authors suggested that tranquilizers may facilitate memory of painful
experiences, leading to an anxiogenic-like effect. However, there would appear to be little
clinical support for this idea. At the present time, this study stands out as a potentially
important exception in the literature, and it is not clear how to explain these results in
view of the rest of the data. However, the use of the experimental context as the CS and
training off the baseline with a single shock presentation differ considerably from the
more traditional paradigms. In addition, the inability to evaluate what the drug does to
baseline consummatory behavior in the absence of the CS, because context was the only
CS, makes it difficult to evaluate the specificity of the particular drug effects on condi
tioned suppression.
2.2.3. Effects of Barbiturates and Meprobamate

With the exception, once again, of Stein and Berger (1969), sodium amytal and phe
nobarbital have been reported to attenuate the CER (Lauener, 1963; Tenen, 1967). How
ever, hexobarbital and pentobarbital did not (Lauener, 1963; Hill et al., 1967). On the
other hand, Miczek and Luttinger (1978) found that pentobarbital caused a dose-related
attenuation of the CER when the CS had been associated with shock, but no systematic
change in responding when bar pressing was suppressed by a stimulus previously paired
with food. With meprobamate, the literature indicates that it blocks the CER after
chronic administration (Hunt, 1957; Corson and Corson, 1967; Xhenseval, 1964b), but
not after acute administration (Hunt, 1957; Ray, 1964; Lauener, 1963; Xhenseval,
1964a). (References to Corson and Corson, 1967 and Xhenseval, 1964a, 1964b in Mil-
lenson and Leslie, 1974.)
2.2.4. Effects of Opiates

Hill and his associates found that opiate agonists such as meperidine, methadone, and
moφhine (Hill et al., 1966, 1967) attenuated the CER (i.e., led to more bar pressing),
even though they decreased baseline bar-press rates in the absence of CS. However, this
was not found by Lauener (1963) in the same series of studies in which several other
compounds did have positive effects. In Hill's studies, the conditioned stimulus was
either a 60- or 523-Hz tone that was presented 2 dB above a constant noise level of 44
dB. The tone was presented once, for 4 minutes during the test session. It is interesting
to note, however, that if the auditory CS was much louder (30 dB above background),
then moφhine was no longer capable of attenuating its suppressive effect. In Lauener's
study, a buzzer (of unspecified intensity) and a red lamp lasting 10 to 60 seconds was
presented every 3 to 4 minutes over a 15- to 25-minute session. Because the doses and
injection-test intervals were similar in both studies, it is possible that greater salience of
the conditioned stimulus in Lauener's study precluded seeing an effect of moφhine on
the CER.
2.2.5. Effects of Chlorpromazine

In a variety of studies, acute administration of chloφromazine has not been found to
attenuate the CER (Hill et al., 1967; Lauener, 1963; Cicala and Hartley, 1967; Kinnard
et al., 1962; Ray, 1964; Stein and BeiBer, 1969), and Tenen (1967) found that it actually
lengthened recovery time. De Vietti and Porter (1969) found a similar lack of effect on
the CER, despite the fact that chloφromazine attenuated the change in heart rate pro
duced by the same CS used to produce the change in bar pressing. With chronic admin
istration of chloφromazine (1.5 mg/kg for 5 days), Appel (1963) reported a modest
blockade of the CER, and another study showed that chloφromazine given chronically
for 25 days (0.4 mg/kg) decreased the change in heart rate normally observed when rats
were placed into a context previously associated with foot shock (Lepore et al., 1974).
2.2.6. Effects of Reserpine

While acute reseφine has not been reported to attenuate the CER (Kinnard et al.,
1962), several studies have shown that chronic administration (e.g., 0.2 mg/kg for 5 days)
consistently attenuates the CER, even though it concomitantly depresses baseline levels
of operant behavior (Appel, 1963; Brady, 1956; Ray, 1964).
2.2.7. Effects of Amphetamine, Cocaine, and LSD

Finally, several studies have found that amphetamine (Lauener, 1963; Miczek, 1973;
Brady, 1956; Hill et al., 1967; Tenen, 1967) or cocaine (Hill et al., 1967) do not affect
the CER. However, Miczek and Luttinger (1978) reported that ^/-amphetamine (0.5-3.0
mg/kg) enhanced the CER when the CS had previously been associated with foot shock,
but actually increased bar-press rates when the CS had been associated with food. On the
other hand, Cappell et al. (1972) reported that amphetamine depressed baseUne levels of
bar pressing and attenuated conditioned suppression. This occurred when the two dif
ferent shock intensity levels were used, which produced differential levels of suppression.
It is not clear why the Cappell et al. study found an effect of amphetamine when so many
other studies have not, and Miczek and Luttinger (1978) found it to actually increase the
CER. Compared to most other studies, animals in the Cappell et al. study received exten
sive fear conditioning (40 1-hour sessions, two pairing per session). This was because they
initially tried to establish a within-subjects discrimination between low and high shocks
and then used the same animals in a between-subjects design. It is possible, therefore,
that this led to a good deal of context conditioning, or perhaps a good deal of CS-context
discrimination, which altered the subsequent effects of amphetamine in the CER test
session.
Finally, LSD was reported to attenuate the CER when suppression was produced by a
60-Hz tone, which produced relatively weak suppression, but not when a stronger level
of suppression was produced by a 523-Hz tone (Hill et al., 1967).
2.3. SUMMARY A N D CONCLUSIONS
On balance, the CER technique used in animals has been generally successful in dif
ferentiating drugs that are anxiolytic or nonanxiolytic in humans. Thus, benzodiazepine,
barbiturates, and opiates generally attenuate the CER, whereas chlorpromazine and
amphetamine do not. However, exceptions to this generalization can be found for every
drug class, and the general impression in the hterature is that the CER is too variable to
be used for screening novel anxiolytics. Instead, punished operant or consummatory
measures have almost completely replaced the CER as a method of screening drugs. This
is because these paradigms are more tightly under stimulus control and because of the
general opinion that responding suppressed by response-contingent shock is more sen
sitive to benzodiazepines than suppression to response-independent shock (Huppert and
Iversen, 1975; Hymowitz and Abramson, 1983; McMillan and Leander, 1975; but see
RawUns et al., 1980; Salmon and Gray, 1986). Because of this, new anxiolytics will most
probably be ones that release punished behavior (i.e., in the general benzodiazepine class,
as these compounds have high efficacy in the conflict test). One wonders, however,
whether there are fundamental differences between conditioned fear and behavioral con
flict and whether the general abandonment of the CER procedure might overlook some
promising new drug classes. For example, opiates are generally claimed to be ineffective
in conflict tests (e.g.. Geller et al., 1963; Kelleher and Morse, 1964; but see Brady and
Barrett, 1986; Leaf and Muller, 1965), yet they generaUy are effective in the CER (Table
8.2). While those who work with conflict conclude that this means CER picks up a false-
positive (i.e., a drug that works in an animal behavioral test, but is not effective in peo
ple), morphine, in fact, can be an effective clinical anxiolytic in certain circumstances
(cf. Redmond, 1977), as well as showing efficacy in other animal tests sensitive to anx-
196 Μ . DAVIS
iolytic drugs such as the potentiated startle test after systemic administration (Davis,
1979b) or the social interaction test after local infiision into the amygdala (File and Rod-
gers, 1979). In addition, because the CER is typically tested in extinction (i.e., no shocks
given in testing), positive effects of moφhine in the CER paradigm cannot be attributed
to analgesia as they can in the conflict procedure. More recently, nonbenzodiazepine
compounds such as buspirone have been claimed to be anxiolytic in humans, yet they
generally lack efficacy in rat conflict models (e.g., McCloskey et al., 1987; Pich and
Samanin, 1986; Riblet et al., 1982), although they do work well in pigeons (e.g., Weiss
man et al., 1984; Witken and Barrett, 1986). Curiously, at the time of this writing, I could
not find any study looking at the effects of buspirone on the CER.
It is probable, therefore, that conflict tests and the CER procedure measure different
aspects of anxious or fearful behavior, each of which may be relevant to human anxiety.
Because of this, the large scale abandonment of the CER may have been premature.
While it is clear that inconsistencies exist on the effects of drugs on the CER, it is almost
as certain that a careful deUneation of the relevant variables would produce a more reli
able behavioral model. For example, several examples exist that suggest that CS intensity
may be an important variable for either seeing or missing a drug effect, yet drug inter
actions with this variable have received no systematic attention and often CS intensity is
not even reported. While anxiolytics have been reported to attenuate the CER when
training is carried out both on and off the baseUne, shock presentation still typically takes
place in the same chamber in which testing occurs. However, little work has been done
on the possible interaction between drug effects on context conditioning versus drug
effects on fear conditioning to an explicit CS. Hence, it would be useful to reopen the
CER as a potential model for screening putative anxiolytics, so as to potentially broaden
our understanding of this important phenomenon as well as to possibly improve drug
development.
3. THE FEAR-POTENTIATED STARTLE EFFECT

A common feature of almost all widely used animal models of fear or anxiety is that
fear is inferred from a cessation or inhibition of behavior. This is true for (1) conflict
tests, (2) the CER, (3) the social interaction test, (4) the light-dark box test, (5) the ele
vated plus maze, or (6) measuring freezing itself. While this is a highly important, valid
measure of fear or anxiety, it is possible that common neural circuits are used in each of
these measures to allow an animal to essentially stop what it is doing normally. Because
of this, it is equally probable that drugs that act on neural processes that mediate behav
ioral inhibition will have similar actions in each of these tests. In some cases, this could
lead to false-positives in every one of these tests, because a drug could potentially alter
distal elements of response inhibition circuits (e.g., intemeurons in the spinal cord) that
might have little relationship to the perception or experience of anxiety in humans. It
would be useful, therefore, to have an alternative behavioral measure that did not depend
on response inhibition to infer a state of fear or anxiety. In addition, in order to carry
out a neural analysis of both the acquisition and expression of fear conditioning, which
ultimately will greatly improve our understanding of the neurotransmitters involved in
anxiety and hence drug development of anxiolytic compounds, a measure of fear using
a simpler behavioral end point would be important. The fear-potentiated startle effect
fulfills many of these requirements.
Capitalizing on anecdotal evidence that people startle more when they are afraid.
Brown et al. (1951) demonstrated that the amplitude of the acoustic startle reflex in the
rat can be augmented by presenting the eliciting auditory startle stimulus in the presence
of a cue (e.g., a light) that has previously been paired with a shock. This phenomenon
has been termed the "fear-potentiated startle effect" and has been replicated using either
an auditory or visual conditioned stimulus and when startle has been elicited by either a
loud sound or an aiφuff (cf, Davis, 1986).
In this test, a central state of fear is considered to be the conditioned response (cf.,
McAllister and McAllister, 1971). Conditioned fear is operationally defined by elevated
startle amplitude in the presence of a cue previously paired with a shock. Thus, the con
ditioned stimulus does not elicit startle. Furthermore, the startle eliciting stimulus is
never paired with a shock. Instead, the conditioned stimulus is paired with a shock, and
startle is elicited by another stimulus either in the presence or absence of the conditioned
stimulus. Fear-potentiated startle is said to occur if startle is greater when elicited in the
presence of the conditioned stimulus. Potentiated startle only occurs following paired,
rather than unpaired or "random," presentations of the conditioned stimulus and the
shock, which indicates that it is a measure of conditioned fear (Davis and Astrachan,
1978). Potentiated startle occurs using either foot shocks or back shocks (Davis and
Astrachan, 1978), indicating that augmented startle probably results from increased fear
and not because the animal makes a postural adjustment (e.g., crouching) that is espe
cially conducive to startle (e.g., Kurtz and Siegel, 1966). In fact, the magnitude of poten
tiated startle correlates highly with the degree of freezing, a very common measure of
fear (Leaton and Borszcz, 1985).
3.1. ADVANTAGES OF FEAR-POTENTIATED STARTLE FOR STUDYING THE

PHARMACOLOGY OF FEAR OR ANXIETY
Fear-potentiated startle offers a number of advantages for analyzing how drugs affect
fear of anxiety. First, potentiated startle is defined as a within-subjects difference in startle
amplitude in the presence (light-noise trials) versus the absence (noise-alone trials) of the
visual conditioned stimulus. This makes it a sensitive measure, because it reduces prob
lems caused by between-subjects variability in startle. Second, it allows an evaluation of
specific effects (reduction of startle on light-noise trials) versus nonspecific effects (reduc
tion of startle on noise-alone trials), so that qualitative, as well as quantitative, drug pro
files can be compared. Third, different intensities of the auditory stimulus can be used
within the same test session to elicit startle. This allows potentiated startle to be assessed
at several points on the measurement scale, circumventing problems of inteφretation
that can arise when markedly different parts of the measurement scale are involved (e.g.,
rate-dependent drug effects in operant paradigms and percent figures used with very dif
ferent baseUnes). Fourth, no shocks are given in testing. Thus, drug effects observed in
testing cannot be explained in terms of changes in sensitivity to shock. Fifth, the sepa
ration between training and testing sessions allows one to evaluate whether a drug alters
original leaming or performance, and tests for state-dependent leaming can be easily
evaluated (e.g., Davis, 1979a). Sixth, potentiated startle does not involve any obvious
operant. Thus, the animal is not required to make or withhold a voluntary response to
indicate fear or lack of fear and, consequently, dmg-induced effects that might be
expected to alter operant performance (e.g., rate-dependent, motivational, disinhibitory
motor effects) are circumvented. As indicated above, most animal tests of fear or anxiety
involve a suppression o f ongoing behavior in the presence o f a fear stimulus. Hence,
certain treatments (e.g., decreases in serotonin transmission) might appear anxiolytic in
these tests if they interacted with neural systems common to each o f these tests (e.g.,
response inhibition), even though they mi¿it not reduce anxiety clinically. Because fear
in the potentiated startle paradigm is reflected by enhanced response output, it may pro-
vide an important altemative test with which to analyze potential anxiolytic compounds.
3 . 2 . EFFECTS OF DIFFERENT DRUGS ON FEAR-POTENTIATED STARTLE
Table 8.3 shows that a variety of dmgs that reduce fear or anxiety in humans decrease
potentiated startle in rats. Dmgs Uke Clonidine, moφhine, diazepam, and buspirone,
which differ considerably in their mechanism of action, all block potentiated startle. In
most cases, these treatments do not depress startle levels on the noise-alone trials.
198 Μ. DAVIS
TABLE 8.3. Effects of Various Drugs on Fear-Potentiated Startle
Effect on
Dose Effect on potentiated
Drug (mg/kg) baseline startle Reference
Alcohol 10% solution None Block Williams, 1960 (cited in Miller

and Barry, 1960)
Diazepam 0.3-2.5 None Block Davis, 1979a
Diazepam 0.6-2.5 Decrease Block Berg and Davis, 1984
Flurazepam 2.5-20 None Block Davis, 1979b
Midazolam 0.5-2.0 None Block Hijzen and Slangen, 1989
Lindane 7.5-30 None Increase Hijzen and Slangen, 1989
DMCM 0.1-0.4 None Enhance Hijzen and Slangen, 1989
RO-15-1788 1 None None Berg and Davis, 1984
Sodium amytal 10-40 None Block Chi, 1965
Moφhine 2.5-10 None Block Davis, 1979b
Naloxone 2.0 None None Davis, 1979b
Nicotine 0.4 None Attenuate Sorenson and Wilkinson, 1983
Clonidine 0.01-0.04 Depress Block Davis etal., 1979
Piperoxane 0.25-1 None Enhance Davis etal., 1979
Yohimbine 0.125-0.25 None Enhance Davis etal., 1979
CRF9-41 5-25 μ% icv None Block Swerdlow et al., 1989
WB-4101 1 None None Davis etal., 1979
Propranolol 20 None Attenuate Davis etal., 1979
Imipramine (chronic 5-10 None None Cassella and Davis et al., 1985
or acute)
Buspirone 0.6-10 Increase Block Kehneetal., 1988
Buspirone 1.25-5.0 Increase Attenuate Mansbach and Geyer, 1988
Gepirone 0.6-10 Increase Block Kehneetal., 1988
Gepirone 3-10 None Attenuate Mansbach and Geyer, 1988
Ipsapirone 10-40 None None-attenuate Davis et al., 1988a
Ipsapirone 3.10 Increase Attenuate Mansbach and Geyer, 1988
Ipsapirone 0.15-2.5 Increase None Davis Μ. and Rasmussen Κ.,
unpublished data, 1987
8-OHDPAT 2.5-10 Increase None E)avisetal., 1988a
8-OHDPAT 0.12-0.5 None Block Mansbach and Geyer, 1988
8.0HDPAT 0.12-0.25 Increase None Davis M. and Rasmussen K.,
Ketanserin 2 None None I>avis M. and Rasmussen K.,
Methysergide 0.3-10.0 Increase Attenuate Mansbach and Geyer, 1988
Cinanserin 10 None None Davis et al., 1988a
Cyproheptadine 5 None None Davis et al., 1988a
p-chloroamphetamine 5 None None Davis et al., 1988a
p-chlorophenylalanine 400 X 2 Increase Enhance Davis et al., 1988a
SCH 23390 0.03-0.50 Decrease Attenuate Davis M. and Rasmussen K.,
SCH 23390 + 0.03 + 0.25, None Block Davis M. and Rasmussen K.,
ipsapirone 5 unpublished data, 1987
SCH 23390 + 8- 0.03 + 0.25, None Block Davis M. and Rasmussen K.,
OHDPAT 0.25 unpublished data, 1987
Sulpiride 20 None None Davis M, and Rasmussen K.,
Sulpiride + ipsapirone 20 + 5 Increase None Davis M. and Rasmussen K.,
Sulpiride + 8- 20 + .25 Increase None Davis M. and Rasmussen K.,
OHDPAT unpublished data, 1987
MK-801 0.03-1.0 Increase Block Davis, M., unpublished data,
1988
Pertussis toxin 1 Mg icv None Attenuate Melia, K., Falls, W. Α., and
Davis, M., unpublished
data, 1988
although drugs like Clonidine do have marked depressant effects on both types of trials.
In addition, drugs like yohimbine and piperoxane, which induce anxiety in normal peo
ple and exaggerate it in anxious people (Chamey et al., 1984; Goldenberg et al., 1947;
Holmberg and Gershon, 1961; Soffer, 1954), actually increase potentiated startle in rats.
Thus, at very low doses, these drugs increase startle amplitude on the light-noise trials
without having any effect on startle on the noise-alone trials and this only occurs in rats
conditioned to fear the light (Davis et al., 1979). On the other hand. Table 8.3 shows
that a variety of treatments that alter serotonin (5-HT) transmission do not affect poten
tiated startle. This is important because treatments that decrease 5-HT transmission have
an anxiolytic profile in several animal tests of fear or anxiety (e.g., operant-conflict test,
lick-suppression test, social interaction test), perhaps by interfering with response inhi
bition (cf., Soubrie, 1986). These effects may represent false-positives in these tests
because treatments Uke /H^hlorophenylalanine do not appear to be anxiolytic when
tested cUnicaUy (e.g., Shopsin et al., 1976) and low levels of serotonin are not generally
associated with cUnical anxiety reduction (cf., Soubrie, 1986). In addition, although the
anxiolytic effects of buspirone have been suggested to be mediated through the serotonin
system, Table 8.3 indicates that the ability of buspirone to selectively decrease fear-
potentiated startle does not seem to be attributable to its actions at either pre- or post
synaptic 5-HT receptors.
Because very few studies have evaluated the effects of drugs on fear-potentiated startle,
it is difficuh to determine at this time whether this model wiU produce consistent data
across laboratories. Recentiy, Mansbach and Geyer (1988) confirmed the finding that
buspirone and gepirone attenuate potentiated startle. However, they found that ipsapi
rone and 8-hydroxy-2-(di-n-propylamino)-tetralin ( 8 - O H D P A T ) also showed efficacy,
counter to data from our laboratory. While Mansbach and Geyer used lower doses of
these compounds than those reported by Davis et al. (1988a), we have not found effects
with ipsapirone and 8 - O H D P A T over the same dose range used by Mansbach and Geyer.
On the other hand, when ineffective doses of these drugs (in our hands) are combined
with only partially effective doses of the dopamine antagonist SCH-23390, we find a
complete blockade of potentiated startle with no decrease in baseUne startle levels (Davis
and Rasmussen, unpublished). In view of these data, it is possible that serotonin and
dopamine are both very important in modulating fear-potentiated startle and that dif
ferences in the efficacy of 5-HT,A drugs on potentiated startle across laboratories resuh
from different tonic levels of dopamine tone across different strains of animals or under
different laboratory test conditions.
3.3. NEURAL SYSTEMS INVOLVED IN FEAR-POTENTIATED STARTLE
Another advantage of the potentiated startle paradigm is that fear is being measured
by a change in a simple reflex. Hence, with potentiated startle, fear is expressed through
some neural circuit that is activated by the conditioned stimulus and ultimately impinges
on the startle circuit. Figure 8.2 shows a schematic summary diagram of the convergence
of the neural pathways that we believe are required for fear-potentiated startle.
3.3.1. The Acoustic Startle Pathway

In the rat, the latency of acoustic startle is 6 milliseconds recorded electromyographi-
cally in the foreleg and 8 milliseconds in the hindleg (Ison et al., 1973). This is an extraor
dinarily short latency and indicates that only a few synapses could be involved in medi
ating acoustic startle. Using a variety of techniques (Davis et al., 1982; Cassella and
Davis, 1986), we have determined that the acoustic startle reflex is mediated by the ven
tral cochlear nucleus, an area just medial to the ventral nucleus of the lateral lemniscus,
an area just dorsal to the superior oUves in the nucleus reticularis pontis caudalis, and
motor neurons in the spinal cord. Bilateral lesions using ibotenic acid in each of these
nuclei eUminate startle, whereas lesions in a variety of other auditory areas do not. Star
tle-like responses can be elicited electrically from each of these nuclei, with progressively
shorter latencies as the electrode is moved down the pathway.
3.3.2. Determining the Point Within the Startle Pathway Where Fear Alters Neural
Transmission
Having deUneated the startle reflex circuit involved in fear-potentiated startle, the next
task was to determine the point within the startle circuit where the visual conditioned
stimulus modulates transmission following conditioning. To do this, startle-like
200 Μ . DAVIS
Startle
Pathway
Ear
VCN
CS Pathway
Latera) Perirhinal / VLL

Retina Geniculate
~ ^ Insular Cortex Output Pathway
Amygdala
Central n. RPC
Cord
Muscles
FIG. 8.2. Proposed neural pathways involved in fear-potentiated startle using a visual conditioned
stimulus. After being paired with a shock, the light may activate the amygdala via a projection
involving the ventral lateral geniculate nucleus and the perirhinal cortex. Activation of the amyg
dala may be both necessary and sufficient to facilitate startle through a direct, perhaps monosyn
aptic, connection to the nucleus reticularis pontis caudalis, an obligatory part of the acoustic
startle pathway. CS = conditioned stimulus; VCN = ventral cochlear nucleus; VLL = ventral
nucleus of the lateral lemniscus; RPC = nucleus reticularis pontis caudalis.
responses have been elicited electrically from various points along the startle pathway
before and after presentation of a light that was either paired or not paired with a shock
in different groups of rats (Berg and Davis, 1985). These experiments have shown that
startle elicited electrically from either the ventral cochlear nucleus or from the paralem-
niscal zone is potentiated by a conditioned fear stimulus, whereas elicitation of startle in
the nucleus reticularis pontis caudalis or beyond is not. Potentiation of electrically elic
ited startle can be blocked by diazepam at doses that have no effect whatsoever on
baseline levels of electrically elicited startle (Berg and Davis, 1984). Based on these and
other data, we have concluded that fear ultimately alters transmission at either the par-
alemniscal zone or the nucleus reticularis pontis caudalis.
3.3.3. Lesions of the Central Nucleus of the Amygdala Block Fear-Potentiated Startle
Because the amygdala has been implicated in innate and conditioned fear in a variety
of behavioral tests (cf, Davis et al., 1988b), we hypothesized that lesions of the amygdala
would block fear-potentiated startle. In fact, lesions of the central nucleus of the amyg
dala following fear conditioning were shown to completely eliminate potentiated startle
(Hitchcock and Davis, 1986). In contrast, transection of the cerebellar peduncles or
lesions of the red nucleus had no effect on potentiated startle. Another experiment using
a visual prepulse test indicated that the blockade of potentiated startle observed in ani
mals with lesions of the amygdala could not be attributed to visual impairment. Taken
together, the results of these experiments support the hypothesis that the amygdala is
involved in potentiated startle, a measure of conditioned fear. The results are also con
sistent with the hypothesis that the cerebellum is involved in motor conditioning, rather
than fear conditioning (Thompson et al., 1987). It is still possible, however, that the cer
ebellum could modulate potentiated startle because electrical stimulation of the cerebel
lum has been reported to increase the magnitude of potentiated startle (Albert et al..
1985), and recent studies indicate that lesions of the vermis may alter heart rate condi
tioning in rats (Supple and Leaton, 1986).
3.3.4. Enhancement of Acoustic Startle by Electrical Stimulation of the Amygdala

At the present time, it is not clear how the amygdala participates in fear-potentiated
startle. It is possible that the light, after being paired with shock, activates the amygdala,
which would then increase startle. If so, then electrical stimulation of the amygdala might
be expected to increase acoustic startle. Consistent with this, we have recently found that
low level electrical stimulation of the amygdala (e.g., 40-400 μΑ, 25-miUisecond trains
of 0.1-miUisecond square wave cathodal pulses) markedly increases acoustic startle
amplitude (Rosen and Davis, 1988a). This excitatory effect has occurred in every rat that
we have tested so far in which electrodes were found to be placed in the central, inter
calated, or medial nucleus of the amygdala or in the area just medial to the amygdaloid
complex. At these stimulus currents and durations, electrical stimulation of the amyg
dala did not produce any signs of behavioral activation except for an enhancement of
startle, indicating that startle is an extremely sensitive index of amygdala stimulation.
Moreover, the duration of stimulation is well below that used to produce kindling in rats
(Handforth, 1984), so that the effects on startle are not associated with convulsions. In
addition, electrical stimulation of the amygdala alone does not elicit startle even at high
currents. Finally, electrical stimulation of several other nearby brain areas such as the
endopiriform nucleus, fundus striati, internal capsule or some sites in the basolateral
nucleus of the amygdala did not increase startle.
The excitatory effect on startle occurs within a few milUseconds from the onset of
amygdala stimulation. By varying the interval between the onset of amygdala stimulation
and the onset of the startle eliciting stimulus, we have estimated that the transit time
from the amygdala to the startle circuit is 4 to 5 milliseconds (Rosen and Davis, 1988b).
This very rapid effect means that the increase in startle is not secondary to autonomic or
hormonal changes that might be produced by amygdala stimulation, because these
would have a much longer latency. In addition, it suggests there must be a relatively
simple neural pathway that connects the amygdala to the startle circuit.
3.3.5. The Role of Different Amygdala Efferent Projections in Fear-Potentiated Startle

Using both retrograde and anterograde tracing techniques, we have found a direct,
perhaps monosynaptic, connection between the central nucleus of the amygdala and the
exact part of the nucleus reticularis pontis caudalis known to be critical for startle (Rosen
et al., 1991). This pathway involves the caudal division of the ventral amygdalofugal
pathway which also sends collaterals to many brainstem target areas involved in the
somatic and autonomic symptoms of fear and anxiety (cf. Davis et al., 1988b). Lesions
at a variety of levels along this pathway completely block potentiated startle (Hitchcock,
1988; Hitchcock and Davis, 1991). In contrast, lesions of other major projections from
the central nucleus of the amygdala such as the stria terminalis or the bed nucleus of the
stria terminalis or knife cuts of the rostral part of the ventral amygdalofugal pathway,
which would interrupt its projections to the rostral lateral hypothalamus and substantia
innominata, do not block potentiated startle.
Finally, the top part of Fig. 8.2 shows the possible visual pathway that could carry
visual information to the amygdala, thus allowing a visual conditioned stimulus to ele
vated startle. Although we are still unclear about the details of this pathway, we believe
it involves a connection between the ventral lateral geniculate nucleus and the perirhinal
cortex, which projects to various nuclei within the amygdala.
In conclusion, we believe that potentiated startle results when a visual stimulus, after
it is paired with foot shock, activates the central nucleus of the amygdala. Activation of
the central nucleus of the amygdala is both necessary and sufficient to increase acoustic
202 Μ . DAVIS
startle by its direct, perhaps monosynaptic, connection to the nucleus reticularis pontis
caudalis, a critical part of the acoustic startle pathway.
3.4. FEAR-POTENTIATED STARTLE AS A MODEL OF ANTICIPATORY ANXIETY
3.4.1. Animal Studies

Recently, we have found that fear-potentiated startle in rats shows a good deal of tem
poral specificity, because the magnitude of fear-potentiated startle in testing is maximal
at the time after light onset in which the shock would have occurred in training (Davis
et al., 1989). This suggests that fear-potentiated startle may be a sensitive measure of
anticipatory anxiety. To show this, three different groups of rats were used. In training,
two groups received 30 light-shock pairings, using a 200-millisecond light-shock interval
in one group and a 51,200-millisecond interval in the other group. The third group had
Hghts and shocks presented in a random relationship to each other. Several days later,
all groups were tested identically by presenting startle stimuli at different intervals after
light onset. Figure 8.3 shows the change in startle of the two paired groups, relative to
the random group, at various times after light onset. Figure 8.3 shows that the group
trained with a 200-millisecond light-shock interval had maximum potentiation at a 200-
millisecond light-noise test interval with no potentiation at much longer intervals. In
contrast, the group trained with the 51,200-millisecond light-shock interval had maxi
mum potentiation at the 51,200-millisecond light-noise test interval, with little or no
potentiation at much shorter intervals. These data suggest, therefore, that fear-potenti
ated startle was maximal at the time when the animal was anticipating receipt of shock,
making it a sensitive measure of anticipatory fear or anxiety.
3.4.2. Human Studies

Early studies indicated that the eye-blink component of air-puff-elicited startle in
humans could be potentiated when elicited at various intervals after presentation of a
visual stimulus previously paired with shock (Spence and Runquist, 1958; Ross, 1961).
In addition, it has been reported that the eye-blink component of acoustic startle in
humans is elevated when subjects view unpleasant slides and reduced when they view
pleasant ones (Vrana et al., 1988).
100 1000 10000 100000

T E S T INTERVAL (ms)
FIG. 8.3. A possible measure of anticipatory fear in rats. Mean change in startle amplitude at
various light-noise test intervals following either 200- or 51,200-millisecond light-shock intervals
in training.
350-
• SAi=E
• ANXIETY
)^ 300-
Ζ
m
m 250-
LJJ
S 200-
EL 150-
<
ζ 100-
50-
FIRST BEFORE DURING — A F T E R —
BLOCKS OF 4 TRIALS
FIG. 8.4. Fear-potentiated startle in humans. Anticipatory anxiety in humans produces potenti
ation of the eyeblink. Mean amplitude of the eyeblink during periods in which subjects were told
they either would not get a shock (safe) or might get a shock (anxiety), before, during, or after a
single shock to the median nerve.
Recently, we have been using the eye-bUnk component of startle in humans to

measure anticipatory anxiety (Grillon, et al., 1991). Subjects have been fitted with
an electrode attached to the wrist through which they are told they might receive
a painful shock. During alternate blocks, the acoustic startle reflex was elicited,
either during a period in which the subjects were told they might get a shock or during
a period when they were told they definitely would not get a shock. In each of the nine
subjects tested so far, the eye-blink component of the startle reflex has been larger during
periods in which subjects anticipated shock (anxiety) than in periods when they did not
(safe) (Fig. 8.4). Importantly, this effect occurred in each of the periods when they were
anticipating shock, before the actual shock was given. Potentiation during shock antici
pation then persisted after presentation of the single shock, which, in fact, was not very
painful (Fig. 8.4). These data indicate, therefore, that anticipation of a shock is suflScient
to elevate the eye-blink component of startle in humans, making it a sensitive measure
of anticipatory anxiety. Currently, we are trying to make the human paradigm more
similar to the one we use in rats, by turning a Ught on and instructing the subjects that
"the longer the light is on, the higher the probability wiU be that a shock will occur." By
probing the startle reflex at various times after light onset, we hope to get a gradient of
anticipatory anxiety in humans, similar, perhaps, to the one obtained in rats using the
long light durations in training and testing shown in Fig. 8.3.
3.5. SUMMARY A N D CONCLUSIONS
The potentiated startle paradigm measures conditioned fear by an increase in the

amplitude of a simple reflex (the acoustic startle reflex) in the presence of a cue previ
ously paired with shock. In humans, potentiated startle can be produced simply by antic
ipation of shock. This paradigm offers a number of advantages as an alternative to most
animal tests of fear or anxiety, because it involves no operant and is reflected by an
enhancement rather than a suppression of ongoing behavior. Lesion and electrical stim
ulation studies on fear-potentiated startle and startle increased by electrical stimulation
of the amygdala are being used to define the neural pathways necessary for a visual con
ditioned stimulus to alter the acoustic startle reflex. The current working hypothesis is
204 Μ. DAVIS
that the conditioned stimulus activates the central nucleus of the amygdala through a
pathway involving the ventral lateral geniculate and perirhinal cortex, which project to
the amygdala. The central nucleus of the amygdala then projects directly to the acoustic
startle pathway so as to modulate the startle response. More work has to be done to define
conclusively the relevant neural pathways involved in fear-potentiated startle. Nonethe
less, we think that, by combining behavioral, anatomical, physiological, and pharmaco
logical approaches, it will be possible to determine each step along the pathway that
mediates the ability of a stimulus signaling fear to alter behavior. Once the extract struc
tures are delineated, it should be possible to determine the neurotransmitters that are
released during a state of fear and how this chemical information is relayed along these
pathways so as to affect behavior. Eventually, this approach should allow us to determine
where plastic changes take place along these pathways which mediate the conditioned
effects that are being measured and the biochemical processes that are involved.
4. ANXIETY, FEAR, AND THE AMYGDALA

An important insight gained from work in several different laboratories is that the cen
tral nucleus of the amygdala appears to play a crucial role in conditioned fear and prob
ably anxiety. Thus, the amygdala and its many efferent projections may be the anatom
ical substrate of the central fear system hypothesized in the introduction to account for
the complex pattem of behaviors produced by a light previously paired with an aversive
shock.
4 . 1 . ANATOMICAL CONNECTIONS BETWEEN THE AMYGDALA AND BRAIN STEM AREAS

INVOLVED IN FEAR
Similar to suggestions of several previous reviews (Gloor, 1 9 6 0 ; Kapp and Pascoe,

1986; Kapp et al., 1 9 8 4 ; LeDoux, 1 9 8 7 ; Sarter and Markowitsch, 1 9 8 5 ) , Fig. 8.5 sum
marizes work done in many different laboratories indicating that the central nucleus of
the amygdala has direct projections to a variety of brain stem areas that might be
FEAR PROVOKING STIMULI • CONDITIONED OR SYMBOLIC STIMULI

CENTRAL NUCLEUS OF THE AMYGDALA
CENTRAL DORSAL MOTOR PARABRACHIAL NUCLEUS TRIGEMINAL VENTRAL TEGMENTAL AREA

GREY NUCLEUS OF NUCLEUS RETICULARIS FACIAL TO FRONTAL CORTEX AND
THE VAGUS PONTIS MOTOR NUCLEUS LOCUS COERULEUS
HYPOTHALAMUS CAUDALIS
ARREST OF CHANGE IN CHANGE IN INCREASED MOUTH OPEN INCREASE DOPAMINE

ONGOING HEART RATE RESPIRATION REFLEX JAW
BLOOD MOVEMENTS AND NOREPINEPHRINE
BEHAVIOR EXCITABILITY
PRESSURE
FREEZING CARDIOVASCULAR PANTING STARTLE FACIAL INCREASED VIGILANCE

CONFLICT TEST CONDITIONING RESPIRATORY INCREASE EXPRESSIONS
CER DISTRESS OF FEAR
SOCIAL INTERACTION IN PANIC
FIG. 8.5. Schematic diagram showing direct connections between the central nucleus of the amyg
dala and a variety of brain-stem target areas that may be involved in different animal tests of fear
and anxiety. CER = conditioned emotional response.
expected to be involved in many of the signs of fear or anxiety. Thus, the central nucleus
of the amygdala projects to a region of the central gray (Beitz, 1982; Post and Mai, 1980)
that has been implicated in fear in a number of behavioral tests (LeDoux et al., 1988;
Liebman et al., 1970). Projections to the lateral hypothalamus (Iwata et al., 1986b) or
direct projections to the dorsal motor nucleus of the vagus (Hopkins and Holstege, 1978;
Schwaber et al., 1982; Takeuchi et al., 1983; Veening et al., 1984) may be involved in
several autonomic measures of fear or anxiety. Projections of the central nucleus of the
amygdala to the parabrachial nucleus (Krettek and Price, 1978; Price and Amaral, 1981;
Takeuchi et al., 1982) may be involved in respiratory changes during fear, and electrical
stimulation of the parabrachial nucleus is known to alter respiratory rate (Cohen, 1971;
Bertrand and Hugelin, 1971). Direct projections to the trigeminal (Post and Mai, 1980)
and perhaps the facial motor nuclei may mediate some of the facial expressions of fear.
As outlined earlier, projections of the amygdala to the nucleus reticularis pontis caudalis
(Rosen et al., 1991; Inagaki et al., 1983) may be involved in fear potentiation of the
startle reflex. Finally, projections from the central nucleus of the amygdala to the ventral
tegmental area (Phillipson, 1979) may mediate stress-induced changes in dopamine turn
over in the frontal cortex (e.g., Thierry et al., 1976). Moreover, this projection might also
Hnk the central nucleus of the amygdala to the locus coeruleus (Deutch et al., 1986),
which itself has been implicated in fear and anxiety (Redmond, 1977) or increased vig
ilance and attention (e.g., Aston-Jones and Bloom, 1981).
4.2. FEAR PRODUCED BY ELECTRICAL STIMULATION OF THE AMYGDALA
Importantly, it has also been shown that electrical stimulation of the central nucleus
of the amygdala can produce a complex pattern of behavioral and autonomic changes
that, taken together, constitute a state that highly resembles a state of fear. Thus, electri
cal stimulation of the central nucleus of the amygdala produces a cessation of ongoing
behavior (Applegate et al., 1983; Gloor, 1960). As mentioned earlier, cessation of ongo
ing behavior is the critical measure of fear or anxiety in several animal models such as
the operant conflict test (Geller and Seifter, 1960), the CER (Estes and Skinner, 1941),
the social interaction test (File, 1980), and freezing itself (e.g., Fanselow and Bolles,
1979). Stimulation of the amygdala can also alter heart rate (Applegate et al., 1983; Kapp
et al., 1982) and blood pressure (Iwata et al., 1987; Morgenson and Calaresu, 1973), both
measures used to study cardiovascular conditioning. Electrical stimulation of the central
nucleus of the amygdala also alters respiration (Applegate et al., 1983; Harper et al.,
1984), a prominent symptom of fear especially in panic disorders. Electrical stimulation
of the amygdala also elicits jaw movements (Applegate et al., 1983; Gloor, I960; Ohta,
1984), which often accompany the fear response. Amygdala stimulation can also pro
duce gastric ulceration (Henke, 1980a, 1980b; Innes and Tansy, 1980; Sen and Anand,
1957), which may result from chronic fear or stress. As outlined earlier, electrical stim
ulation of specific parts of the amygdala increases the acoustic startle reflex, which is
elevated during fear. Finally, it has been reported in humans that electrical stimulation
of the amygdala elicits feelings of fear or anxiety, as well as autonomic reactions indic
ative of fear (Chapman et al., 1954; Gloor et al., 1981).
Taken together, the highly correlated set of behaviors seen during fear may result from
activation of a single area of the brain (the central nucleus of the amygdala), which then
projects to a variety of target areas, which themselves are critical for each of the specific
symptoms of fear or anxiety. Moreover, it must be assumed that all of these connections
are already formed in an adult organism, because electrical stimulation produces these
effects in the absence of prior explicit fear conditioning. Given this innate wiring dia
gram, it would seem most parsimonious to assume that a neutral stimulus will elicit a
state of fear when that stimulus comes to activate the amygdala after being paired with
an aversive stimulus. Thus, fear conditioning probably involves neural plasticity afferent
to, or in, the amygdala, rather than a change in its efferent target areas.
206 Μ . DAVIS
4.3. THE ROLE OF THE AMYGDALA IN FEAR ELICITED BY A CONDITIONED STIMULUS
Consistent with this interpretation, several studies have shown that a neutral stimulus
paired with aversive stimulation will now alter neural firing in the amygdala, especially
the central nucleus of the amygdala (Henke, 1983; Pascoe and Kapp, 1985). Moreover,
lesions of the central nucleus are known to eliminate or attenuate conditioned changes
measured by a cessation of ongoing behavior such as freezing (Iwata et al., 1986a),
reduced bar pressing in the operant conflict test (Shibata et al., 1986), or the conditioned
emotional response paradigm (KelUcut and Schwartzbaum, 1963; Spevack et al., 1975).
Lesions of the central nucleus also block conditioned changes in heart rate (Cohen, 1975;
Gentile et al., 1986; Kapp et al., 1979), blood pressure (Iwata et al., 1986a), or ulceration
induced by immobilization stress (Henke, 1980a, 1980b). Data outlined earlier indicate
that lesions of the central nucleus of the amygdala block fear-potentiated startle (Hitch
cock and Davis, 1986). Lesions of the amygdala are known to block several measures of
innate fear in different species (cf. Blanchard and Blanchard, 1972; Ursin et al., 1981).
This, along with a large literature implicating the amygdala in many other measures of
fear such as active and passive avoidance (for reviews, see Kaada, 1972; Sarter and Mar-
kowitsch, 1985; Ursin et al., 1981) and evaluation and memory of emotionally signifi
cant sensory stimuH (Bennett et al., 1985; Bresnahan and Routtenberg, 1972; Ellis and
Kesner, 1983; Gallagher and Kapp, 1978, 1981; Gold et al., 1975; Handwerker et al.,
1974;Kesner, 1982; Liang et al., 1985, 1986; Mishkin and Aggleton, 1981)compellingly
indicate a crucial role of the amygdala in fear.
4.4. CONDITIONED FEAR VERSUS ANXIETY
Clinically, fear is regarded to be more stimulus specific than anxiety, despite very sim
ilar symptoms. Figure 8.5 suggests that spontaneous activation of the central nucleus of
the amygdala would produce a state resembling fear in the absence of any obvious elic
iting stimulus. In fact, fear and anxiety often precede temporal lobe epileptic seizures
(Gloor et al., 1981), which are usually associated with abnormal electrical activity of the
amygdala (Crandall et al., 1971). An important implication of this distinction is that
treatments that block conditioned fear might not necessarily block anxiety. For example,
if a drug decreased transmission along a sensory pathway required for a conditioned
stimulus to activate the amygdala, then that drug might be especially effective in blocking
conditioned fear. However, if anxiety resulted from activation of the amygdala not
involving that sensory pathway, then that drug might not be especially effective in reduc
ing anxiety. On the other hand, drugs that act specifically in the amygdala should affect
both conditioned fear and anxiety. Moreover, drugs that act at various target areas might
be expected to provide selective actions on some, but not all, of the somatic symptoms
associated with anxiety. It is noteworthy in this regard that the central nucleus of the
amygdala is known to have a high density of opiate receptors (Goodman et al., 1980),
whereas the basolateral nucleus, which projects to the central nucleus (Smith and Mill-
house, 1985), has a high density of benzodiazepine receptors (Niehoff' and Kuhar, 1983).
In fact, local infusion of opiate agonists into the central nucleus of the amygdala blocks
conditioned bradycardia in rabbits (Gallagher et al., 1981), ulcers produced by cold-
restraint stress in rats (Ray et al., 1988a) and has anxiolytic effects in the social interac
tion test (File and Rodgers, 1979). Furthermore, local infusion of benzodiazepines into
the amygdala has anxiolytic effects in the operant conflict test (Nagy et al., 1979; Petersen
and Scheel-Kruger, 1982; Petersen et al., 1985; Scheel-Kruger and Petersen, 1982; Shi
bata et al., 1982; Thomas et al., 1985). Perhaps similarly, midazolam injected into the
amygdala antagonized pentylenetetrazol-induced stimulus discrimination (Benjamin et
al., 1987), another test thought to measure anxiety in animals (Lal and Emmett-Oglesby,
1983). Interestingly, anticonflict effects of benzodiazepines only seem to occur after local
infusion into the basolateral nucleus (the nucleus of the amygdala that has a high density
of benzodiazepine receptors) and not after local infusion into the central nucleus (Peter-
sen and Scheel-Kruger, 1982). Finally, endogeneous levels of several biogenic amines in
the amygdala may play a critical role in fear and anxiety, since treatments that alter local
amygdala levels of serotonin (File et al., 1981), dopamine (Ray et al., 1988b), or norepi
nephrine (Gallagher and Kapp, 1981; Liang et al., 1986) each can alter various measures
of fear in animals. Taken together, therefore, these results suggest that drug actions in
the amygdala may be sufficient to explain both fear-reducing and anxiety-reducing effects
of different drugs. Future studies employing local infusion of benzodiazepine or opiate
antagonists into the amygdala, coupled with systemic administration of various agonists,
may be able to determine whether local binding to receptors in the amygdala is necessary
to explain their anxiolytic effects. Eventually, local infusion of various drugs into specific
target areas may be used to evaluate whether highly specific anxiolytic actions are pro
duced. These results could then serve as a guide for eventually producing more selective
anxiolytic compounds.
Acknowledgments—This research was supported by NIMH Grants MH-25642 and MH-41298. NINCDS
Grant NS-18033, Research Scientist Envelopment Award MH-00004, Grant AFOSR-87-0336 from the Air
Force Office of Scientific Research and the State of Connecticut. My sincere thanks are extended to Lee Schles
inger, who tested many of the animals used for these studies, to Janice Hitchcock who evaluated the role of the
amygdala and its afferent and efferent projections on fear-potentiated startle, and to Jeffrey Rosen, who eval
uated the effect of electrical stimulation of the amygdala on startle. Thanks are also extended to Ariel Deutsch
and Robert Roth for collaboration on the PHA-L studies, and to Leslie Fields for help in typing the paper.
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File.S. Ε., editor.
(1991) Psychopharmacology cfAnxiolytics and Antidepressants.
PeiBunon Press, Inc. (New York), pp. 213-230
CHAPTER 9
EFFECT OF ANXIOLYTICS A N D ANTIDEPRESSANTS

ON EXTINCnON A N D NEGATIVE CONTRAST
CHARLES F . FLAHERTY
Department of Psychology, Rutgers University, New Brunswick, NJ USA
1. INTRODUCTION
The effects of anxiolytics and antidepressants on the behaviors that result from
removal of positive reinforcement (extinction) or from the reduction in the amount of
positive reinforcement (negative contrast) will be considered in this review. Typically, a
reduction in reinforcement to some nonzero value leads to an abrupt decline in respond
ing to a level below that of animals that have experienced only the lower value of rein
forcement. The difference in performance between animals for which reward has been
decreased and the unshifted control group, termed a successive negative contrast effect,
is usually transitory. That is, the shifted animals eventually recover to the level of the
shifted control group. Extinction, as befits the name, is relatively permanent.
There are a number of behavioral similarities between extinction and successive neg
ative contrast. For example, extinction generally proceeds more rapidly, and contrast is
larger, the greater the acquisition reward magnitude. Also, intermittent reinforcement in
acquisition retards extinction (Mackintosh, 1974) and reduces degree of negative con
trast (Flaherty, 1982). There are also similarities in drug action. In general, tranquilizers
slow the rate of extinction and reduce or eliminate negative contrast. There are, however,
differences in detail as a function of drug, drug administration routine, and behavioral
paradigm. The effects of antidepressants have been much less studied, but current evi
dence indicates that these agents have relatively little effect on extinction or negative
contrast.
Although there are many inteφretations of extinction and contrast, a common theme
is that an emotional response is a correlate of, and possibly causally related to, behaviors
consequent to reward elimination or reduction (e.g., Amsel, 1958, 1967; Gray, 1969,
1977, 1982, 1987; Mackintosh, 1974). To some extent, the study of the effects of drugs
on extinction and negative contrast is the study of drug effects in animal models of frus
tration, disappointment, and possibly anxiety.
2. EXTINCTION
2.1. ANXIOLYTICS
2.1.1. Continuous Reinforcement

The administration of benzodiazepines (BZ) or barbiturates retards extinction follow
ing a period in which each response has been reinforced. This result has been obtained
in a variety of circumstances. For example, chlordiazepoxide (CDP) (7.5 and 15 mg/kg)
increased responding in extinction after discrete-trial, lever-press training (Heise et al.,
1970) and diazepam (2 mg/kg) increased lever pressing in extinction after free-operant,
lever-press training (Thiebot et al., 1983). Similarly, extinction of a panel-pushing
response by pigs is retarded by diazepam (1 mg/kg; Dantzer, 1977a); and extinction of
runway behavior is retarded by CDP (5 mg/kg; Buckland et al., 1986; Feldon and Gray,
1981a; McNaughton, 1984). Extinction of bar pressing for brain-stimulation reward
213
214 C.F.FLAHERTY
(medial forebrain bundle) is also retarded by CDP (15 mg/kg; Gandelman and Trowell,
1968).
The effect of diazepam (2 mg/kg) on extinction is blocked by the BZ antagonist flu
mazenil (Ro 15-1788) (Thiebot et al., 1983), but flumazenil alone apparently has little
effect on extinction, except for a possible tendency for high doses (10 mg/kg or 32
mg/kg) to increase resistance to extinction (Hawkins et al., 1988; Thiebot et al., 1983).
Some evidence for the involvement of gamma-amino butyric acid ( G A B A ) in the
antiextinction effects of the BZs was provided by the finding that both Picrotoxin and
bicucuUine tended to block the effects of CDP on extinction. Picrotoxin administered
alone tended to facilitate extinction, possibly indicating an anxiogenic effect (Buckland
et al, 1986). However, the same report failed to find effects of the G A B A A agonist mus
cimol and the G A B A B agonist baclofen on extinction. These drugs were administered
systemically, however, and muscimol, at least, is known to penetrate the brain poorly in
such circumstances (Baraldi et al., 1979). Given this, muscimol (.00125 mg/kg, IP, one
of the doses used in the Buckland et al., study) was found to retard extinction in another
study (Fernandez-Teruel et al., 1985). However, these investigators also reported that
diazepam (2 mg/kg, IP) reduced responding in extinction, a result attributed by the
authors to a sedative effect. The diazepam effect, but not the muscimol effect, was
blocked by flumazenil (2 mg/kg).
The reason for the discrepancy between these two studies in regard to the effectiveness
of muscimol is not clear. Both used operant procedures, but there were many other pro
cedural differences, including the use of repeated acquisitions and extinctions in the
Buckland et al. paper, but only a single acquisition and extinction session in the 1985
study by Fernandez-Teruel et al.
In a variation on the usual procedure, Shemer and colleagues (Shemer et al., 1984)
administered CDP (5 mg/kg) for 12 days before the start of runway acquisition training,
at which time drug administration was discontinued. In extinction, animals that had the
prior experience with CDP administration were more resistant to extinction than con
trols. Thus, the effects of CDP administration were long-lasting and the "removal" of
the drug led to effects opposite to what would have been expected from administration
of the drug.
The effect of barbiturates on extinction generally parallels that of the BZs. Thus,
sodium amobarbital (17.5 or 20 mg/kg) increases resistance to extinction in the runway
(Barry et al., 1962; Dudderidge and Gray, 1974; Feldon et al., 1979; Ison and Rosen,
1967; McNaughton, 1984). However, barbiturates may not be as effective in operant
situations. Heise et al. (1970) reported that the phenobarbital (20 and 30 mg/kg) and
pentobarbital (5 and 10 mg/kg) did not increase resistance to extinction of a discrete-
trials lever-press response, and Griffiths and Thompson (1973) found that pentobarbital
(20 mg/kg) led to fewer total responses in extinction following training on an FR-20 bar-
press sechedule. However, these effects are probably not characteristic of lever pressing
per se, since Weissman (1959) reported that pentobarbital (8, 16, and 24 mg/kg)
increased responding during nonreinforced (5—) periods of a multiple schedule.
Ethanol also increases resistance to extinction (e.g., Barry et al., 1962; Devenport,
1984). Devenport suggested one possible mechanism underlying this increased persist-
ance, one that would seem to be maladaptive under natural circumstances. Rats shifted
to extinction or to a lower level of reward may normally enhance their exploration of
the environment, perhaps searching for the ''missing" reward (e.g., Elliott, 1928; Flaherty
et al., 1978, 1979). Evidence from Devenport's ethanol study suggested that the drug
(1.5, 2.0 g/kg of a 10% solution, but not a 0.75 g/kg dose) reduces exploratory behavior
in extinction and enhances perseveration to previously rewarded locations. Somewhat
similar results for both ethanol and diazepam were reported by Beck and Loh (1990). In
this study, both agents tended to increase arm reentries in extinction and both also
increased immobility, although neither drug had this effect in acquisition of the radial
maze task.
Extinction and negative contrast 215
Although ethanol, barbiturates, and the BZs have generally similar effects on the
extinction of positively reinforced responses, ethanol may differ from the other two drug
classes in its effects on the extinction of aversively motivated behaviors (see Mason
[1983] for a review of this literature; and see Dantzer [1977b] for a review of the effects
of BZs in these, and related, paradigms).
2.1.2. Intermittent Reinforcement

Animals trained with intermittent reward in acquisition are more resistant to extinc
tion than animals given continuous reinforcement in acquisition. This partial reinforce
ment extinction effect (FREE) is one of the most pervasive and most investigated find
ings in the behavioral literature (cf, Mackintosh, 1974; Gray, 1982, 1987). Although
there are many inteφretations of the FREE, one that has maintained currency over the
years is Amsel's frustration theory. In brief, the theory holds that the withdrawal of
reward is frustrating, frustration is aversive, and this aversiveness promotes a decline in
responding. Animals with a history of intermittent reinforcement experience frustration
during acquisition of an instrumental response, but the tendency to avoid the goal region
in anticipation of frustration is counterconditioned. That is, animals learn to persist in
responding because reward is sometimes present when frustration was anticipated and,
thus, anticipation of frustration itself becomes a cue that controls the instrumental
approach behavior. This learned tendency to continue responding in the presence of frus
tration then prolongs responding in extinction (Amsel, 1958, 1967).
Some of the evidence taken to support the hypothesis that extinction is frustrating or
aversive includes the observations that the stress hormone corticosterone is elevated in
extinction and that this elevation is related to the loss of reward and not to changes in
responding per se (Davis et al., 1976; Coe et al., 1983). In addition, animals will learn to
escape from a cue paired with nonreward when reward had been expected, but not from
a cue simply paired with nonreward in the absence of a reward expectation (Daly, 1974;
Mackintosh, 1974).
Evidence relevant to the presence of frustration in the acquisition of a partially rein
forced response is derived from the effect on the FREE of tranquilizers administered
during acquisition. This evidence suggests that CDP administered only during the acqui
sition period will facilitate extinction (Demarest and MacKinnon, 1978) and reduce or
eliminate the FREE (e.g., Feldon and Gray, 1981a, 1981b; Willner and Crowe, 1977).
The administration of CDP in both the acquisition and extinction phases will also reduce
or eliminate the FREE (Feldon and Gray, 1981a, 1981b; McNaughton, 1984). When
CDP is given only during the extinction period following intermittent reward in acqui
sition, the drug has an overall effect of increasing resistance to extinction (Demarest and
MacKinnon, 1978), but it does not eliminate the FREE (i.e., the drug has no differential
effect on continuously reinforced and partially reinforced groups (Feldon and Gray,
1981a, 1981b).
The BZ antagonist flumazenil (1,5, 10 mg/kg) had no effect on the FREE when given
in acquisition, extinction, or both (Hawkins et al., 1988).
As was the case with CDP, the administration of sodium amobarbital during acquisi
tion reduced or eUminated the FREE (Feldon et al., 1979; Gray, 1969; Gray and Dud-
deridge, 1971; McNaughton, 1984). Capaldi and Sparling (1971) varied the sequences of
rewarded and nonrewarded trials experienced by the rats during acquisition and reported
that the FREE was eliminated by sodium amobarbital (20 mg/kg) only when it was
administered under conditions in which a rewarded trial followed a nonrewarded trial,
within the same day, in acquisition. In this study, there were six trials per day with a
short (1.5-minute) intertrial interval. It should be noted that the Feldon et al. study found
clear effects of sodium amobarbital with a 24-hour intertrial interval.
Not all results conform to a pattern consistent with the conditioned frustration inter
pretation. Ison and Pennes (1969) provided evidence that the effects of sodium amobar-
216 C.F.FLAHERTY
bital may be state-dependent in that the FREE was reduced in animals that were given
the drug in acquisition and saline in extinction and in animals that were given saline in
acquisition and the drug in extinction. Somewhat different results were reported by Gray
(1969), who found that sodium amobarbital administered during the acquisition period
completely eliminated the FREE, whereas the change from saline in acquistion to drug
in extinction reduced, but did not eliminate, the FREE. Gray's study differed from that
of Ison and Pennes in that more acquisition trials were given by Gray.
Another divergence from the frustration account was reported by Ziff and Capaldi
(1971) who found no effect on the FREE of sodium amobarbital given in acquisition.
This experiment differed from the typical study in that there were only six acquisition
trials (three in the continuously rewarded group) and the intertrial interval was very short
(1.5 minutes).
An overview of the various effects of anxiolytics on the FREE suggests that the drugs
are particularly likely to be effective when there is extensive acquisition training and
when the intertrial interval is long (24 hours), and that CDF, particularly in doses higher
than 5 mg/kg, might have clearer and more consistent effects than the barbiturates.
The importance of the intertrial interval is further suggested by a comparison of the
results obtained by McNaughton (1984), who reported a large FREE in animals given
sodium amobarbital both in acquisition and extinction, and the results of Feldon et al.
(1979), who reported the complete absence of the FREE in animals that received the
drug both in acquisition and extinction. The McNaughton study employed an intertrial
interval of between 2 and 10 minutes, whereas the Feldon et al. experiment used a 24-
hour intertrial. McNaughton also reported that CDF given both in acquisition and
extinction would eliminate the FREE in a discrete-trial, lever-press task (FR-5) when
there was a 24-hour intertrial interval.
A further complication to this already complicated paradigm is that different drug-
related results are often obtained with different measures of performance. In particular,
the degree to which sodium amobarbital diminishes the FREE seems to be greater in the
goal region of a runway than in the start region (Feldon et al., 1979, Exp. 2). This out
come probably reflects the greater control of behavior by the anticipation of aversive
events as the time/place of occurrence of those events approaches (Miller, 1959; Gray,
1987). Probably related to this factor is the finding that animals given intermittent rein
forcement in acquisition often run more slowly than continuously reinforced animals in
the goal region, but not in earlier sections of the runway, where they may even run faster
(Goodrich, 1959; Haggard, 1959). Also, successive negative contrast effects are more
likely to be obtained in the goal region of the runway than in the start region (Raherty,
1982). The effects of intertrial interval and number of acquisition trials probably reflect
different degrees and kinds of leaming that take place during acquisition. For example,
long intertrial intervals and extended acquisition training may favor the development of
control over responding by cues associated with anticipated emotional events rather than
by cues associated with the rewarding event that occurred on the previous trial. In this
regard, it is interesting to note that lesions of the septum will eliminate the FREE if a
short acquisition period is given, but not if there is extensive acquisition training (Henke,
1974). Extensive discussions of the behavioral aspects of the FREE are presented by
Mackintosh (1974, chapter 8), Capaldi (1966, 1974), and Gray (1982,1987).
Finally, it should be noted that nonanxiolytics may have anxiolytic-like effects on the
FREE. For example, amphetamine given in acquisition on nonrewarded trials or on all
trials, abolished the FREE, exactly the pattem of results expected from an anxiolytic on
the basis of the fmstration account of extinction. In addition, amphetamine (1.5 mg/kg)
given in extinction increased resistance to extinction in animals with a history of inter
mittent reward, again an anxiolytic-hke effect (Weiner et al., 1985, 1987). However,
unlike an anxiolytic, amphetamine did not increase resistance to extinction in animals
with a history of continuous reinforcement (see also Dudderidge and Gray, 1974). The
inteφretation of the amphetamine data offered by Weiner et al. is that the dmg enhances
control over behavior by stimuU associated with reinforcement (thereby minimizing, in

the partial reinforcement procedure, control by stimuli associated with
nonreinforcement).
2.1.3. Energized Behaviors

Immediately after the onset of extinction, there is sometimes a temporary increase in
rate or force of responding. Fowler (1974) reported that CDP (5 mg/kg) actually
increased the response force in extinction, but had no effect in acquisition.
Another example of the invigorating effects of nonreward is the Amsel frustration
effect (FE). If rats are given random 50% reinforcement in the initial goal box and 100%
reinforcement in the terminal goal box of a double runway, they run faster in the second
alley after nonrewarded trials in the initial goal box than after rewarded trials in the
initial goal box. This difference in running speed is referred to as the FE and is usually
interpreted as reflecting an energized effect of frustrative nonreward (Amsel and Roussel,
1952; Gray, 1982). The FE is not influenced by sodium amobarbital (Freedman and
Rosen, 1969; Gray, 1969; Ison et al., 1967).
2.1.4. Consummatory Behavior

Mixed results have been found with the extinction of consummatory behavior. Soub
rie et al. (1978) reported increased licking on an empty tube that had previously con
tained water, if diazepam (2 and 4 mg/kg), CDP (8, but not 16 mg/kg), and lorazepam
(0.25 and 0.50 mg/kg) were administered. Similar results were obtained with CDP (8
mg/kg) by Bialik et al. (1982). Soubrie (1979) reported that these extinction reducing
effects of CDP were not blocked by Picrotoxin (1 mg/kg). Discrepant results were
obtained by Miczek and Lau (1975), who reported that empty tube licking was not
affected by CDP (5, 10, 20 mg/kg) when repeated daily cycles of 15 minutes of an empty
tube and 15 minutes of access to water were given. This latter result may be due to the
extended training given in this procedure and/or to the length of access to the empty
tube. Another study reported that licks on an empty tube that had previously contained
a 32% sucrose solution for 10 days were increased by CDP (8 mg/kg), but reliably so only
in the first 5 minutes, and most particularly in the first minute, of a 20-minute access
period (Haherty et al., 1989).
2.1.5. Discrimination Learning

The differential responding that develops when one stimulus ( S + ) is paired with
reward and another stimulus (S—) is paired with nonreward has been traditionally ana
lyzed in terms of the concurrent acquisition of a response tendency to S-h and extinction
of a generalized response tendency to S - (e.g., Flaherty, 1985; Mackintosh, 1974;
Spence, 1936), with perhaps the additional effects of emotional responses elicited by
nonreward under conditions similar to those in which reward is received (Amsel, 1962).
The effect of BZs is consistent with this interpretation and with the effects of anxiol
ytics on extinction. That is, discrimination learning is disrupted by the BZs, which gen
erally elevate responding to the nonreward stimulus (e.g.. Cole, 1988; Wedeking, 1969;
reviewed by Cole, 1986). The effect of CDP on S— responding is reversed by flumazenil
(Cole, 1983). Buckland et al. (1986) reported dose dependent effects of CDP on S -
responding in an operant discrimination. A 10 mg/kg dose of CDP elevated S— respond
ing, whereas a 5 mg/kg dose was ineffective (this same dose did enhance resistance to
extinction in a runway task) and 15 to 20 mg/kg doses had suppressive effects on
responding. The effect of the 10 mg/kg dose was not altered by the concurrent admin
istration of bicucuUine (1.5 or 1.75 mg/kg). Paradoxically, a 2 mg/kg dose of bicucuUine
enhanced the rate-increasing effects of CDP to both S + and S—. Picrotoxin also
enhanced the rate increasing effects of CDP, but in this case, only to responding under
218 C.F.FLAHERTY
S + conditions. These effects of Picrotoxin and bicucuUine, which differ quahtatively

from those obtained in the case of extinction in the straight runway, remain unexplained.
Muscimol (0.00125 and 0.25 mg/kg) had no effect on discrimination performance, but
baclofen (1 mg/kg) suppressed S— responding whether the drug was administered alone
or in combination with muscimol. Thus, baclofen, in this case, had anxiogenic-like
effects rather than anxiolytic effects.
Sodium amobarbital interferes with discrimination leaming in a mnway by elevating
S— responding without influencing S-h responding (Ison and Rosen, 1967). Fentobar-
bital has been shown to dismpt discrimination leaming in a number of operant tasks
(Weissman, 1959), including the acquisition of a conditional discrimination (Blough,
1957). Caul (1967) reported no interference with the acquisition of a mnway choice
leaming task by sodium amobarbital (20 mg/kg), but the dmg substantially interfered
with reversal leaming.
The susceptibility of discriminative behavior to dismption by anxiolytics may vary as
a function of stage of training. Dantzer (1977a) reported that when pigs were trained on
an FR-10 schedule that altemated with a time out (TO) period during which no rein
forcement was available, diazepam (1 mg/kg) enhanced TO responding early in training,
but not later in training. Similarly, Dantzer and Baldwin (1974) reported only a small
effect of CDF (5 and 10 mg/kg) on extinction responding during a multiple FR-8 extinc
tion schedule, but the dmg was administered after stable responding had been attained.
In his review, Cole noted that the dismptive effects of the BZs on discrimination training
were more apparent when the dmg was administered from the beginning of acquisition
than when the dmg was administered after a discrimination had developed.
Anxiolytics may be less likely to impair discriminative performance when both S +
and S— are available at the same time (see Cole, 1986; Gray, 1982, for reviews; the Caul
[1967] study mentioned above is an exception to this pattem). The failure of anxiolytics
to interfere with performance in these "simultaneous" discrimination tasks may be
because there is less of a demand that the subject inhibit responding.
2.2. ANTIDEPRESSANTS
2.2.1. Continuous Reinforcement

Studies investigating the effects of antidepressants on extinction of appetitive behavior
are sparse indeed. One early study compared the effects of two tricyclic antidepressants,
protriptyline (7 mg/kg) and chlorimipramine (7 mg/kg), and a monoamine oxidase
inhibitor, nialamide (100 mg/kg). None of the dmgs had an effect in the first extinction
session, but in a subsequent extinction session, given 3 days later without dmg admin
istration, the former protriptyline group, but no other dmg group, showed enhanced
resistance to extinction compared to vehicle controls. In a third extinction session, in
which protriptyline was again administered, the dmg group again showed enhanced
resistance to extinction compared to vehicle controls. There is no clear explanation for
this pattem of results (Ellison et al., 1975).
Amitriptyhne (10 mg/kg) and mianserin (15 mg/kg) administered chronically (12
days) increased resistance to extinction in a mnway task (Egan et al. 1979). In this exper
iment, there were 8 acquisition days and 4 extinction days—the effects of amitriptyline
were reliable on the last two extinction days, those of mianserin, on the first three extinc
tion days. Amitriptyline reduced food intake, but the effects of the dmg on extinction
were probably not due to altered food motivation since there were no effects of the dmg
on mnway behavior in acquisition, and mianserin had the opposite effect on food intake,
but the same effect on resistance to extinction.
Contrary to the above results, a different antidepressant, desmethylimipramine (DMI)
(7.5 mg/kg), was reported to have had no effect in the extinction of mnway or lever-
pressing tasks when the dmg was administered in both acquisition and extinction. How-
ever, resistance to extinction was increased when the drug was withdrawn 3 days before
the start of extinction (Willner et al., 1981). In a subsequent experiment it was found
that the DMI effect was obtained if animals were treated with the drug between acqui
sition and extinction (14 days), but not if they were treated during the 14-day acquisition
phase (Wilner and Towell, 1982). The explanation for these results is not obvious.
However, these effects of DMI were not replicated in a study by Lucki and Frazer
(1985), who reported no effects of DMI (10 mg/kg) when administered chronically dur
ing both acquisition and extinction of a bar-press response or when the drug (7.5 mg/kg)
was withdrawn 3 days before the start of extinction, as in the Willner et al. (1981) study.
The overall impression given by these few studies is that antidepressants have no sim
ple or orderly effects on extinction or that they have no replicable effect at all.
2.2.2. Differential Reinforcement for Low-Rate Responding

The paucity of data and the absence of systematic results concerning the effects of
antidepressants on extinction is relieved somewhat by a coherent series of studies in
which the effect of antidepressants on differential reinforcement for low-rate (DRL)
responding has been investigated. On a DRL schedule, animals must learn to withhold
responding until a specific interval of time has elapsed since their last response. After this
interval has passed, the next response is reinforced. Responses with a shorter interres-
ponse time (IRT) than the criterion value reset the interval timer. Conventional inter
pretation of performance on this schedule assumes that the animals must learn to dis
criminate the passage of time since their last response and to inhibit responses with
shorter IRTs.
A series of studies by Seiden and his colleagues (Seiden and O'Donnell, 1985) has
indicated a degree of sensitivity of DRL schedules to antidepressants. For example, imip
ramine, desipramine, and chlorimipramine, administered acutely, decreased response
rate and increased reinforcement rate when the rats were responding on a DRL 18-sec-
ond schedule (McGuire and Seiden, 1980). Similar effects were obtained on a DRL 72-
second schedule with a long list of tricyclic antidepressants, including desipramine (2.5-
20 mg/kg), imipramine (2.5-20 mg/kg), and amitryptyUne (2.5-20 mg/kg). Inspection
of the distribution of IRTs suggested that the drugs lengthened the average interresponse
time without changing the fundamental profile of the distribution, indicating that tem
poral discrimination was not degraded by the drug (O'Donnell and Seiden, 1983; Seiden
and O'Donnell, 1985).
Antidepressants from the monoamine oxidase (MAO) inhibitor group (e.g., iproniazid
5-40 mg/kg) and atypical antidepressants such as mianserin, zimeUdine, and trazadone
also improve efficiency in the DRL 72-second schedule. Recently, the MAO-A inhibitors
clorgyline and CGPl Γ305Α were found to enhance DRL 72-second performance, but
the MAO-B inhibitor (—) deprenyl did not have a performance enhancing eflfect (Marek
and Seiden, 1988).
It has also been recently reported that centrally acting jS-adrenergic agonists clenbu-
terol and prenalterol have an antidepressant profile in DRL 72-second performance,
reducing response rate and increasing reinforcement rate. These eflfects were blocked by
the iS-adrenergic antagonist propranolol (O'Donnell, 1987). Using a DRL 60-second
schedule. Sanger (1988) was unable to demonstrate eflfects of imipramine and mianserin
on DRL eflSciency. However, the a2-adrenoceptor antagonists idazoxan and yohimbine
increased responding and thereby reduced reinforcements, not an "antidepressant pro
file" in this paradigm.
Other agents have also been found to be without eflfect, or to interfere with, DRL
responding. The atypical antidepressants nomifensine and bupropion interfered with
DRL performance by increasing response rate and reducing the number of reinforce
ments obtained. Also, neuroleptics (e.g., chloφromazine 1-4 mg/kg, haloperidol (0.025-
2 mg/kg) and anxiolytics (e.g., CDP 2.5-20 mg/kg, pentobarbital 2.5-20 mg/kg) do not
220 C. F. FLAHERTY
improve performance on the DRL 72-second schedule (Seiden and O'Donnell, 1985).
On a shorter DRL 15-second schedule, the anxiolytics CDF (3 and 10 mg/kg) and phe
nobarbitone (30 mg/kg) interfered with performance by increasing response rate,
whereas chloφromazine tended to decrease response rate (Sanger and Blackman, 1975).
Given that antidepressants lower the level of spontaneous motor activity in rats
(Tucker and File, 1986), it would be tempting to attribute the improved efficiency pro
duced by these drugs on DRL performance to this motor effect. However, a number of
drugs that also lower response rate (e.g., chloφromazine, pimozide, haloperidol, ethanol,
and clozapine) did not increase the number of reinforcements obtained (O'Donnell and
Seiden, 1982, 1983; Seiden and O'Donnell, 1985). Thus, there is not a simple relation
ship between a drug's motoric effects and its effects on DRL behavior. It is possible that
antidepressants reduce level of responding without also disrupting the animals' timing
ability, and it is this combination that is necessary for the enhanced efficiency of DRL
responding.
2.2.3. Discrimination Learning

Imipramine (1,3, 10, and 17 mg/kg) was found to increase S— responding in pigeons
trained in a typical discrimination paradigm in which many S— responses were made
during the course of leaming, but the dmg had no effect on S - responding in birds that
leamed the discrimination without making errors (Terrace, 1963). Further effects of anti
depressants in discrimination leaming paradigms are considered below under the topic
of behavioral contrast.
3. CONTRAST EFFECTS
The term "contrast effect" refers to an exaggeration of the effect of reward on behavior
when an animal experiences two or more rewards (Dunham, 1968). Contrast effects are
pervasive in animal behavior, occurring in operant tasks, mazes, mnways, electrical stim
ulation of the brain, and consummatory behavior. All that seems to be necessary is the
opportunity for animals to experience two or more reward levels, within certain para
metric constraints, and an experiment appropriately designed to measure the contrast
effects (Bitterman, 1975, 1988; Flaherty, 1982; Mackintosh, 1974; Mellgren, 1972; Wil
liams, 1983).
As described above, successive negative contrast involves a downshift in reward (quan
tity, quality, or frequency—shifts in reward delay have provided equivocal results; Flah
erty, 1982; Mackintosh, 1974). Rats shifted from a large to a small reward in a mnway
generally show an abmpt decrement in mnning speed to a level slower than that of ani
mals that have experienced only the lower level of reward (e.g., Crespi, 1942; Mellgren,
1972). It is the slower mnning by the shifted animals, as compared to the unshifted ani
mals, that is referred to as a successive negative contrast effect (SucNCE). The degree of
SucNCE varies directly with the degree of difference between preshift and postshift
reward (Flaherty, 1982) and inversely with the time between the last experience with the
preshift reward and the first experience with the postshift reward (e.g., Gonzalez et al.,
1973).
Successive negative contrast also occurs in consummatory behavior. For example, rats
given access to 32% sucrose for 5 minutes a day consume more than rats given access to
4% sucrose. If the 32% group is shifted to 4% sucrose, there is a precipitous decline in
lick frequency to a level substantially below that of animals maintained on the 4%
sucrose solution. This negative contrast effect typically diminishes in degree over a 3 or
4 day postshift period (Lombardi and Flaherty, 1978; Vogel et al., 1968). As is the case
with contrast obtained in mnway behavior, degree of contrast in consummatory behav
ior varies directly with the degree of difference between preshift and postshift sucrose
(Flaherty et al., 1983) and inversely with the length of the retention interval between the
preshift and postshift periods (Ciszewski and Flaherty, 1977; Flaherty and Lombardi,
1977). Contrast is obtained in fi-ee feeding as well as deprived animals. The principal
difference between the two conditions seems to be faster recovery from contrast in
deprived animals (Riley and Dunlap, 1979).
Negative contrast in consummatory behavior also occurs if a sucrose solution is adul
terated with quinine (Flaherty and Rowan, 1989b) and with shifts in the concentration
of saccharin solutions. But, whether or not contrast is obtained in the latter procedure
may critically depend on the degree of difference between the two solutions and also
possibly on the absolute values of the solutions selected (Raherty and Rowan, 1986;
Vogel et al., 1968). Particularly robust contrast effects may be obtained by shifting rats
from sucrose solutions (20% or 32%) to a saccharin solution (0.15%) (C. F. Raherty, M.
Weaver, G. A. Rowan, and P. S. Grigson, unpublished data).
3.1. ANXIOLYTICS
3.1.1. Contrast in Instrumental Behavior

Negative contrast in the runway is reduced by CDP (5 and 10 mg/kg administered
chronically, Rosen and Tessell, 1970) and by sodium amobarbital (20 mg/kg; Rosen et
al., 1967; Ridgers and Gray, 1973).
3.1.2. Contrast in Consummatory Behavior

Chlordiazepoxide reduces negative contrast in consummatory behavior in a dose-
dependent manner, but there are paradoxes in the effectiveness of this drug. First of all,
the drug is largely ineffective when administered acutely on the first postshift day, but
doses of 6 or 8 mg/kg will eUminate contrast when administered acutely on the second
postshift day (Raherty, et al., 1980, 1990b). This difference in the effectiveness of CDP
across the first 2 postshift days is not due to a differential retention interval between the
last preshift day and the first or second postshift days, because the drug was found to be
ineffective when the first postshift day was given either 24 or 48 hours after the last pre
shift day (Raherty et al., 1986).
However, the animals must have some experience with the postshift solution before
the CDP becomes effective. When the animals were allowed access to the postshift 4%
sucrose for 20 minutes on the first postshift day, instead of the usual 5 minutes, CDP (8
mg/kg) became effective during the second 5 minutes, a period that would normally cor
respond to the second postshift day (Raherty et al., 1986).
This differential effectiveness of the drug early and late in the postshift period may be
related to a developing stress response. Based on the measurement of corticosterone lev
els, it seems that negative contrast is not stressful on the first position day, but it is stress
ful on the second postshift day (Raherty et al., 1985). One possibility is that the elevated
corticosterone on the second postshift day reflects an anticipatory frustration response,
or uncertainty, or anxiety, based on the experience on the previous day. Such an inter
pretation would leave the differential effects of CDP on negative contrast in this para
digm consistent with Gray's model, wherein it is assumed that anxiolytics reduce antic
ipatory or conditioned forms of anxiety, but not primary responses to aversive events
(e.g., Gray, 1982).
Another possibility is that the pattern of CDF's effectiveness and corticosterone ele
vation reflects the operation of different psychological processes underlying negative con
trast early and late in the postshift period. For example, the initial response to reward
reduction may involve the activation of a search process to locate the "missing" reward
(EUiott, 1928; Raherty et al., 1978, 1979). Subsequently, as the missing reward is not
located, the animal may enter a conflict period triggered by the drive to consume the
222 C. F . F L A H E R T Y
postshift reward (the animals are food-deprived) balanced against the preference for the
remembered preshift award. It may be this convict stage that is stressful and during this
stage that CDF is effective in reducing contrast.
Freliminary data have shown that rats downshifted in sucrose concentration in an
eight-arm radial maze show marked increases in entries to the seven arms that did not
contain sucrose during the acquisition period (Flaherty, 1991).
Two BZs other than CDF have been tested in the standard consummatory contrast
paradigm. Midazolam was found to reduce contrast in a dose-dependent manner
(Becker, 1986; Flaherty et al., 1990a) and, in an unpublished study, flurazepam had small
contrast-reducing properties, but only at relatively high doses (10 and 20 mg/kg).
A question might be raised regarding the role that the appetite-stimulating effects of
the BZs play (e.g., Cooper and Estall, 1985) in the recovery from consummatory negative
contrast. The evidence suggests that recovery from contrast is not simply related to appe
tite stimulation because (1) CDF is relatively ineffective in reducing contrast on the first
postshift day, even though it often has an appetite-stimulating effect on that day (e.g.,
Flaherty et al., 1986, 1990b); and (2) CDF has appetite-stimulating effects in two other
contrast procedures, anticipatory contrast and simultaneous contrast, but the drug does
not reduce contrast in either of these procedures (Flaherty et al., 1977; Flaherty and
Rowan, 1988).
Chlordiazepoxide (8 mg/kg) was found to reduce contrast in the Syracuse low-avoid
ance rats, but not in the Syracuse high-avoidance rats (Flaherty and Rowan, 1989a). It
was ineffective (4 and 8 mg/kg) in both the Maudsley reactive and nonreactive strains
(Rowan and Flaherty, in press).
Ethanol has a pattem of effectiveness much like that of CDF. That is, it reduces neg
ative contrast in a dose-dependent manner when administered on the second postshift
day, but is ineffective on the first postshift day (Becker and Flaherty, 1982). Ethanol and
CDF have additive contrast-reducing effects when administered conjointly (Becker and
Flaherty, 1983).
The contrast-reducing effect of ethanol was also potentiated by valproate, which by
itself, did not reduce contrast (Becker and Anton, 1990). This study also found that the
effects of valproate plus ethanol and the effects of ethanol alone were antagonized by
Picrotoxin (2 mg/kg). The effect of ethanol on contrast was also blocked by Ro 15-4513,
which by itself, did not influence contrast except at a high dose (3 mg/kg), where it tended
to enhance contrast (Becker, 1989).
Sodium amobarbital (17.5 mg/kg) also reduces SucNCE when given acutely during
the postshift stage (Flaherty et al., 1982; Flaherty and Driscoll, 1980). Unlike CDF, amo
barbital was equally effective when administered for the first time on either the first or
second postshift day, but the degree of contrast reduction was considerably less than that
produced by CDF. Amobarbital was not effective when given during both the preshift
and postshift periods.
Moφhine (4 and 8 mg/kg but not 0.5, 1, 2, and 16 mg/kg) reduced contrast and, like
sodium amobarbital, moφhine was equally effective on the first and second postshift
days. Also like sodium amobarbital, the degree to which moφhine reduced contrast was
small. Naloxone blocked the contrast-reducing effects of moφhine, but did not itself
influence contrast (Rowan and Flaherty, 1987).
The novel anxiolytic and 5-HT,A agonist buspirone (0.125, 0.250, 0.5, 1, 2, and 15
mg/kg) had no effect on negative contrast. The ineffectiveness of buspirone was dem
onstrated in acute studies, with the dmg administered on either the first postshift day,
the second postshift day, or both, and in chronic studies (24 days). A 15 mg/kg dose of
buspirone administered acutely suppressed sucrose intake, as did a chronic 2 mg/kg dose.
The buspirone analog gepirone (2.5, 5.0, 10.0 mg/kg), a more selective 5-HT,A antago
nist, also had no effect on contrast, but the highest dose reduced sucrose intake overall.
These data, plus the failure of the 5-HT2 antagonists ritanserin and ketanserin to influ-
ence contrast suggest the lack of a serotonergic involvement in this paradigm (Flaherty
etal., 1990a).
Clonidine, an «2 noradrenergic agonist, has been reported to have anxiolytic effects in
the potentiated-startle paradigm (Davis et al., 1988) and in the Geller-Seifter conflict test
(Kruse et al., 1980). However, it does not replicate the typical pattern of anxiolytics in
the partial reinforcement extinction effect (Halevy et al., 1986), and it has no effect at all
(3.12, 6.25, 12.5, 25.0, and 50.0 Mg/kg) on negative contrast in consummatory behavior
(Flaherty et al., 1987). Clonidine is also ineffective as an anxiolytic in the elevated plus-
maze (Johnston et al., 1988).
3.1.3. Discrimination Learning—Behavioral Contrast

The effects of several anxiolytics on S— responding during discrimination learning
were considered above. A corollary of the development in discriminative responding in
certain operant schedules is behavioral contrast. In the prototypical experiment, the ani
mals are initially trained with two cues, that are equally reinforced (e.g., variable interval
[VI] = 1 minute). After apparent asymptotic responding is reached, an extinction sched
ule is introduced in the presence of one of the cues; the reward associated with the alter
native schedule is left unchanged. Often, as responding declines in the presence of the
cue associated with extinction, it increases in the presence of the unchanged component.
This increase, relative to the preshift baseline, is referred to as positive behavioral con
trast. Much less frequently studied is negative behavioral contrast, wherein reinforce
ment density in one component is increased (made more favorable) and, as a conse
quence, responding in the unchanged component declines (for reviews, see Halliday and
Boakes, 1972; Dickinson, 1972; Mackintosh, 1974; McSweeney and Norman, 1979; Wil
liams, 1983).
Baltzer et al., (1979) used a variant of the typical behavioral contrast procedure and
found a substantial effect of anxiolytics. Thus, CDP (6 mg/kg), diazepam (6 mg/kg), and
amylobarbitone (15 mg/kg) substantially reduced both positive and negative behavioral
contrast, suggesting that these drugs may have a "mood leveling" effect and not just an
anxiolytic effect.
3.2. ANTIDEPRESSANTS
3.2.1. Contrast in Consummatory Behavior

Imipramine (8 and 16 mg/kg) has no effect on successive negative contrast when
administered acutely on the second postshift day or when administered subchronically
(7 days; H. C. Becker and C. F. Flaherty, unpublished data). The same doses also have
no effect on simultaneous contrast (Flaherty et al., 1977). To the extent that a seroto
nergic mechanism is involved in depression (e.g.. Cowan, 1990; WiUner, 1990; Price et
al., 1990), the evidence cited above regarding the effects of serotonergic agents on con
trasts further suggests that antidepressants are unlikely to influence negative contrast.
A summary of the relative effects on negative contrast in consummatory behavior of
a variety of drugs is presented in Fig. 9.1 in terms of a drug effectiveness ratio (DER).
The DER reflects the extent to which contrast was reduced by the most effective dose of
each drug, relative to vehicle control and relative to the preshift baseline lick frequencies
of each group.
3.2.2. Discrimination Learning—Behavioral Contrast

The administration of DMI (5 mg/kg) completely prevented the occurrence of positive
behavioral contrast when pigeons were shifted from a multiple VI — Γ, VI — Γ to a
multiple VI - Γ extinction schedule (Bloomñeld, 1972). However, this effect was not
specific to antidepressant action because chloφromazine (10 mg/kg) had exactly the
224 C. F. FLAHERTY
Ineffective Effective
III
CE
LU
Q
lifllisp
FIG. 9 . 1 . Drug effectiveness ratios (DERs) obtained in a series of dose/response studies utilizing
the consummatory negative contrast procedure. The values plotted represent the results obtained
with the most effective dose of each drug. The DER illustrates degree of contrast reduction by
obtaining the ratio of the difference between shifted and unshifted vehicle groups and the differ
ence between shifted and unshifted drug groups: the larger the DER value, the greater the contrast
reduction. The CDP plus ethanol values were obtained with 4.0 mg/kg CDP (an ineffective dose)
and 0 . 5 0 g/kg of a 15% EtOH injection (an ineffective dose). Scop = scopolamine; Pyr = pyril-
amine; Clon = Clonidine; Amph = amphetamine; Prop = propranolol; Meth = methysergide;
Imp = imipramine; Busp = buspirone; Morp = moφhine; Flur = flurazepam; ABS = amo
barbital sodium; ETOH = ethanol; CDP = chlordiazepoxide; MDZ = midazolam.
same effect. Blough (1957) also reported that chloφromazine (10 and 30 mg/kg) reduced
responding in a discrimination task. From an inspection of the published data, it appears
that the two drug groups responded at a consistently lower rate than the saline controls.
The failure of response elevation (behavioral contrast) to occur may have been related
to this motor-depressant function of the drugs rather than to any contrast-specific action
(cf Ortiz et al., 1971).
Baltzer et al. (1979), in their variant of the behavioral contrast paradigm mentioned
above, also investigated antidepressants. They found that pargyline (1 mg/kg) had no
effect on either positive or negative behavioral contrast, imipramine (3 mg/kg) had no
effect on positive contrast and a "weakly significant" effect in reducing negative behav
ioral contrast, and maprotiline (3 mg/kg) had a small, but reliable, effect in enhancing
positive behavioral contrast. Given that all of these effects were small and inconsistent
and that the profile produced by imipramine did not differ substantially from that pro
duced by chloφromazine (0.5 mg/kg), the most reasonable conclusion would seem to
be that antidepressants, at least those tested, do not have a particularly robust or mean
ingful effect on behavioral contrast.
4. SUMMARY AND CONCLUSIONS

By and large, anxiolytics retard the course of extinction and alleviate successive neg
ative contrast. The effects of antidepressants are less certain, but the little evidence that
is available suggests minimal effects in both paradigms.
Although anxiolytics retard extinction, extinction does take place under the influence
of the drugs and, in fact, the retardation is often not a major effect. Even the moderate
effect demonstrated in these studies may overstate the case, because a consistently non-
reinforced group injected with the drug is usually not included in these experiments. The
inclusion of such a control group would allow for an estimate of the rate-enhancing effect
of the drugs independently of any specific effects that the agents may have on extinction.
The moderate effect of emotion-related drugs no doubt reflects the likelihood that
extinction is largely an associative process—the disconñrmation of the expectancy that
a speciñc response will lead to reward (e.g., Mackintosh, 1974), or will lead to reward in
the speciñc extinction context (Bouton, 1991). The emotional response that accompanies
extinction may serve to hasten the process, but it is not the prime mover. In a naturalistic
setting, the animals would be expected to go elsewhere in search of sustenance and, in
this sense, an anxiolytic effect would be maladaptive if it enhanced persistence in an
unproñtable endeavor. This might be demonstrated by the use of multiple reward loca-
tions, such as in a radial-arm maze, as suggested by Devenport's results obtained with
ethanol (Devenport, 1984).
The procedural similarity between extinction and negative contrast is paralleled in the
effectiveness of the anxiolytics in moderating both and in the failure of antidepressants
to substantially influence either. A difference between extinction and negative contrast
is that there is stiU a reward available in the contrast paradigm, and in the usual restricted
experimental setting, an orderly endogenous recovery process in deprived animals seems
to drive them to the acceptance of the postshift reward (Flaherty, 1982 1991; Klinger,
1975, 1977). Anxiolytics accelerate this process, with varying degrees of effectiveness and
at varying points in the recovery cycle (Flaherty, 1991). Although there is no direct evi-
dence, inferences based on the interactions between BZs, barbiturates, and ethanol on
the one hand, and GABAergic mechanisms on the other hand (e.g., Biggio and Costa,
1986) would suggest a role for a GABAergic system in this endogenous recovery process.
Given the relative ineffectiveness of CDP in alleviating negative contrast in the period
immediately following the downshift, it may be that agents that are effective through the
modulation of a GABAergic system are inert until the endogenous recovery process is
initiated (Flaherty, 1991). Several of the procedural and empirical characteristics of the
successive negative contrast paradigm (i.e., the reduction in reward, the availability of
one or more less desirable alternatives, an orderly recovery process) suggest that it may
be useful as an animal model of disappointment.
The data showing that antidepressants do not systematically affect either contrast or
extinction complement other data showing that such agents also do not influence either
exploratory behavior or the reward value of food and water (see review by File and
Tucker, 1986). If it may be concluded that the current review also suggests a lack of a
disinhibitory effect of antidepressants in extinction, contrast, DRL responding, and dis-
crimination training (although the evidence is less clear in the latter case), then three
elements important for extinction and contrast (reward value, tendency to explore when
reward value is decreased, and inhibition of behaviors leading to the reduced reward) are
all apparently unaffected by antidepressants.
However, given that extended chronic treatment (e.g., 25 days) with imipramine (2.5
mg/kg), DMI (10 mg/kg), or amitriptyline (10 mg/kg) yields some, perhaps small, degree
of anxiolytic activity in other animal models of anxiety such as punished drinking and
novelty suppressed feeding (e.g., Fontana and Commissaris, 1988; Bodnoff et al., 1988),
perhaps more chronic studies in the extinction paradigm are needed before the book is
closed on this issue.
Other conclusions suggested by this review are as follows:
1. The extinction of consummatory behavior seems to share a common pharmacologi-
cal proñle with the extinction of instrumental behavior.
2. The evidence suggests that anxiolytics administered during acquisition of an inter-
mittent reinforcement schedule reduce or eUminate the partial reinforcement extinc-
tion effect, but these effects are not simple—being dependent on parametric consid-
erations such as the number of acquisition trials, the intertrial interval, and the
location in a runway in which the dependent measures are taken.
3. Behaviors that are "energized" in extinction have a different pharmacological proñle
from the behaviors that decUne in extinction.
226 C . F. FLAHERTY
4. Responding to the unrewarded stimulus in a discrimination leaming paradigm is

affected by anxiolytics in a fashion similar to standard extinction, but there are not
enough data to make a clear statement regarding the effects of antidepressants in this
paradigm.
5. Anxiolytics reduce behavioral contrast, possibly both positive and negative behavioral
contrast, and antidepressants have minimal and possibly nonspecific effects in this
paradigm. However, the conclusions regarding antidepressants are based on scanty
data.
6. Responding on a DRL schedule, particularly a DRL 72-second schedule, has a very
different profile from that of extinction or contrast, with antidepressants facilitating
behavior and anxiolytics being ineffective or dismptive of behavior. The facilitatory
effects of antidepressants may be related to a slowing of motor behavior without con
comitant dismptions of timing behavior and without the occurrence of response dis
inhibitory effects. There is some evidence that centrally acting /8-adrenergic agonists
have an antidepressant profile in this task.
Acknowledgments^PrepaTííXxon of this chapter was aided by a grant from the National Institute of Mental
Health (MH-40489) and a Charles and Johanna Busch Memorial Grant. The comments of Patricia Grigson,
Sandra File, and Kathleen Krauss on an earlier drañ are appreciated.
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File,S. Ε, editor.
(1991) Psychopharmacology ofAnxiolytics and Araidepressants.
PWeamon Press, Inc. (New York), pp. 231-250
Printed in the United Sutes of America
CHAPTER 10
EFFECTS OF PSYCHOTROPIC DRUGS ON THE BEHAVIOR AND

NEUROCHEMISTRY OF OLFACTORY BULBECTOMIZED RATS
H. VAN RIEZEN* AND B. E. L E O N A R D f
^Department of Pharmacology, CIBA-GEIGY Ltd., Basel, Switzerland; fDepartment of Pharmacology,
University College, Galway, Republic of Ireland
1. INTRODUCTION
One of the problems facing the neuropharmacologist attempting to identify potential
antidepressant drugs lies in the difficulty in obtaining an animal model capable of iden
tifying "antidepressant activity" rather than a specific property of the drug that may not
be causally related to its therapeutic eflfect (Leonard, 1975). The limitations of the first
rodent model of depression, in which the ability of a compound to reverse the hypo
thermia or ptosis induced by an acute dose of reseφine is considered an indication of
antidepressant activity, are legion (Spencer, 1977). The more recently introduced behav
ioral models, based on "learned immobility" (behavioral despair) (Porsoh et al., 1977),
have successfully shown that antidepressants of diverse chemical structure may specifi
cally motivate rodents to attempt to escape from an adverse environment (water). While
such tests oflfer many advantages over the reserpinized rodent test, they suflTer from the
disadvantage that they will also detect centrally acting drugs with anticholinergic and
antihistaminergic properties that lack antidepressant activity. Furthermore, it is well
established that such animal models suflfer the major disadvantage of selecting antide
pressants following acute administration, whereas in the clinical situation, most antide
pressants must be given for a period of several days, or even weeks, before the full clinical
advantage may become apparent. It is against this background that the search for behav
ioral models of depression must be considered. It is not the purpose of this short review
to consider the various types of animal models that have been developed to detect the
activity of antidepressants following their chronic administration. These have been the
subject of a monograph (Willner, 1984, 1985) and review (Leonard, 1985, 1989). This
review will concentrate on a description of the olfactory bulbectomized rat model that
was developed by Cairncross and colleagues (Cairncross and King, 1971; Cairncross et
al., 1975, 1977, 1979; Cairncross, 1984) to detect potential activity following the chronic
administration of antidepressants.
Hanin and Usdin (1977) have suggested that a relevant animal model of a psychiatric
disease should (1) simulate and reproduce the human syndrome; (2) show behavioral
changes that are similar to the symptoms of the disease; and (3) that these symptoms
should respond to drug treatment in a similar manner to that observed in man. There is
experimental evidence to suggest that the olfactory bulbectomized rat model may fulfill
most of these criteria. Evidence in favor of this view will form the content of this review.
Jesberger and Richardson (1986b) have implied that only the bilaterally olfactory bul
bectomized rat is an appropriate model for depressive illness in that the consequences of
the disruption in neuronal functioning caused by the lesion may be largely reversed by
chronic antidepressant treatment. An assessment of this view will be considered under
the following topics:
1. changes in behavior following bilateral olfactory bulbectomy;

2. behavioral tests that may be used to evaluate the effects of antidepressants in the bul-
231
232 Η . VAN RIEZEN A N D B. E. LEONARD
bectomized rat, and an assessment of the effects of chronic antidepressant treatment

in reversing these changes;
3. changes in central neurotransmission following bulbectomy.
2. CHANGES IN BEHAVIOR FOLLOWING BILATERAL OLFACTORY

BULBECTOMY
2.1. SURGICAL FROCEDURE
For optimal behavioral responsiveness, adult male rats (300-320 g) were used, because
experience has taught that stable behavioral changes, lasting at least 72 days postopera
tively, are found in animals of this weight range (O'Connor, 1986; Shibata et al., 1980;
Thompson and Thome, 1975). Before surgery is undertaken, it is essential that the ani
mals be handled by the experimenter several times daily for at least 3 to 4 days. It is also
essential to continue the daily handling of the animals throughout the postoperative
period in order to minimize the hyperirritability of the animals. The body weights of the
animals should be recorded at 3-day intervals. As can be demonstrated in Fig. 10.1, ani
mals lose weight following bulbectomy but generally recover within 2 to 4 weeks of
surgery.
Bilateral olfactory bulbectomy may be performed under tribromoethanol (250 mg/kg,
IF) or chloral hydrate (375 mg/kg, IF) anesthesia. While other investigators have used
pentobarbitone sodium as the general anesthetic (see Caimcross, 1984), we have found
the shorter-acting nonbarbiturate anesthetics to be superior to barbiturates or volatile
anesthetics in that the recovery rate following surgery is rapid and complications arising
from prolonged anesthesia or from the irritant effects of a volatile anesthetic are negli
gible (O'Connor and Leonard, 1988; Jancsar and Leonard, 1983). Approximately 96%
of all animals survive surgery using these nonbarbiturate anesthetics.
The surgical procedure is relatively simple and has been described in detail by Caim
cross (1984). The procedure may be summarized as follows. Following anesthesia, a mid
line longitudinal incision is made from 1 centimeter posterior to 1 centimeter anterior
to the bregma. Burr holes, 2 millimeters in diameter, are then drilled through the skull
8 millimeters anterior to the bregma and 2 millimeters to either side of the midline. The
olfactory bulbs are removed by suction (e.g., by means of a blunt hypodermic needle
attached to a water pump), care being taken to avoid damage to the frontal cortex. The
burr holes are then filled with hemostatic sponge, tetracycline powder applied to the
wound, and the skin closed with 7.5 millimeter surgical clips. Sham-operated rats are
treated in the same way, but the olfactory bulbs are not removed. Figure 10.2 illustrates
the position of the burr hole relative to the main anatomical features of the rat
skull.
After completing the behavioral studies on the bulbectomized rats, it is essential to
ensure that the surgical procedures have been correctly implemented. Thus, following
the decapitation of the animal at the end of an experiment, it is important to check that
the olfactory bulbs have been completely removed and that no damage to the frontal
cortex has occurred. The results obtained from any rats showing incomplete removal of
the bulbs or damage to the frontal cortex should be excluded from subsequent evalua
tion. It is worthy of note that the hyperirritability and aggressiveness of the bulbectom
ized rats is probably a consequence either of lack of handling of the animals prior to, and
following, surgery and/or due to damage to the frontal cortex (Leonard and Tuite, 1981;
Richman et al., 1972; Thome et al., 1973).
Following surgery, the animals are allowed to recover for at least 14 days before the
commencement of any experimental procedure. O'Connor et al. (1985) have shown that
the characteristic hyperactivity in the "open field" apparatus is present 7 days following
surgery. However, it is our experience that complete recovery of the animals (as shown
Drug efiects o n olfactory bulbectomized rats 233
Grams Adutt rats

Body Weight
Sham Operated
450 Η
Olfactory
Bulbectomized
425 Η
400 Η
375 Η
Sham Operated
350 -\ Olfactory
Bulbectomized
325 Η
300 4
275 Η
250 Η
225 Η
-Τ 1 1 r
5 10 15 20 25
Days after surgery
FIG. 10.1. Weight development after olfactory bulbectomy or sham operation of young and adult
rats.
by the wound healing and regaining of body weight) is generally attained only 14 days
following surgery.
In order to minimize the variance in the behavioral parameters being studied, it is
recommended that only four rats be consigned to a cage of dimensions 19 X 23 X 14
centimeters, each cage consisting of two bulbectomized and two sham-operated rats. This
procedure allows four treatments to be administered to each cage of rats, namely, sham
-h vehicle, sham H- drug, bulbectomized H- vehicle, bulbectomized H- drug.
It must be emphasized that good postoperative care of the animals is essential if valid
behavioral studies are to be undertaken. In addition to controUing for any wound infec
tion, it is necessary to maintain the ambient temperature at about 20**C, thereby ensuring
that the animals do not become hypothermic (Forster et al., 1980). Most experiments
reported by Leonard and co-workers (see references) have used animals that are kept
under a 12-hour light-dark cycle (lights on at 7 AM).
Following the procedures outUned above, it has been our experience over the last 10
years that over 90% of rats subjected to surgery may be used for experimental studies.
234 Η . VAN RIEZEN AND B . E . LEONARD
Olfactory bulbs
FIG. 10.2. Schematic illustration of the location of the burr holes relative to the main anatomical
features of the rat skull and position of the olfactory bulb in the cranial cavity.
2 . 2 . RELEVANT NEUROANATOMICAL CONNECTIONS BETWEEN THE OLFACTORY

BULBS AND THE LIMBIC REGIONS OF THE RAT BRAIN
The olfactory bulb is the first processing station of olfactory information. Olfactory
bulbectomy completely deprives the animal of olfactory signals. It is clear that this must
have important consequences for food finding, orientation in its environment, and risk
assessment, as well as partner and enemy identification; thus, olfactory bulbectomy
greatly disturbs the way in which the animal experiences itself and its world.
The centripetal fibers in the bulbs connect them to olfactory structures such as the
paraolfactory area, the uncus, the amygdala, and habenular nuclei. Moreover, there are
connections to the hypothalamus, the hippocampus, the reticular formation and to the
entorhinal, parahippocampal, and pyriform cortex (Shepherd, 1 9 7 2 ) . Olfactory bulbec
tomy, therefore, will produce an abrupt loss of input to these systems. However, also the
centrifugal fibers running toward the olfactory bulbs are damaged by bulbectomy, and
this causes widespread retrograde degeneration. A more detailed description of the con
nections of the olfactory bulb has been made by Jancsar and Leonard ( 1 9 8 3 ) and Leon
ard and Tuite ( 1 9 8 1 ) . They are illustrated diagrammatically in Fig. 1 0 . 3 .
Only an outline of the relevant features will be presented here. There are three general
aspects of the anatomy of the olfactory bulbs in the rat that are worthy of comment,
namely: ( 1 ) the primary connections of the olfactory and accessory bulbs; ( 2 ) the projec
tion field of the olfactory areas and amygdaloid nucleus complex via the stria terminalis
and ventral and dorsal amygdalofugal pathway; and ( 3 ) the medial forebrain bundle and
the amygdalo-tegmental projection.
Drug effects on olfactory bulbectomized rats 235
Olfactory System N. Accumbens Globus

main and accessory é>
GABA Pallidus
bulbs
GABA
GABA DA
Habenulae
5HT
Amygdaloid Complex 5HT, NE Ventral Tegmental Raphe Nuclei

Area
GABA DA
Mesencephalon
FIG. 10.3. Schematic representation of the afferent and efferent connections of the olfactory bulb.
GABA = gamma-amino butyric acid; DA = dopamine; NE = norepinephrine; 5-HT =
serotonin.
1. Centripetal fibers from the main olfactory bulbs project through the lateral olfactory
tract and pass to the dorsal and external parts of the anterior olfactory nucleus, the
antero-lateral part of the olfactory tubercle, the pyriform cortex, the nucleus of the
lateral olfactory tract, the cortical and medial amygdaloid nuclei and parahippocam-
pal cortex, and the lateral entorhinal area (Kosel et al., 1981). The accessory bulb
sends efferents to the amygdaloid nuclei. The fibers of the main bulb pass through the
accessory bulbs en route to the lateral olfactory tract. Thus, lesions of the accessory
bulb will inevitably involve eflFerents from the main olfactory bulbs. The accessory
olfactory tract at the rostral end of the amygdaloid region enters the stria terminalis
to terminate in the bed nucleus of the stria terminalis.
2. Neurons from the olfactory amygdala project to the medial and posteromedial cor
tical nucleus of the amygdala. This indicates that the amygdala is an area of olfactory
and vomeronasal integration, where the dual olfactory system proposed by Raisman
(1972), becomes partially integrated. These connections represent some of the prin
cipal efferent and afferent connections of the olfactory projection system that are in
direct association with the amygdaloid cortex.
The stria terminalis is the major eflFerent pathway to the telencephahc and dience
phalic structures from the amydaloid nuclei complex. The dorsal component of the
stria terminalis sends fibers to the bed nucleus of the stria terminaUs, the lateral septal
nucleus, the nucleus accumbens septi, the medial preoptic area, and the ventromedial
nucleus of the hypothalamus, and also carries afferent fibers from the amygdaloid
nuclear complex to the olfactory area. The ventral component of the stria terminaUs
projects to the bed nucleus of the stria terminaUs, the preoptic areas, the hypothala
mus, and the premammiUary areas. The commissural component connects the amyg
dalae of the two hemispheres through the anterior commissure.
3. The medial forebrain bundle (MFB) represents a dkect Unk between the paracentral
reticular nuclei of the mesencephalon, representing the primary mesoUmbic areas and
the forebrain Umbic system and is the major source of noradrenergic and serotonergic
terminals in the olfactory bulb. The MFB is in close association with the stria termin
aUs. The ventral portion of the descending MFB contains axons originating in the
anterior olfactory nucleus, the olfactory tubercle, the amygdala, and the piriform cor-
tex. Fibers from the piriform cortex and olfactory tubercle course medially through
the amygdala to enter the MFB, in which they continue to the dorsal mesencephalon.
The dorsal MFB, on the other hand, consists of fibers derived from the hippocampus,
septum, nucleus of the diagonal band of Broca, frontal cortex, and nucleus accum
bens. While the detailed distribution of these tracts is uncertain, there is evidence that
they project to the preoptic areas.
Although this is only a superficial account of the main connections of the olfactory
and the limbic system and its relationship with other areas of the brain, it does serve to
illustrate the complexity and diversity of the projection fields from the main and acces
sory bulbs.
The removal of the main olfactory bulbs and its disruptive effect on the efferent inputs
to the accessory bulb, as well as possible damage to the accessory bulb itself, will inevi
tably alter and disrupt the function of these areas.
Surgically induced lesions have traditionally been used to study the localization of
brain functions with little or no reference to secondary changes occurring as a conse
quence of the lesions being made. Schoenfield and Hamilton ( 1 9 7 7 ) have suggested that
surgical lesions (e.g., removal of the olfactory bulbs) produce more than just a disruption
of localized neuronal mechanisms and can initiate a series of secondary moφhological
and functional alterations that can occur at several levels of brain organization. Experi
mental evidence has clearly established that the disruption in behavior that occurs fol
lowing bulbectomy is unlikely to be due to anosmia per se, but probably involves mal
functioning of amygdalae and cortical areas (Tiffany et al., 1 9 7 9 ) .
Two main difficulties arise in relation to the removal of the olfactory bulbs. First, it is
not possible to clearly separate the main olfactory bulb and the anterior olfactory nucleus
unless the lesion is made rostrally in the bulb, in which case not all the projections from
the bulb would degenerate. Second, the projection pathways of the main bulb pass
through the accessory bulb en route to the main olfactory tract. Thus, following bilateral
bulbectomy, fibers emanating from the main and accessory olfactory bulbs degenerate.
In the future, progress in understanding the role of each of these systems in the bulbec
tomy model can only be expected from the use of methods similar to the ones used by
Cairncross et al. ( 1 9 7 9 ) , who produced more selective lesions by injecting specific neu
rotoxins in the olfactory bulbs and analyzing the resulting behavioral and neurochemical
changes.
2 . 3 . EFFECTS OF THE STRAIN OF RODENT U S E D ON THE BEHAVIORAL EFFECTS OF

BILATERAL OLFACTORY BULBECTOMY
Hyperactivity and irritability have been used to assess the differences in the behavioral
responsiveness of olfactory-bulbectomized rats of different strains (Leonard and Tuite,
1 9 8 1 ) . For irritable reactions, female rats are less reactive than males. Of the three strains
of rats studies. Hooded rats show less behavioral effects than Wistar and Wistar less than
Sprague-Dawley. For most of the experiments reported in this review, male Sprague-
Dawley rats were used. In our experience, this strain showed the most consistent behav
ioral changes in passive avoidance, exploratory behavior, etc., of the three strains that
were compared (Organon Scientific Development Group, internal report). Although,
probably, genetic differences between strains are reflected in the expression of the behav
ioral deficit after bulbectomy, the differences in social behavior of the three strains inves
tigated are likely to play an important role in this respect (see also Thome et al., 1973;
Thompson and Thome, 1 9 7 5 ) ,
2 . 4 . CHANGES IN BEHAVIOR FOLLOWING OLFACTORY BULBECTOMY
The North American psychologist Watson ( 1 9 0 7 ) was probably the first investigator
to comment on the behavioral effects of bilateral bulbectomy in rats. Like him, most
experimenters have reported an increased irritability and aggressiveness in rats following
bulbectomy. More recent evidence suggests that hyperemotionality and aggressiveness

do not appear in rats handled frequently before and during the postoperative period and
are not primary effects of the lesions per se (Richman et al., 1972; Thome et al., 1973;
Leonard and Tuite, 1981).
An increased incidence of muricidal (mouse-kiUing) behavior has also been reported
to occur following bilateral bulbectomy (Albert et al., 1981). While such behavior is rel-
atively uncommon in most strains of intact rats the frequency of such behavior is
reported to increase dramatically following bulbectomy. However, the pattem of the
mouse-kiUing behavior is quite different to that seen in spontaneously muricidal strains.
Thus, the latency to attack the mouse following bulbectomy is much longer and the
killing is less efficient (e.g., more bites are inflicted by the bulbectomized rat compared
to the spontaneously muricidal rat). This behavior of the bulbectomized rat has been
termed "irritable aggression" and has been discussed in more detail elsewhere (Leonard
and Tuite, 1981). Moreover, when spontaneously muricidal rats are bulbectomized,
muricidal behavior is considerably attenuated.
An increased incidence of cannibalism in female rats following bulbectomy, intermale
aggression, territorial aggression, and an alteration in sexual behavior (Lumia et al.,
1987; Tyler and Gorski, 1980) all suggest that such behavioral abnormalities are caused
by the absence of more than just the essential olfactory cues following the lesions (see
Leonard and Tuite, 1981, for details).
In rodents, bulbectomy is followed by an increase in locomotor activity in a novel
environment (Sieck, 1972; Richman et al., 1972; Jancsar and Leonard, 1983). These
changes in locomotor activity and in other types of behavior are not likely to be due to
anosmia, because the reversible inhibition of the functioning of the olfactory nerve in
the nasal cavity by zinc sulfate solution does not result in such behavioral changes. The
hyperactivity shown by bulbectomized animals in the open held must be considered as
a failure of adaptation and risk assessment (Blanchard and Blanchard, 1983) and this
"deñcit" can be attenuated by the chronic administration of antidepressants (Table
10.1). It should be noted that dmgs lacking antidepressant activity do not attenuate this
behavior and neither wiU antidepressants following their acute administration (Leonard,
1984).
Differences have been found between the active and passive avoidance performance
of the bulbectomized rat; thus, bulbectomized rats have been shown to be deficient in
the acquisition of a passive avoidance task, but superior to controls in the acquisition of
a two-way active avoidance task (shuttle box) (Thomas, 1973; Sieck and Gordon, 1972;
Sieck et al., 1974). However, other investigators have shown that one-way active avoid-
ance leaming is deficient after bulbectomy (Caimcross and King, 1971; King and Caim-
cross, 1974; Marks et al., 1971). Deficits in mnway behavior for a food reward (Egan et
al., 1979; Jancsar and Leonard, 1984) and in conditioned taste aversion also occur in the
bulbectomized rat. The main differences between the bulbectomized rat and its sham-
operated control are summarized in Table 10.2.
Changes in social (e.g., increased aggression, both territorial and, in general, irritabil-
ity), sexual (e.g., matemal and mating behavior), and nonsocial behaviors such as explor-
atory activity of a novel environment have been shown to occur following bulbectomy
(see Leonard and Tuite, 1981 for details). Recently, O'Connor and Leonard (1988) have
shown that, when placed on a holeboard apparatus, the ambulation score, head dipping
(a measure of the exploratory activity indicated by the animal investigating a hole), and
grooming were significantly reduced in bulbectomized rats when compared with their
sham-operated controls.
Furthermore in a novel test for neophobia, bulbectomy caused a total elimination of
the neophobia response when compared with sham-operated controls. In this test, which
was developed by Broekkamp et al. (1986b), glass marbles are introduced into the home
cage of the bulbectomized or sham-operated animal. The marbles provide the aversive
stimulus and the time taken for the animal to displace and/or scatter the marbles from
their initial position in the cage was used as a measure of the neophobic response.
238 Η. VAN RIEZEN AND B. E. LEONARD
TABLE 10.1. Summary of Chronic Effects of Various Antidepressants and Other Psychotropic Drugs on
Behavior of the Bulbectomized Rat in the Open-Field Apparatus
Dose Dose
Drug (mg/kg IP) Activity Drug (mg/kg IP) Activity
Antidepressants Novel or putative antidepressants

Desipramine 5.0 Progabide 150.0
Imipramine 5.0 Sulpiride 20.0
Amitriptyline 5.0 E-flupenthixol 2.0 -1-
Lofepramine (1) 30.0 — Alprazolam 1.5
Nomifensin 3.0 -H Adinazolam 2.5
4-hydroxy nomifensin 5.0 4- Salbutamol 10.0 -1-
Mianserin racemate 5.0 Other compounds
enantiomer 5.0 + Gamma vinyl GABA 75.0
(—) enantiomer 5.0 — Baclofen 5.0
Desmethyl mianserin 5.0 — Diazepam 2.5 —
8-hydroxy mianserin 5.0 — Reseφine 0.1 —
Mepirzepine racemate 2.0 -h Clonidine 0.2 —
(-f) enantiomer 2.0 THIP 10.0 —
( —) enantiomer 2.0 — Yohimbine 1.0
Sertraline 5.0 Methysergide 3.0
Fluvoxamine 10.0 — Phenobarbitone 20.0 —
Fluoxetine 10.0 — Chlorpromazine 10.0 —
Trazodone ( 2 )
Moclobemide ( 3 )
50.0
10.0 -1-
Atropine 5.0
-
Pargyline 20.0 —
All drugs given at doses indicated in parentheses once daily for at least 14 days prior to behavioral testing. (1) at the dose of
30 mg/kg it is possible that the activity is due to the desipramine metabolite, lower doses inactive; (2) not active at lower doses;
(3) animals were treated twice daily with this dose; + drug active, attenuation of hyperactivity; - drug does not attenuate
hyperactivity.
For references see Jancsar and Leonard (1983); O'Connor and Leonard (1987, 1988); O'Connor et al. (1985); Earley and
Leonard (1985); Butler et al. (1988); Jesbeiger and Richardson (1985, 1986a, 1986b).
Whereas sham-operated animals either completely, or almost completely, buried most

of the marbles, bulbectomized animals did not attempt to bury any of the marbles. From
this experiment, it would appear that the neophobic response is markedly attenuated
following bilateral bulbectomy.
In addition to the gross behavioral changes that occur following bulbectomy, physio
logical changes have also been reported. Such changes include decreased heart rate and
blood pressure (Kawasaki et al., 1980a, 1980b), decreases in the frequency of REM sleep
(Sakurada et al., 1976; Sakurada and Kitara, 1977), altered thermoregulation (Forster et
al., 1980), and polydipsia (Wren et al., 1977). The changes in thermoregulation and pos
sibly in fluid intake may be indicative of altered hypothalamic function, which has also
been implicated by Kawasaki et al. (1980a, 1980b); these investigators reported that bul-
TABLE 10.2. Some Behavioral Tests in Which Bulbectomized Rats Have Been Shown To Differ from Sham-
Operated Controls
Type of Change by
Type of Behavior Bulbectomy Reference
Muricidal behavior Cain, 1 9 7 4

Cannibalism Fleming and Rosenblatt, 1 9 7 4
Sexual behavior — Larsson, 1971
Exploratory behavior -1- Sieck, 1 9 7 2
Passive avoidance learning — Thome etal., 1973, 1 9 7 6
Active avoidance learning ± Thome etal., 1 9 7 3
Extinction of conditioned taste aversion — Jancsar and Leonard, 1 9 8 4
Eating pattern* ± La Rue and Le Magneu, 1 9 7 2
Rapid eye movement sleep — Sakurada et al., 1976
*Eatíng pattern disrupted following bulbectomy: quantity of food consumed reduced, but frequency of eating is increased.
( + ) Increased and ( - ) decreased relative to sham-operated controls.
bectomized rats exhibited enhanced emotional response to stimulation of the posterior

hypothalamus. It is not without interest that this altered hypothalamic responsiveness
developed gradually over a period of 10 days, which may be suggestive of the develop-
ment of neurotransmitter receptor hypersensitivity, in this and/or other brain regions, as
a consequence of the lesion.
O'Connor and Leonard (1986) have also reported changes in other physiological vari-
ables as a consequence of bilateral bulbectomy. Thus, hypematremia and hyperchlor-
hydria occur in both young (7-week old) and adult (12-week old) rats following bulbec-
tomy. This effect is maintained for at least 4 weeks after surgery and is unaffected by
chronic antidepressant treatment. Serum potassium and calcium levels were unaffected.
Decreased sensitivity of the osmoreceptors to elevated sodium (Chiaraviglio, 1969), tha-
lamic malfunctioning due to the brain lesion (Sweet et al., 1949), blood loss, or hypoten-
sion may help to explain these changes in electrolyte balance.
3. BEHAVIORAL TESTS USED TO EVALUATE EFFECTS OF FSYCHOTROFIC

DRUGS ON BULBECTOMIZED RATS
3.1. IRRITABILITY
Irritability and an enhanced reactivity to sudden handling or environmental change

have been reported to occur in bulbectomized rats soon after surgery (Douglas et al.,
1969). Such behavior often makes experimentation difficult and occasionally leads to the
experimenter being bitten by the animal. However, as has already been stated, regular
handling of the animal by the experimenter completely removes this abnormal behavior.
In order to undertake a study of the effects of psychotropic drugs on the irritability of
the bulbectomized rat, the animal should be isolated following surgery and subjected to
the minimum of handling. The degree of irritability may be evaluated by placing the rat
on a flat surface and scoring its response to a puff of air blown sharply through a straw
onto the back of its neck, prodding the animal with a blunt rod, and subjecting it to a
sudden loud noise (e.g., hand clap). The response, as well as the subsequent freezing time,
can then be scored according to the schedule of Caimcross et al. (1978a).
3.2. M u R i c i D E BEHAVIOR
Most strains of rats seldom spontaneously kill mice placed in their home cage. Follow-
ing bilateral bulbectomy, however, most aduh male rats wiU develop mouse-killing (mur-
icidal) behavior during the week following surgery (Ueki, 1982; Lloyd et al., 1982). Stud-
ies by Albert et al. (1985) have shown that the behavior pattem exhibited by the
bulbectomized rat when kiUing a mouse is quite different to that shown by spontaneously
muricidal strains and has been referred to previously in this review. Furthermore, stim-
ulation of the locus coemleus of spontaneously muricidal rats signiñcantly attenuates the
behavior, whereas such stimulation in bulbectomized animals has little effect (Yama-
moto et al., 1982). This suggests that the neurological substrate subserving muricide
behavior differs between the surgically induced and spontaneously muricidal rat.
In an attempt to deñne the neuroanatomical site that is responsible for muricidal
behavior in bulbectomized rats, Ueki (1982) and Horovitz (1967) have shown that
lesions of the cortico-medial amygdala attenuated such behavior, as did the localized
administration of antidepressants to this region of brain. In spite of this, there is some
doubt that muricidal activity is a useful parameter of the bulbectomized rat model of
depression or for the detection of antidepressants, since anxiolytic dmgs are also active
in attenuating the muricidal response of bulbectomized rats (Takaoka et al., 1988).
Muricidal behavior that occurs foUowing bilateral olfactory bulbectomy has been cor-
related with a deficit in central noradrenergic transmission. In support of the hypothesis
is the evidence that desipramine, which shows some selectivity in inhibiting noradrena-
line uptake, blocks muricidal behavior in bulbectomized rats (Hara et al., 1983). Fur-
thermore, Yamamoto et al. (1982) showed that electrical stimulation of the locus coe
ruleus suppressed muricidal behavior in bulbectomized rats, which led to the conclusion
that noradrenergic stimulation of the hypothalamus probably occurs via the medial fore
brain bundle. This was confirmed by finding that noradrenaline concentrations in the
medial amygdala, ventromedial, and lateral hypothalamus were normalized by desipra
mine treatment of those bulbectomized rats that exhibited muricidal behavior (Iwasaki
et al., 1986b). More recently, Iwasaki et al. (1989) showed that 7 days following bulbec
tomy, most rats exhibited muricidal behavior that was correlated with a decrease in the
release of noradrenaline from the lateral hypothalamus and an increase in the release of
dopamine from the ventromedial hypothalamus. Whether these changes in the bulbec
tomized rat exhibiting muricidal behavior are also connected to other forms of abnormal
behavior following bulbectomy is unclear, although there is experimental evidence to
show that bulbectomy results in a reduction in the turnover of noradrenaline in the lim
bic region of the brain (Janscar and Leonard, 1984) and a reduction in noradrenaline
levels in the piriform cortex, which was reversed by subchronic treatment with amitrip
tyline (Cairncross et al., 1979).
3.3. PASSIVE AVOIDANCE DEFICITS
Several groups of investigators have shown that bulbectomized rats are superior to
sham-operated animals in the acquisition of a two-way active avoidance task but inferior
to the control group in acquiring a passive avoidance task.
Two experimental approaches have been used to demonstrate a passive avoidance def
icit in bulbectomized rats. The most common one utilizes a shock box, whereby the rat
has to remain on a wooden platform in order to avoid a mild foot shock (Broekkamp et
al., 1980). An alternative procedure utilizes a two-compartment box where the rat has to
learn to remain in one compartment in order to avoid a foot shock (van Riezen et al.,
1976). Another modification of the passive avoidance test is to determine the number of
shocks delivered to the tongue of a fluid-deprived rat before it ceases to lick the electrified
spout of a drinking bottle (Wren et al., 1977). In all these experimental situations, the
bulbectomized rat shows a passive avoidance deficit, an effect clearly demonstrable
within 7 days of surgery, but likely to be reduced within 4 weeks of the operation (Broek
kamp et al., 1986a). These changes were found to be more marked in the young (3-week
old) than in the aduh (3-month old) animals. In general, little correlation was found
between the hyperactivity of the bulbectomized animals in the open-field apparatus and
their deficit in passive avoidance behavior.
3.4. ACTIVE AVOIDANCE DEFICITS
King and Cairncross (1974) demonstrated that bulbectomized rats showed a deficit in
the acquisition of a one way active avoidance task by using a two-compartment box, the
rat being placed in one chamber and required to move into the second chamber within
a predetermined time in order to avoid a mild foot shock. This deficit in active avoidance
behavior was found to be corrected by chronic amitriptyUne treatment; untreated bul
bectomized rats required more foot shocks before they learned to move into the second
chamber (Cairncross et al., 1973). By contrast, bulbectomized rats perform better than
their sham-operated controls in a two-way avoidance task (shuttlebox).
3.5. DEFICITS IN APPETITE-MOTIVATED CONDITIONING
In these tests, food peUets or water may be used to motivate food- or fluid-deprived
rats to learn a novel task. In general, olfactory bulbectomized rats show a deficit in acqui
sition of the learned response.
Leonard et al. (1979) and Wren (1978) showed that food-deprived bulbectomized rats
were slower than their controls in running down a straight alleyway to reach a food
reward. In some experiments the extinction of the leamed response by the bulbectomized
rats was slower than that of the sham-operated controls. Phillips (1970) showed that bul
bectomized rats performed poorly in a four-choice visual discrimination box in which
the animals had to leam to discriminate between different black-white patterns in order
to obtain a food reward. Bulbectomized rats even following 10 sessions of 100 trials,
failed to leam the correct responses, whereas their controls made 80 to 90% correct
choices.
3.6. EFFECTS ON SPATIAL LEARNING
The Morris water maze has been widely used as a test of spatial leaming in rats. In
this experimental procedure, rats must leam to utilize the visual cues provided in order
to locate a partially submerged platform; the water temperature is maintained at ITC to
prevent hypothermia. Hippocampal lesions or prior treatment with such amnestic dmgs
as scopolamine significantly delay the ability of the rat to locate the platform. Jancsar
(unpubUshed) has shown that bulbectomized rats do not differ from sham-operated ani
mals in the acquisition or retention of this leaming task. However, bulbectomized rats
are deficient when tested in the radial maze (Hall and Macrides, 1983).
3.7. HYPERACTIVITY IN THE OPEN-FIELD APPARATUS
Hyperactivity following bilateral olfactory bulbectomy was first reported by Watson

(1907), and although later investigators suggested that this was due to damage to non-
olfactory stmctures (Cain, 1974), studies by Caimcross et al. (1979) and ourselves (Janc
sar and Leonard, 1983) have shown that the hyperactivity can occur in the absence of
damage to any stmctures other than the main and accessory olfactory bulbs.
Olfactory bulbectomized rats consistently show a hyperactivity when they are sub
jected to a stressful novel environment such as the open-field apparatus (O'Connor et
al., 1985; Jancsar and Leonard, 1983). No consistent changes in the behavior of bulbec
tomized rats can be detected when they are placed in a novel, but relatively unstressful,
environment such as the holeboard apparatus or the Y-maze (O'Connor et al., 1985).
Thus, it would appear that bulbectomized rats have a stronger stress responsiveness than
their sham-operated controls.
No changes have been reported in the activity of bulbectomized rats compared with
their sham-operated controls when their activity is measured in activity cages (Jancsar,
unpubhshed).
3.8. CONDITIONED TASTE AVERSION
Animals leam to avoid a novel food or liquid if they become ill within a short period
of samphng (bait shyness). The time it takes to extinguish the association between the
aversive stimulus and the food or fluid reward can be used as a measure of leaming
(Gaston, 1978). Following bilateral ablation of the olfactory bulbs, a dismption of this
associative leaming occurs. Thus, bulbectomized rats extinguish the leamed aversion
towards the conditioning stimulus much faster than sham-operated controls (Jancsar and
Leonard, 1981a, 1981b).
Changes in the activity of the serotonergic system in limbic regions of the brain, par
ticularly in the amygdaloid cortex, may underlie the deficit in conditioned taste aversion
behavior of the bulbectomized rat. Lorden and Margules (1977) have shown that changes
in the activity of the serotonergic system, particularly in Umbic regions, may subserve
such leaming behavior.
242 Η . VAN RIEZEN A N D B. E. LEONARD
3.9. OTHER BEHAVIORS
Bulbectomized rats do not show consistent differences from their sham-operated con
trols with regard to changes in their behavior in the Y-maze (O'Connor et al., 1985),
holeboard (O'Connor and Leonard, 1988), or in the learned immobility test (Gorka et
al., 1985), which was devised by Porsolt et al. (1977) to detect potential antidepressants.
As has been mentioned earlier, bulbectomized rats fail to show an aversive response
to a novel object placed in their cage (i.e., diminished neophobia) (O'Connor and Leon
ard, 1988). When given a choice of different types of novel food, satiated bulbectomized
rats also showed a different food choice from their sham-operated controls (O'Connor et
al., 1985).
3.10. EFFECTS OF ANTIDEPRESSANTS ON THE BEHAVIOR OF BULBECTOMIZED RATS
Cairncross and King (1971) were the first to show that the deficit in passive avoidance
learning by bulbectomized rats could be corrected by the chronic administration of ami
triptyline. These findings were confirmed in a later study (Cairncross et al., 1975), and it
was also shown by these investigators that the behavioral deficit was associated with a
decrease in the concentration of noradrenaline in the frontal cortex. Van Riezen et al.
(1976) extended the approach of Cairncross et al. (1975) by comparing the effects of
amitriptyline with the novel tetracylic antidepressant mianserin and with a diverse group
of psychotropic drugs that included chloφromazine, amphetamine, chlordiazepoxide,
and lithium chloride in a varied battery of tests such as the open-field test, avoidance
learning in a two-compartment box, irritability, and appetitive learning. These investi
gators found that only the antidepressants produced a normalization in the behavior of
the bulbectomized rats. Amphetamine also normalized the avoidance behavior, but
enhanced the irritability of the animals and further increased their hyperactivity in the
open field. Van Riezen et al. (1976) concluded that the bulbectomized rat was a useful
model for the detection of antidepressant activity, but that a battery of tests should be
used to distinguish antidepressants from drugs with stimulant properties.
Cairncross (1984) reported that the chronic administration of viloxazine (see also
Greenwood et al., 1982), nomifensin, and iprindole improved the passive avoidance def
icit of bulbectomized rats, while Noreika et al. (1981) reported that both buproprion and
doxepine were also effective in this test. Joly and Sanger (1986) reported that fluoxetine
was effective in normalizing the passive avoidance deficit following a single dose. This
effect of fluoxetine was blocked by prior administration of the serotonin receptor antag
onists metergoline. Other investigators have also reported that fluoxetine was active in
normalizing the passive avoidance deficit following a single dose (Broekkamp et al.,
1980), an effect shared by the serotonin releasing agent fenfluramine and by the serotonin
uptake inhibitor clomipramine (Lloyd et al., 1982). Butler et al. (1988), however, have
found fluoxetine to be inactive in reversing the hyperactivity of bulbectomized rats in
the open-field apparatus. It is not without interest that Joly and Sanger (1986) also failed
to find fluoxetine active in the open-field test, even though it exhibited activity in the
passive avoidance test following a single dose.
Of the putative antidepressants studied, Lloyd et al. (1987) have shown that the GABA
(gamma-amino butyric acid)-mimetic agent fengabine will correct the passive avoidance
deficit of bulbectomized rats after a single administration. It should be noted that these
investigators confirmed the findings of Cairncross et al. (1975) and van Riezen et al.
(1976) that imipramine and mianserin were only active following their chronic
administration.
The results of these studies in which some drugs facilitating the serotonergic and
GABAergic systems are active in normalizing passive avoidance behavior following acute
administration conflict with the actions of actual, or putative, antidepressants on the
behavior of the bulbectomized rat in the open-field apparatus. Detailed studies over the
Drug effects o n olfactory bulbectomized rats 243
last 10 years have failed to detect any antidepressant that will attenuate the hyperactivity
of bulbecotmized rats in the open field following acute administration (see Table 10.1).
In addition to the passive avoidance and open-field tests that have been used routinely
to assess drugs for their possible antidepressant activity in bulbectomized rats, there is
experimental evidence to show that other abnormal behaviors elicited by the lesion (e.g.,
muricidal behavior, conditioned taste aversion, and active avoidance behavior) are cor
rected by chronic antidepressant treatment.
From the results of these studies, it may be concluded that a combination of passive
avoidance and open-field tests would normally be sufficient to estabHsh the possible anti
depressant activity of a novel compound following its chronic administration to bulbec
tomized rats.
3.11. APPLICATION OF THE BULBECTOMIZED MODEL TO ASSESS THE

ANTIDEPRESSANT POTENTIAL OF A NOVEL COMPOUND: LEVOPROTILINE
Previous studies on the R(-)enantiomer of the selective noradrenaUne uptake inhibitor

oxaprotiUne showed the compound to be devoid of any action on the noradrenergic sys
tem after either acute or chronic administration (DeUni-Stula et al., 1983; Mishra et al.,
1983). Furthermore, levoprotiUne was largely devoid of any effect on the serotonergic
system; the only activity it appeared to have at central neurotransmitter receptors
appeared to be related to its antagonism of H, receptors. Interest in levoprotiUne would
undoubtedly have waned had the early cUnical studies not indicated that the drug had
antidepressant activity (DeUni-Stula et al., 1983). More recent cUnical studies have con
firmed the antidepressant activity of the compound (Wendt, 1988).
In most animal models used to select putative antidepressants, levoprotiline would
appear to be inactive. However in the behavioral "despair" test in rats, levoprotiline
acted like most other clinically effective antidepressants, in that it reduced the immobility
of rats subjected to forced swimming after both acute and subchronic (7 days) adminis
tration; the effect foUowing acute administration only occurred after a relatively high
dose (20 mg/kg/day) of the drug (Delini-Stula et al., 1988).
Janscar and Leonard (unpublished) have found that the chronic oral administration
of levoprotiline (10 mg/kg b.i.d. for 21 days) to sham-operated and bulbectomized rats
resulted in an attenuation of the hyperactivity of the lesioned animals when they were
subjected to the stress of the open field. LevoprotiUne was also shown to be effective in
reversing the deficit in the retention of bulbectomized rats in the passive avoidance test.
In both the open-field and passive avoidance tests, most "classical" antidepressants show
similar activity to levoprotiUne. The finding of effects of levoprotiline in the bulbectom
ized depression model underUnes its potential to identify antidepressant activity inde
pendent of "biochemical mechanism of action."
4. NEUROCHEMICAL CHANGES FOLLOWING BULBECTOMY AND THE

EFFECTS OF ANTIDEPRESSANTS
4.1. EFFECT OF SELECTIVE NEUROTOXINS ADMINISTERED TO THE OLFACTORY BULBS
ON THE BEHAVIOR A N D NEUROTRANSMITTER CHANGES
Caimcross et al. (1979, 1981) investigated the possible importance of the retrograde
degeneration of specific neurotransmitter systems in causing the behavioral deficits seen
in the bulbectomized rats by injecting specific neurotoxins into the bulbs. These inves
tigators showed that only 5,7- or 5,6-dihydroxytryptamine, which selectively destroy
serotonergic nerves, could produce the complete repertoire of behavioral deficits that
were corrected by chronic treatment with amitriptyhne or mianserin. The effects of these
specific neurotoxins could be prevented by prior treatment with specific serotonin uptake
inhibitors that prevented the transport of the neurotoxin into serotonergic nerve termi
nals. Histological examination of the brains of the rats showed that retrograde degener
ation occurred in the amygdaloid, hippocampal, stria terminalis, and preoptic regions.
These findings have recently been replicated by Kagashe and Leonard (in preparation),
who also showed that chronic treatment of the animals with mianserin antagonized the
behavioral effects of the lesion.
Cairncross et al. (1979) reported that the selective noradrenergic neurotoxin 6-
hydroxydopamine did not produce the complete bulbectomy syndrome following its
bilateral administration to the bulbs. These results therefore suggest that the lesions of
the serotonergic system play a primary role in causing the behavioral deficits seen in the
bulbectomized rat. Again, Kagashe and Leonard (unpublished observations) have repli
cated this experiment and found that no change occurred either in open-field or passive
avoidance behavior following the lesion of dopaminergic/noradrenergic fibers.
Kainic acid lesions (1 Mg injected into each bulb) have also been studied for their pos
sible behavioral effects following injection into the bulbs by Kagashe and Leonard (in
preparation). The preliminary results of these studies demonstrate an abnormality in
open-field and passive avoidance behavior which may be prevented by prior chronic
treatment with desipramine. Unlike 5,7-dihydroxytryptamine and 6-hydroxydopamine,
kainic acid causes lesions of several types of cell bodies including glutamate, GABA, and
acetylcholine. Since the behavioral effects of the kainic acid lesion could not be differ
entiated from those of the serotonergic neurotoxin-induced behavior, this suggests that
the changes in the serotonergic systems are primary and that the changes in other trans
mitter systems (such as described in Section 3.2) are secondary and possibly contribute
little to the bulbectomy syndrome. Moreover, it demonstrates that lesions of the acces
sory olfactory bulbs are not necessary to produce the model behavior.
4.2. CHANGES IN CENTRAL NEUROTRANSMITTERS FOLLOWING BULBECTOMY
Pohorecky et al. (1969) were the first to report that the concentration of noradrenaline
in the telencephalon was decreased in the bulbectomized rat, while Edwards and col
leagues (Edwards, 1974; Edwards et al., 1977) subsequently showed that the extent of the
depletion of this transmitter was related to the degree of brain damage to the regions
caudal to the bulbs through which the noradrenergic fibers ascend to terminate in the
olfactory bulbs. Cairncross et al. (1979) reported that bulbectomy causes a reduction in
the noradrenaline content of the frontal cortex, which was reversed by treatment with
amitriptyline (Cairncross and King, 1971) or desipramine (Iwasaki et al., 1986a). Leon
ard et al. (1979) showed that the turnover of noradrenaline in the frontal cortex was also
reduced in this brain region following bulbectomy. Reviewing the early studies, Hirsch
(1980) showed that findings varied with time after lesions, brain region, and method of
turnover determination. So, for instance, O'Connor and Leonard (1986) showed that a
similar reduction in noradrenaline turnover occurred in the amygdaloid cortex and in
the midbrain, but more recent studies of the changes in the noradrenaline content of the
amygdaloid cortex following bulbectomy do not confirm these findings (Broekkamp et
al., 1986a). One possibility for these differences may rest with the methods used to quan-
titate noradrenaline. Thus, the studies by Broekkamp et al. (1986a) used a high-perfor
mance liquid chromatography (HPLC) technique to determine noradrenaline, whereas
all the previous studies utilized a fluorimetric procedure. Iwasaki et al. (1986a) found
differences depending on whether the rats were muricidal or not, implying that behav
ioral reactions were responsible for the changes in catecholamine turnover.
Jancsar and Leonard (1984) reported that serotonin turnover was decreased in the
amygdaloid cortex following bulbectomy, whereas dopamine was unchanged and GABA
was increased. While it is difficult to relate these changes to the behavior of the rat, it
may be speculated that the reduction in the turnover of serotonin could be associated
with the increased irritability (Di Chiara et al., 1971) and possibly the hyperactivity
observed in the open-field apparatus. Other studies have shown that lesions of the olfac
tory tubercle caused by the selective neurotoxin 5,7-dihydroxytryptamine result in
hyperactivity in the open field, whereas those of the medial raphe do not (Jancsar,
unpubUshed).
Other changes in neurotransmitter function that might be relevant to depression have
also been found in the brain of the bulbectomized rat. Thus the density of both « ι and
/3-adrenoceptors in the cortex are increased foUowing bulbectomy (Earley, unpublished);
it is not without interest that the densities of these receptors on the blood cells of
depressed patients are also increased, which lends support to the view that the bulbec
tomized rat may be considered as a model of depression. Further evidence is provided
by recent studies on the transport of [ΉΙ-serotonin into platelets of bulbectomized rats.
Studies undertaken by us (Healy et al., 1983, 1985; Butler and Leonard, 1986) and by
others (Coppen and Wood, 1980; Tuomisto et al., 1979) have shown that the uptake of
[ΉΙ-serotonin into platelets from depressed patients is significantly impaired, but gen
erally returns to control values in patients who respond to treatment. Butler et al. (1988)
have shown that platelets from bulbectomized rats show a similar deficit in serotonin
transport to depressed patients, and that, following chronic treatment with either the
noradrenaline uptake inhibitor desipramine or the novel serotonin uptake inhibitor ser-
traUne, the uptake of [^]-serotonin returns to the value found in the sham-operated
animals. These changes in serotonin transport appear to parallel the normalization in
behavior as indicated by an attenuation in the hyperactivity of the bulbectomized rat in
the open-field apparatus. There is evidence that «i-acid glycoprotein (AGF) may be
responsible both for defective [^]-serotonin transport in depressed patients (Nemeroff
et al., 1990) and also in the bulbectomized rat (Arnold and Meyerson, 1990). AGF has
also been implicated in suppressing lymphocyte responsiveness in patients (Chiu et al.,
1977). It is therefore possible that AGF is responsible for the changes observed in sero
tonin transport in bulbectomized rats.
In addition to these changes in platelet function, which are associated with a normal
ization of [Ήΐ-serotonin transport into cortical synaptosomes in bulbectomized rats
chronicaUy treated with desipramine or sertraline, O'NeiU et al. (1987) have shown that
bulbectomy is associated with a suppression of neutrophil phagocytosis that is partially
reversed by chronic desipramine treatment. It was previously shown that neutrophil
function is subnormal in depressed patients, but normalized foUowing effective antide
pressant treatment (O'NeiU and Leonard, 1986). The results of these studies therefore
suggest that abnormalities in some aspects of immune function, in addition to those
abnormalities in central neurotransmission, are qualitatively similar in the depressed
patient and the bulbectomized rat.
Studies of the changes in neurotransmitter function in the bulbectomized rat have so
far been largely restricted to those neurotransmitters that are readily amenable to anal
ysis. The findings that the density of muscarinic receptors is increased in Umbic regions
of brains from suicide victims (Meyerson et al., 1982) suggests that an abnormality in
the central cholinergic system might be associated with the iUness. Broekkamp et al.
(1986a) investigated possible changes in choUne acetyltransferase activity in the olfactory
tubercles of the bulbectomized rat brain and compared the results with the sham-oper
ated controls. In general, no change in the activity of this enzyme was found in the adult
rat foUowing bulbectomy, although 15 days after surgery, a 26% increase in the activity
of choline acetyltransferase was detected. Since some 14 days later the activity of this
enzyme had returned to control levels, it seems unUkely that this change was causally
related to the behavioral deficits that are apparent for at least 3 months foUowing surgery.
However, there is preliminary evidence to suggest that central cholinergic function, as
shown by an increased responsiveness to a physostigmine challenge (tongue protrusion),
is also enhanced some 2 to 3 weeks following surgery, which suggests that changes in
central choUnergic transmission may play an ancillary role in the bulbectomy syndrome
(Leonard, 1988). On the other hand, Millan et al. pubUshed that olfactory bulbectomized
rats are less sensitive to pilocarpine-induced convulsions (Millan et al., 1986).
4.3. CHANGES IN PLASMA CORTICOSTERONE FOLLOWING BULBECTOMY
Depression is characterized by a number of neuroendocrine disturbances ( A P A Task

Force, 1987), the most widely studied undoubtedly being plasma Cortisol. There is good
clinical evidence to show that many depressed patients lose the circadian periodicity of
Cortisol secretion and hypersecrete the hormone throughout the 24-hour period. Many
depressed patients also show a resistance to the Cortisol depressant effect of the synthetic
glucocorticoid dexamethasone; this forms the basis of the dexamethasone suppression
test, which is a commonly used, although very much debated, biological marker of
depression ( A P A Task Force, 1987).
Cairncross and co-workers (1977) have reported that bilateral olfactory bulbectomy
results in an elevation of both basal and stress-induced plasma corticosterone concentra
tions and that chronic antidepressant treatment reverses this hypersecretion (Cairncross
et al., 1978b, 1979). Similar findings were reported by Cattarelli and Demael (1986).
Studies by O'Connor and Leonard (1984) failed to confirm these findings, however. Fur
thermore, no difference in the sensitivity of the bulbectomized rats to the corticosterone
suppression effect of an acute dose of dexamethasone could be detected when compared
to the sham-operated controls. In a more extensive study of possible changes in corti
costerone in bulbectomized rats by Broekkamp et al. (1986a), the lack of change in the
plasma concentration of this hormone was noted in both young (7-week old) and adult
12-week old rats. It therefore seems unlikely that changes in the pituitary-adrenal axis,
as indicated by the plasma corticosterone concentration, are abnormal in well-handled
bulbectomized rats. It is not without interest that O'Connor (1986) reported that no
differences could be found in the concentration of adrenal asorbic acid in bulbec
tomized and sham-operated rats when the animals were subjected to a mild foot shock
stress.
5. CONCLUSIONS
A major criterion for judging the validity of animal models of psychiatric disease lies
in their responsiveness to chronic drug treatment known to be therapeutically effective.
In addition, the utility of such models depends on their ability to predict the therapeutic
activity of novel compounds undergoing development. To some extent, the olfactory
bulbectomized rat is a useful model fulfilling both these criteria. However, it must be
emphasized that the bulbectomized rat is an inexact model of depression. For example,
the hypothalamic-pituitary axis of the "well-handled" bulbectomized rat would appear
to be normal despite the preliminary suggestion of Cairncross et al. (1977) that the bul
bectomized rat behaves like the depressed patient and hypersecretes adrenal glucocorti
coids in response to stress. The lack of change in the pituitary-adrenal axis following
bulbectomy suggests that the noradrenergic control of corticotropin (ACTH) release is
normal in these "well-handled" bulbectomized animals, even though the turnover of the
biogenic amine neurotransmitters in the limbic regions is reduced.
The results of the behavioral studies suggest that an essential feature of the bulbectom
ized rat lies in its reduced ability to adapt to sudden environmental changes. This is
shown by the delay with which the bulbectomized rat habituates to a stressful novel envi
ronment and the delay in both acquisition and extinction of a learned response. Such
changes are apparently not due to motor impairment or a deficit in feeding behavior, as
the bulbectomized rat eventually performs its behavioral tasks as efficiently as the sham-
operated control. Thus, bulbectomy appears to be associated with a deficit in adaptation
that may resemble the behavior of the depressed patient whose symptoms may be trig
gered by a significant life event.
It may be concluded that the olfactory bulbectomized rat resembles the depressed
patient (1) in response to chronic antidepressant drug treatment; (2) regarding many of
Drug effects on offactory bulbectomized rats 247
the behavioral and physiological changes; (3) regarding the abnormalities in peripheral
and central neurotransmitters; and (4) in some immunological functions.
Acknowledgments—The authors thank Drs. Suzanne Jancsar, Bernadette Earley, and Billy O'Connor for per
mission to quote from their unpublished work.
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PeiBamon Press, Inc. (New York), pp. 251-260
CHAPTER 11
VOCAL MANIFESTATIONS OF ANXIETY

A N D THEIR PHARMACOLOGICAL CONTROL
JOHN D . NEWMAN
Laboratory of Comparative Ethology, NICHD, PoolesviUe, Md, USA
1. INTRODUCTION
The prominent use of spoken language in our own species as a means of communi
cating about emotional and mental state has tended to overshadow the presence of non
verbal cues associated with the expression of affect. It is only after considerable excursion
into the experimental literature that we begin to appreciate the widespread existence of
species-specific, nonverbal communication signals employed by other animal species, as
well as both the occurrence of nonverbal signaling and the modulation of speech sounds
(through changes in pitch, tempo and prosody) associated with changes in human affec
tive expression. While the terminology associated with the assessment and treatment of
anxiety-related syndromes is relatively precise in human clinical cases (cf., the Diagnostic
and Statistical Manual of Mental Disorders, 3rd ed., rev. [DSM-III-R], APA, 1987), find
ing corresponding syndromes in animal models is frau¿it with a number of problems
(Lister, 1990; see also Chapter 7). Furthermore, "anxiety" as an emotional state in
humans is generally associated with feelings of impending danger or fear (Ninan et al.,
1982). Since what animals feel can only be inferred, the term anxiety in the context of
the present review will be used rather loosely to correspond to stress and distress, as used
by various authors. In most cases, it is the ability of drugs with anxiolytic or anxiogenic
properties (determined from human clinical trials) to modulate animal vocalizations that
is the main justification for associating the vocal behavior in question with anxiety-
related syndromes in humans. The situation with one anxiety-related syndrome, sepa-
ration anxiety disorder, is somewhat clearer. Here, the occurrence of this disorder in
children, its frequent association with temper tantrums and crying, and its amelioration
by the presence of a parent figure have led numerous authorities to conclude that this
disorder is related in an evolutionary sense to the widely represented behavior of devel-
oping offspring in many bird and mammal species when separated from their parent or
family group. Thus, the young of many species emit characteristic vocalizations when
lost or separated, the pituitary-adrenal axis is activated, and both the behavioral and
biochemical manifestations are terminated upon successful reunion. Although the acous-
tic characteristics of the relevant vocalizations vary in pitch and duration (occupying a
frequency range above human hearing in many rodents and referred to as "ultrasounds"
as a consequence), the structure of separation-induced vocalizations is very similar across
species, at least in nonhuman primates (Newman, 1985), suggesting a shared neural sub-
strate underlying production of these sounds in nonhuman primates and the correspond-
ing sounds (crying) in human infants. The clinical association between anxiety and
depression has led to the examination of antidepressants in the pharmacological control
of anxiety. With respect to the study of vocal manifestations of anxiety, imipramine (a
tricyclic antidepressant) has had the longest association with altered vocal behavior. One
study investigated the effect of imipramine on the speech of human adults (Helfrich et
al., 1984). Imipramine in a single dose of 100 mg administered orally produced a reduc-
tion in speech fundamental frequency (fO), as well as an increase in the acoustic energy
below 500 Hz (low-frequency region). These same authors also administered the
251
252 J . D . NEWMAN
a-adrenergic agonist Clonidine to another group of adult humans. Clonidine in a dose of

0.3 mg produced a lowering of systolic blood pressure and a dramatic increase in the
amount of acoustic energy in the low-frequency region, in keeping with the known
reduction of sympathetic arousal associated with this drug. These changes were inter
preted as being related to a reduction in muscle tone associated with the relaxing effects
of imipramine. Although no one, to my knowledge, has reported the effect of anxiolytics
(e.g. diazepam or other benzodiazepines) on crying in human infants, the effect of ben
zodiazepine tranquilizers on voice stress has been measured in patients reporting high
levels of chronic anxiety (Smith, 1982). Unfortunately, there was no significant correla
tion between change in reported anxiety level and change in voice stress following treat
ment, leading the author to conclude that the psychological stress evaluator employed in
that study did not measure anxiety in any sense related to symptom level.
From a clinical perspective, interest in the pharmacological control of anxiety under
standably has focused on reducing or alleviating the symptoms. Consequently, animal
models have been examined primarily for their sensitivity to putative anxiolytics (see
File, 1987; Lister, 1990). However, the difficulties of separating sedative from anxiolytic
effects and finding animal models that spontaneously exhibit the high levels of anxiety-
related symptoms exhibited by human patients require that drugs with potential anxi
ogenic properties also be examined. Although rarely reported in humans (except in the
case of separation anxiety disorder in children), an increase in vocalization has been used
as a measure of increased anxiety in animal models, in association with an increase in
symptoms such as somatic, gastrointestinal, cardiac, and pituitary-adrenal activity.
Undrugged subjects placed in anxiety-provoking situations (such as social separation or
proximity to aversive stimuli) have been reported to increase their level of vocalization
in correspondence to the stressfulness of the procedure. Thus, young monkeys vocalize
more when they are removed from their mother and familiar surroundings than when
the mother is removed and the subject remains in its home cage (e.g. Ritchey and Hen
nessy, 1987; Levine and Wiener, 1988), rat pups vocalize more when removed from
mother and litter mates and placed in a temperature-regulated enclosure at lower than
normal temperatures (Insel et al., 1988), and components are added to the shock-
induced vocalization of aduU rats as shock strength is increased (Levine et al., 1984). In
human infants, pitch and duration of crying increase with the invasiveness of a painful
procedure (Forter et al., 1986). Furthermore, studies have shown that drugs effective in
blocking the action of clinically important anxiolytics can also lead to increased vocali
zation as part of the animal model's reaction (Ninan et al., 1982; Gardner and Budhram,
1987; Harris and Newman, 1987; Insel et al., 1989). Hence, increased anxiety can resuU
from the interaction of environmental stressors and pharmacological manipulation. One
important implication of this finding is that chemical abnormalities in some individuals
may lead them to experience increased anxiety under otherwise mildly stressful
conditions.
2. NEUROCHEMICAL SYSTEMS AND THEIR ROLE IN MEDIATING

FRODUCTION OF ANXIETY-FROVOKED VOCALIZATION
2.1. THE OPIATES
A major role for brain opiatergic systems in mediating the distress of social separation
has been overwhelmingly documented by the extensive studies of Jaak Fanksepp and his
group and, subsequently, by others. Acute, systemic administration of moφhine sulfate
reliably reduces separation-induced vocalizations in a dose-dependent manner, for
example, in puppies over a dose range of 0.125-0.50 mg/kg SC (Fanksepp et al., 1978a)
and infant guinea pigs over a dose range of 0.125-5.0 mg/kg SC (Herman and Fanksepp,
1978). A low dose of morphine (5 Mg) introduced into the fourth ventricle anterior to the
cerebellum is also effective in reducing separation calls in chicks (Fanksepp et al., 1980c).
Anxiety and vocalizations 253
Intracranial administration of 2 /ig /J-chlomaltrexamine (which irreversibly binds to opi

ate receptors in vitro) blocked the ability of low doses of intraventricular moφhine to
reduce separation calls for up to 3 days (Panksepp et al., 1982). As stated in one review
of this work (Panksepp et al., 1985, p. 6), . . all opioid agonists that were tested proved
to be remarkably potent agents for alleviating separation distress" and, referring specifi
cally to separation-induced vocalizations, . . no other behavior is as powerfully and
consistently modified by low doses of opiate receptor agonists."
Comparison of moφhine and naloxone with a wide range of psychoactive drugs
administered both centrally and peripherally led to the conclusion that the opiate system
has the most powerful specific effect on distress vocalization of all systems studied (Pank
sepp et al., 1980c). One major question left unresolved by these studies is to explain the
inconsistent effect of the opiate antagonist, naloxone, in increasing the level of distress
in socially separated individuals. The effectiveness of naloxone appears to vary according
to circadian and seasonal cycles, at least in domestic chicks (data presented in Panksepp
et al., 1985). Systemically administered naloxone has been shown to increase the rate of
separation-induced vocalizations in chicks (2.5 mg/kg; Panksepp et al., 1980c) and in
juvenile and adult guinea pigs (1 mg/kg SC; Herman and Panksepp, 1978). Naloxone
administered centrally (into the cerebral ventricles) increased separation-induced vocal
izations in chicks at a dose (0.3 Mg) ineffective peripherally (Panksepp et al., 1980b).
Intraventricular naloxone (1.0 ^g) increased separation calls in chicks even in the pres
ence of another chick (Panksepp et al., 1980a). Direct administration "of brain opioid
peptides into the fourth ventricle reduced isolation-induced vocalizations in 2- to 4-day-
old chicks (Panksepp et al., 1978b; Vilberg et al., 1984). Vilberg et al. (1984) demon
strated that the endoφhins (α, 7, β) and D-Ala^ Met-enkephalin all were effective in a
dose-dependent manner, while Met-enkephalin was without effect. Beta-endoφhin was
effective at lower doses than the other endoφhins or moφhine. The peptides lost their
effectiveness in reducing vocalizations when the interval between peptide administration
was lengthened from 4 to 15 minutes, approximating the time course of peptide degra
dation. Even endogenous peptides with opioid activity, which normally occur outside of
the brain, reduced separation calls in chicks when injected into the fourth ventricle (caso-
moφhin fragments, 1-100 nmol; Panksepp et al., 1984). The interaction between an
exogenous opioid antagonist and environmental modulation of separation calls was
investigated by Blass et al. (1987). In that study, orally administered sucrose decreased
total separation calls given during an 8-minute test period in rat pups, an effect that was
reversed by naltrexone (0.5 mg/kg IP).
The effects of opiate receptor agonists and antagonists on separation calls have not
gone unnoticed by behavioral pharmacologists working with nonhuman primates. Kalin
et al. (1988) demonstrated that separation calls of infant rhesus macaques are decreased
by moφhine (0.1 mg/kg IM) and increased by naloxone (1.0 mg/kg IM). A lower dose
of naloxone (0.1 mg/kg), while having no intrinsic effect of its own on vocalization, did
block the suppression of calling by moφhine. Essentially the same effects of these opiate
manipulations also occurred with respect to corticotropin (ACTH) levels measured 1
hour after drug administration. A higher dose of naloxone (1.0 mg/kg IM) significantly
increased separation-induced coos in rhesus infants over vehicle trials with the same sub
jects (Kalin and Shelton, 1989). In a study of infant squirrel monkeys, Weiner, et al.
(1988) also demonstrated that moφhine (0.9 mg/kg IV) decreased and naloxone (1.7
mg/kg IV) increased separation calls. However, that study failed to find any correlation
between drug treatment and Cortisol levels measured 4 hours after drug administration.
Investigations with adult squirrel monkeys demonstrated essentially the same effects of
moφhine and naloxone on separation calls found in infant monkeys. Newman (1988)
reported that moφhine (10 mg/kg IM) completely suppressed separation calls (isolation
peeps), an effect that was reversed with 0.5 mg/kg naloxone. Naloxone given alone pro
duced variable results in increasing separation calls over vehicle trials at the lowest dose
(0.4 mg/kg IM), but reliably increased calling at 1.6 mg/kg (Harris and Newman, 1988a).
254 J. D . N E W M A N
At the highest dose used (3.2 mg/kg IM), the calHng rate dropped back to baseline levels.
In a subsequent study, a dose of 0.4 mg/kg naloxone (IM) was administered to the same
group of animals during three experiments, and a significant increase in separation call
ing was demonstrated over all drug trials compared to vehicle control trials during the
same experimental series (Harris and Newman, 1988b).
In a study on the effects of various drugs on tail shock-induced vocalizations in aduh
male rats, Levine et al. (1984) found that moφhine (3.0 mg/kg) preferentially affected
vocalizations occurring after cessation of the shock (the AD component of the vocal reac
tion). Tail shock at threshold levels produced short calls with a rapid rising-then-falling
frequency contour (VI). Stronger shock levels produced louder, longer VI calls and,
additionally, longer latency calls (the AD component) that had longer durations, and
rose slowly in frequency. Moφhine produced a significant elevation in the shock level
required to elicit AD, without affecting VI. This elevation in threshold did not occur
when the rats were restrained in addition to being shocked. Heart rate levels associated
with restraint suggested that the rats were increasingly stressed by this procedure. Essen
tially the same results occurred with a low dose of diazepam (0.1 mg/kg) as with mor
phine. The authors concluded that moφhine and diazepam differentially affect the com
ponent of pain-induced vocalization reflecting the affective state of the animal, rather
than the sensory intensity of the painful stimulus. Details regarding the neural substrate
mediating the production of distress calls and their modulation by opiates have been
investigated in adult guinea pigs. Herman and Panksepp (1981) found that pretreatment
with naloxone (1.0 mg/kg SC) decreased threshold currents for electrical stimulation
through indwelling electrodes of the thalamic periventricular gray region to evoke pain
like screams. Peripherally administered moφhine raised the current threshold, with 10
mg/kg blocking the evoked vocalizations completely. The issue of whether vocalizations
evoked by social separation are mediated by the same central opiatergic system regulat
ing analgesia to pain was addressed by Weiler and Blass (1988). These investigators dem
onstrated that cholecystokinin (sulfated CCK octapeptide) markedly reduced separation
calls in rat pups at doses of 1.0 μ/kg and higher (IP) and counteracted naltrexone's rever
sal of the typical moφhine-induced suppression of separation calls. However, CCK was
ineffective in altering the latency of paw lifting to a hot plate or in blocking moφhine's
analgesic effect on this behavior.
2.2. THE BENZODIAZEPINES
The first study of the effect of benzodiazepines on rate of separation-induced vocali

zation in rodent pups appears to be that of Gardner (1985b). In two stress-inducing par
adigms, rat pup ultrasounds were inhibited by chlordiazepoxide (6-20 mg/kg IP) and
diazepam (2 and 4 mg/kg IP). Unfortunately, these doses also produced muscle relax
ation and sedation, leaving open the issue of the specificity of drug-related changes on
vocalization. Insel et al. (1986) found that lower doses (0.5 and 1.0 mg/kg IP) also
decreased separation-induced ultrasounds in rat pups, but without a concomitant
decrease in motor activity. These authors demonstrated a role for the benzodiazepine
receptor complex in mediating ultrasound production by reversing the diazepam effect
with Ro 15-1788 and by showing that antagonists and inverse agonists for this receptor
complex produced differential effects on vocal behavior. Further evidence for the
involvement of the benzodiazepine receptor in socially separated rat pups was obtained
through the use of in vivo autoradiography. Tritium-labeled Ro 15-1788 bound to ben
zodiazepine receptor in the brain 30% less following 25 minutes of social separation than
in the brains of pups kept with their litter mates (Insel et al., 1988, 1989). The authors
noted that the only brain region showing a significant change in binding in the separated
pups was the neocortex, a region not identified as part of the neural substrate mediating
ultrasound production. As a consequence, the precise role of the benzodiazepine receptor
in mediating separation-induced ultrasounds in rat pups remains unclear. Gardner and
Budhram (1987) also used a variety of benzodiazepine agonists, antagonists, and inverse
agonists to demonstrate their differential effects on ultrasound production in rat pups.
Engel and Hard (1987) compared the effects of ethanol and diazepam on rat pup ultra
sounds and locomotor behavior. Both drugs produced a dose-related decrease in calling
rate (over the dose range 0.25-1.0 mg/kg), but only diazepam produced a significant
change in locomotion. Ro 15-1788 reversed the diazepam-related behavioral effects, but
not those produced by ethanol. Ro 15-1788 also produced a dose-related decrease in
ultrasound production (5-20 mg/kg). Benton and Nastiti (1988) argued that the neuro-
biological mechanisms that modulate separation calling may be implicated in the etiol
ogy of psychiatric disorders. These authors investigated the effect of a range of psycho
tropic drugs on ultrasonic calling by separated mouse pups; the major tranquilizers,
chloφromazine and haloperidol were without effect, as were imipramine, meprobamate,
and amobarbital. On the other hand, suppressed calling occurred with amitriptyline, the
benzodiazepines diazepam and chlordiazepoxide, and the novel anxiolytic ipsapirone.
Carden and Hofer (1989) also found that chlordiazepoxide (2.0 mg/kg) lowered the rate
of ultrasound production in separated rat pups. Equally effective in reducing vocalization
in this context were moφhine (0.125 mg/kg) and a single anesthetized litter mate in the
test chamber. Naltrexone, but not Ro 15-1788, blocked the reduction in vocalization
associated with the presence of a litter mate, and Ro 15-1788, but not naltrexone, facil
itated quiet contact between the subject and the anesthetized litter mate. Gardner
(1985a) presented a procedure involving induction of vocalization by holding rat pups
by the tail as a method for screening anxiolytic drugs. With this screening method, it was
found that chlordiazepoxide (0.2-10 mg/kg IP) and diazepam (0.2-4 mg/kg IP) inhibit
vocalizations. Furthermore, the benzodiazepine receptor ligand CL 218,872 also inhib
ited vocalizations over a dose range of 2 to 20 mg/kg without producing other overt
behavioral changes. Drugs ineffective in changing vocal behavior in this paradigm were
amitriptyline, haloperidol, and naloxone. The only other drug found to inhibit vocali
zation without producing other overt behavioral effects was metergoline (at doses of 5
and 10 mg/kg). Cuomo et al. (1988) studied the effects of diazepam (0.5-1.0 mg/kg IP)
and alprazolam (1.25-2.5 mg/kg IP) on vocalizations associated with foot shocks in aduh
rats. A significant decrease in the length (duration) of the vocalizations occurred follow
ing administration of these benzodiazepines, and Ro 15-1788 blocked the diazepam
effect. Neither calling rate nor frequency measures were affected at these doses. A similar
finding was reported by Sales et al. (1986). Tonoue and colleagues (1987) found that
diazepam (2.5 mg/kg SC) reduced the rate of foot shock-induced vocalization in male
rats. Benzodiazepine antagonists (Ro 15-1788 and CGS 8216) blocked the diazepam
effect. These authors additionally found that intraventricular iS-endoφhin also attenu
ated vocalizing. Naloxone selectively blocked the iS-endoφhin effect, but not that pro
duced by diazepam, whereas the benzodiazepine antagonists were without effect when
administered prior to iS-endoφhin treatment.
The temporal organization of rat ultrasound production proves to be amenable to dif
ferential drug effects. Van der Poel et al. (1989) demonstrated that mild, electrical tail
stimulation causes a conditioned vocal reaction in adult rats consisting of short bouts of
calls. Chlordiazepoxide (2.5-10 mg/kg) produced a dose-dependent increase in longer
calling bouts than when treated with vehicle (saline), although mean calling rate was the
same for the two treatments.
A study using maternally separated infant rhesus monkeys (Kalin et al., 1987) found
that diazepam (0.1 and 1.0 mg/kg IM) decreased vocalization and plasma Cortisol while
increasing locomotor activity. Attempts to make social contact with adjacent subjects
also increased, but did not reach statistically significant levels. The benzodiazepine recep
tor antagonist Ro 15-1788 (5 and 10 mg/kg) given concurrently with diazepam blocked
the increase in activity and the decrease in Cortisol (but not the increase in vocalizations),
and had no intrinsic effects on behavior or pituitary-adrenal activity when administered
alone.
256 J. D . NEWMAN
An earlier study implicated the benzodiazepine-gamma-amino butyric acid (GABA)

receptor complex in a primate model of anxiety (Ninan et al., 1982). The ethyl ester of
/J-carboline-3-carboxylic acid (ß-CCE; 2.5 mg/kg IV) produced elevations in heart rate,
blood pressure, plasma Cortisol, and catecholamines in chair-restrained rhesus macaques.
Initial reactions to this drug included piloerection and increased vigilance, followed
within minutes by significant increases in vocalization, defecation, urination, and penile
erection. Pretreatment with Ro 15-1788 (5.0 mg/kg) or diazepam (1-2 mg/kg) blocked
or markedly attenuated the /3-CCE-induced behavioral and physiological changes. A
study of the vocal alarm response of adult squirrel monkeys presented with a predator
model (Glowa et al., 1988) demonstrated a modestly enhanced vocal response to the
stimulus following 5 successive days of 1 mg/kg diazepam pretreatment, and a markedly
enhanced reaction following this pretreatment schedule when the monkey was injected
with 1.0 mg/kg Ro 15-1788 10 minutes prior to testing.
2.3. THE MONOAMINES
A role for central monoamines in mediating the production of anxiety-related vocal

izations is well documented. Some of the evidence comes from studies with clinically
relevant drugs (e.g., antidepressants) whose mechanisms of action are thought to be pri
marily on monoamine metabolism and receptor availability. The separation calls of
domestic chicks (Lehr, 1989) and rat pups (Mos and Olivier, 1989) are both susceptible
to manipulation by a variety of antidepressent drugs. Mos and Olivier (1989) found that
different antidepressants differed in their profile of action on this behavior. Chlorimipra
mine (10 mg/kg IP) reduced separation call production while the pups were maintained
either in an environment of 37*C (warm-plate condition) or 18*C (cold-plate condition).
Other antidepressants affected calling only in the cold-plate condition (the 5-hydroxy
tryptamine [5-HT] reuptake blocker fluvoxamine and the mixed 5-HT/NA (noradren
ergic) reuptake blocker clovoxamine, both at doses of 5-20 mg/kg IP). A role for the
tricyclic antidepressant imipramine in reducing separation calls has been documented in
puppies (acute dose of 8 mg/kg 1 hour before testing; Scott, 1974), adult squirrel mon
keys (acute dose of 10 mg/kg 1 hour before testing; Harris and Newman, 1987), and
infant rhesus monkeys (daily treatment of 10 mg/kg p.o.; Suomi et al., 1981). One study
reported an increase in separation calls of rhesus macaque infants after an acute dose of
15 mg/kg imipramine IM 30 minutes prior to testing (Porsoh et al., 1984). Implication
of monoamine metabolic pathways in mediating separation call production comes from
the use of inhibitors of the enzyme monoamine oxidase (MAO), which prevent the
breakdown of monoamines. Winslow and Newman (1988) showed that milacemide, a
prodrug whose metabolism in the brain results in competitive inactivation of MAO-B,
produces a dose-dependent decrease (100-400 mg/kg SC) in separation calls and loco
motor activity in adult squirrel monkeys. A subsequent study demonstrated differential
action of selective MAO-A and MAO-B inhibitors on separation calling rate, call struc
ture, and locomotion in this same paradigm (Newman et al., 1989; 1991).
The respective roles of the better-known monoamine neurotransmitters, norepineph
rine, dopamine, and serotonin, have been explored through the use of selective neuro
transmitter receptor ligands, reuptake inhibitors, and neurotoxins. Affecting synaptic
action of norepinephrine through the use of the aj-adrenoreceptor agonist Clonidine has
resulted in altered separation calling in a variety of avian and mammalian models. In
many of these studies, the action of Clonidine on separation calls was blocked by co
administration of a2-adrenoreceptor antagonists (yohimbine or iadoxan). The initial
demonstration of this was by Panksepp et al. (1980c) in domestic chicks. In that study,
Clonidine was the only one of 18 nonopiate drugs tested to prove as effective as moφhine
in reducing separation calls. Subsequent experiments showed that intraventricular doses
as small as 0.08 Mg were sufficient to suppress separation calls (Rossi et al., 1983). In
these latter experiments, 6-hydroxydopamine (6-OHDA) was injected intracerebrally in
some animals prior to testing with Clonidine. Doses of 6-OHDA that significantly
depleted norepinephrine and dopamine failed to alter the suppression of separation calls
by intraventricular Clonidine. The authors concluded from this finding that clonidine's
effect on separation calling is not mediated through the inhibition of norepinephrine
release by prejunctional «j-adrenoreceptors. Interestingly, Clonidine has been shown to
increase separation calling rate in rodent pups (gerbil: 0.1 mg/kg IP; Thiessen and
Upchurch, 1981; Wistar rat 0.1-0.2 mg/kg SC, Hard, et al., 1988; albino rat: 0.05 mg/kg
IP, Kehoe, 1988; 5.0 Mg/kg IP, Insel et al., 1988). The ability of Clonidine to increase
calling rate reverses on postnatal day 17 (Kehoe, 1988). Although Clonidine also
decreases body temperature in neonatal rat pups and the rate of ultrasound production
is temperature-dependent in neonates, the dose-dependent effects of Clonidine on calling
rate could not be explained entirely by alterations in body temperature (Hard et al.,
1988).
Clonidine has also been administered in tests of socially separated primates. In a study
of maternally separated infant rhesus monkeys, separation calls were reduced after 100
Mg/kg IM, a dose that also reduced locomotor activity and environmental exploration
(Kalin and Shelton, 1988). A study of adult squirrel monkeys demonstrated a dose-
related decrease in separation calls after Clonidine (0.05-0.15 mg/kg IM, Harris and
Newman, 1987), the specific dose that produced a significant decrease from control levels
varying between individual subjects. The interaction of a2-adrenergic and opiatergic
mechanisms on anxiety-related vocalization has been studied in adult squirrel monkeys.
Harris and Newman (1986, 1988b) demonstrated that concurrent administration of
yohimbine and naloxone did not increase rate of separation calUng over either drug
alone. Instead, there was the unexpected increase of another vocalization usually asso
ciated with less stressful contexts.
A role for serotonergic receptors in mediating separation calling in rat pups has been
demonstrated. Mos and Olivier (1980) found that 5-HT,A agonists were effective in
reducing separation calls of pups tested in both warm and cold environments, whereas
mixed 5-HT,A/B agonists and the 5-HT,B agonist l-[3-(Trifluromethyl)phenyl]-piperazine
H Q (TFMPP) reduced calling only when rates were increased as a result of testing the
pups on a cold plate. The 5-HT2 agonist (I)-l-(2,5-dim.ethoxy-4-iodophenyl)-2-amino-
propane HCl (DOI) also reduced ultrasounds regardless of the temperature of the testing
environment, but the pups were severely sedated at these doses (0.3-3.0 mg/kg IP). Fur
ther implication of serotonergic mechanisms in mediating separation calls in rat pups
comes from a study by Winslow and Insel (1990a). These authors found that a variety
of 5-HT-selective reuptake blockers reduced calling rates at doses that left locomotor
activity and rectal temperature unchanged. Further, acute and repeated administration
of the serotonin-selective neurotoxin 3,4 methylenedioxymethamphetamine (MDMA)
to rat pups resulted in a lasting decrease in separation calls without altering locomotor
behavior, geotaxis, or weight gain (Winslow and Insel, 1990b).
A role for dopamine receptors in mediating production of anxiety-related vocaliza
tions is less clear. Cuomo et sd. (1987) tested D, and D2 receptor antagonists on ultrasonic
separation calls of neonatal rat pups and found little evidence for changes in calling rate.
On the other hand, these investigators demonstrated that frequency and duration param
eters of the vocalizations did change following administration of a selective D, receptor
antagonist.
2.4. CORTICOTROPIN RELEASING FACTOR
The well-established activation of the hypothalamic-pituitary-adrenal axis following

exposure to stressful situations raises the question of the role of corticotropin releasing
hormone (CRH) in anxiety-related behavior. While this issue has been rather extensively
explored, at least in rodents (e.g., Dunn and File, 1987), the involvement of CRH in
mediating anxiety-provoked vocalization has only recently been examined. Administra
tion of CRH into the cerebral ventricles of infant rhesus monkeys produced a significant
decrease in the level of activity during maternal separation at the highest dose tested (10
258 J. D . NEWMAN
Mg), but had no significant effect on other behaviors, including vocalization (Kalin et al,
1989). The same dose of CRH also reduced rectal temperature, but did not significantly
affect heart rate. Plasma levels of ACTH and Cortisol measured 1 hour after separation
were elevated following the 10 Mg CRH treatment. A study of socially separated aduU
squirrel monkeys found that 10 Mg CRH resulted in a significant increase in locomotor
activity, both during the separation trial and after returning the subject to the colony
(Winslow et al., 1989). The effects of CRH on separation calls was less clear, although in
one animal with a high basehne calUng level, 1.0 and 10 Mg CRH resulted in nearly com
plete suppression of calling. A clearer relationship between intraventricular CRH and
vocal behavior was obtained in a study of rat pups (Insel and Harbaugh, 1989). In that
study, using a lower dose range than employed with primates, a significant reduction in
separation calls occurred following treatment with 0.1 and 0.01 Mg CRH. This change in
calling rate was blocked by concurrent administration of α-helical corticotropin releasing
factor, a CRH antagonist. Pups did not show signs of sedation or changes in locomotor
activity following these treatments.
2.5. CENTRAL CHOLINERGIC MECHANISMS
Cholinergic mechanisms have been given little attention regarding a possible role in
mediating the behavioral manifestations of anxiety. However, since several reports have
documented changes in anxiety-provoked vocalization following administration of cho
linergic drugs, this evidence will be presented here. Panksepp et al. (1980c) reported that
atropine (10 mg/kg IP) resulted in an increase in separation calls of domestic chicks.
Subsequently, Sahley et al. (1981) reported that the muscarinic antagonist scopolamine
(250 nmol total injected dose) introduced into the fourth ventricle increased separation
calling rate by more than 20%, while nicotine suppressed calhng and a peripheral nico
tinic antagonist had no effect. In a study with adult squirrel monkeys, Glowa and New
man (1986) found that a central muscarinic antagonist, benactyzine HCl (0.3-3.0
mg/kg IM), resulted in a dose-dependent increase in alarm calls given in response to a
predator model. Newman (1988) subsequently showed that separation calls were not
increased with this drug, suggesting some context-specificity to the drug's action.
3. CONCLUSIONS
The foregoing review illustrates the wide range of drugs that have been shown to influ
ence vocal behavior in anxiety-provoking contexts. Many of these drug effects are similar
to those described for other behaviors in the same contexts, in increasing or decreasing
rate of occurrence in the expected direction according to the anxiolytic or anxiogenic
profile of the drug in question. Clear roles for opiate, benzodiazepine, and a-adrenergic
receptor mechanisms in mediating vocal behavior evoked by social separation have been
demonstrated. A role for other neurochemical substrates requires further confirmation,
but there are suggestive data implicating serotonergic, CRH, and choUnergic pathways.
As with other behavioral measures of anxiety, the involvement of disparate neurochem
ical mechanisms indicates that complex and heterogeneous neural circuitry underlies the
changes in vocal behavior observed. This neural circuitry will ultimately need to be iden
tified and the interactions of its constituents understood before a full understanding of
the mechanisms regulating anxiety is possible.
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INDEX
Acetylcholine (ACh), 6 3 - 6 4 in future, 1 7 2 - 1 7 5

ACh {see Acetylcholine) other tests of, 1 7 0 - 1 7 2
Acoustic startle pathway, 1 9 9 - 2 0 0 , 201 social behavior tests of, 1 6 8 - 1 7 0
A C T H {see Adrenocorticotropic hormone) o f other anxiety disorders, 1 7 5 - 1 7 7
Active avoidance deficits, 2 4 0 o f panic anxiety
Adrenalectomy, 4 5 background of, 1 5 8 - 1 6 0
Adrenal hormones, 85 exploratory behavior tests of, 1 6 0 - 1 6 8
Adrenocorticotropic hormone (ACTH) in future, 1 7 2 - 1 7 5
and basal corticosterone concentrations, 4 1 - other tests of, 1 7 0 - 1 7 2
42 social behavior tests of, 1 6 8 - 1 7 0
and benzodiazepines, 4 3 o f psychopathology, 132
and Cortisol levels, 3 3 - 3 4 and term "model," 155
and corticotropin releasing factor, 8 o f vocal manifestations o f anxiety, 2 5 1 , 2 5 2
and corticotropin releasing hormone, 29, Animal studies {see also N o n h u m a n primate
31-32,40 social behavior; Olfactory
endocrine role of, 3 0 bulbectomized rats)
and gamma-amino butyric acid release, 45 o f anticipatory anxiety, 2 0 2
and pituitary /9-endoφhin, 6 4 o f chlordiazepoxide, antistress effect of, 4 3
and stress, 2 9 - 3 0 of dominance, 8 7 - 8 9
Aggression, 8 4 - 8 5 {see also Dominance) o n drug discrimination models
Alcoholism, 6 antidepressant cues, 1 2 2 - 1 2 3
Alprazolam, 5, 10, 12, 15 anxiogenic cues, 1 1 8 - 1 2 2
2-Amino-7-phosphonoheptanoic acid (AP7), anxiolytic cues, 1 0 7 - 1 1 8
116 o f g a m m a - a m i n o butyric acid-
Amitriptyline, 13, 76, 218 benzodiazepine receptor complex, 4 5 -
Amphetamines, 99, 138, 141, 195 46
Amsel frustration effect (FE), 217 o f hypothalamic-pituitary-adrenal
Amygdala hormones
and anxiety, 2 0 4 - 2 0 7 in anxiety, 3 4 - 3 9
in conditioned stimulus, 206 in depression, 3 7 - 3 9
electrical stimulation of, 205 in exogenous administration of, 3 9 - 4 1
and fear, 2 0 4 - 2 0 7 o f operant responding suppressed by
and fear-potentiated startle effect, 2 0 0 - 2 0 2 punishment
Analogy, 156 conclusions about, 141
Animal models with monkey, 138, 141
of anxiety, 1 5 5 - 1 5 8 with other species, 141
of conditioned emotional response with pigeon, 138
background of, 187 with rat, 1 3 4 - 1 3 8
conclusions about, 1 9 5 - 1 9 6 o f stressors
conditioned suppression, 1 8 7 - 1 8 9 behavioral changes with, 6 6 - 7 3
drugs in, 189, 1 9 2 - 1 9 5 genetic differences or responses to, 7 4 - 7 6
conclusions about, 177 individual differences or responses to, 7 4 -
o f fear-potentiated startle effect 76
advantages of, for studying pharmacology Anticipatory anxiety, 2 0 2 - 2 0 3
of fear or anxiety, 197 Anticonvulsants, 9
background of, 1 9 6 - 1 9 7 Antidepressant cues, 1 2 2 - 1 2 3
conclusions about, 2 0 3 - 2 0 4 Antidepressants {see also specific names of)
drugs in, 1 9 7 - 1 9 9 in anxiety
as model o f anticipatory anxiety, 2 0 2 - 2 0 3 control of, 2 5 1 - 2 5 2
neural systems involved in, 1 9 9 - 2 0 2 therapeutic issues of, 1 1 - 1 6
function of, 156 in contrast effects, 2 2 3 - 2 2 4
o f generalized anxiety disorder drug discrimination models in, 1 2 2 - 1 2 3
background of, 1 5 8 - 1 6 0 in extinction, 2 1 8 - 2 2 0
exploratory behavior tests of, 1 6 0 - 1 6 8 in generalized anxiety disorder, 1 4 - 1 5
261
262 Index
Antidepressants (cont.) anxiolytics in, 251

on hypothalamic-pituitary-adrenal background, 2 5 1 - 2 5 2
hormones, 4 1 - 4 4 conclusions about, 258
and nonhuman primate social behavior, 9 9 - and neurochemical systems, 2 5 2 - 2 5 8
100 Anxiety disorders
in olfactory bulbectomized rats, 2 4 2 - 2 4 6 alprazolam in, 15
on panic attacks, 2 animal models of, 1 7 5 - 1 7 7
paradox of, 1 7 - 1 9 buspirone in, 1 5 - 1 6
in social phobia, 14-15 Clonidine in, 15
in stressors, 76 diagnostic classification of, 31
Antipsychotics, 137 diazepam in, 15
Anxiety DSM-IIl-R on, 31
and amygdala, 2 0 4 - 2 0 7 and hypothalamic-pituitary-adrenal
animal models of, 1 5 5 - 1 5 8 hormones, 32
anticipatory, 2 0 2 - 2 0 3 nonpanic, 14
antidepressants in obsessive compulsive disorder, 176
control of, 2 5 1 - 2 5 2 phobic disorders, 175
therapeutic issues of, 1 1 - 1 6 post-traumatic stress disorder, 1 7 6 - 1 7 7
buspirone in, 1 1 3 - 1 1 5 Anxiety-provoked vocalization (see
causative factors of, 7 - 8 Vocalization manifestations o f
conclusions about, 2 0 anxiety)
and conditioned fear, 2 0 6 - 2 0 7 Anxiogenic cues
and defensive behaviors, 171 - 1 7 2 background of, 118
with depression, 4 - 6 , 155 FG 7142 cue, 1 2 1 - 1 2 2
diagnostic classification of, 1-2 pentylenetetrazole cue, 118-121
diagnostic issues of, 1-3 Anxiogenic drugs
drug treatment of, 1 9 - 2 0 drug discrimination models in, 1 1 8 - 1 2 2
DSM-III-R on, 131, 155 and nonhuman primate social behavior, 9 1 -
epidemiological studies of, 1 97
and fear, 251 in vocal manifestations of anxiety, 251
F G 7 1 4 2 in, 3 7 - 3 8 Anxiolysis, 1 4 4 - 1 4 5
genetic studies of, 5 - 6 Anxiolytic cues
and head dipping, 1 6 3 - 1 6 4 benzodiazepine cues, 1 0 8 - 1 0 9
and hypothalamic-pituitary-adrenal excitatory a m i n o acids, 1 1 6 - 1 1 8
hormones nonbenzodiazepine benzodiazepine receptor
animal studies of, 3 4 - 3 9 ligands, 1 0 9 - 1 1 3
clinical studies of, 3 0 - 3 4 serotonin cues, 1 1 3 - 1 1 6
exogenous administration of, 3 9 - 4 1 term of, 108
and m o n o a m i n e oxidase inhibitors, 11, 1 3 - Anxiolytics
14 in contrast effects, 2 2 1 - 2 2 3
neurochemistry of, 7, 1 4 4 - 1 4 5 in discrimination leaming, 218
and nonhuman primate social behavior, 9 1 - drug discrimination models in
97 benzodiazepine cues, 1 0 8 - 1 0 9
and operant responding, early models of, excitatory a m i n o acids, 1 1 6 - 1 1 8
133-134 nonbenzodiazepine benzodiazepine
organic origin of, 155 receptor ligands, 1 0 9 - 1 1 3
pharmacological control of, 2 5 1 , 252 serotonin cues, 1 1 3 - 1 1 6
pharmacology of, 197 term of, 108
psychological treatment, 1 9 - 2 0 and extinction
and punished behavior, 1 3 1 - 1 3 2 consummatory behavior, 217
rebound, 10 continuous reinforcement, 2 1 3 - 2 1 5
state, 1, 31, 156 discrimination leaming, 2 1 7 - 2 1 8
and stress, 7 - 8 energized behaviors, 217
symptoms of, 2 - 3 intermittent reinforcement, 2 1 5 - 2 1 7
therapeutic issues of on hypothalamic-pituitary-adrenal
antidepressants, 11-16 hormones, 4 1 - 4 4
benzodiazepines, 10-11 and nonhuman primate social behavior, 9 1 -
drug or psychological treatment, or both, 97
19-20 and punished behavior, 1 3 1 - 1 3 2
pharmacotherapy of depression, 9 - 1 0 and tricyclics, 1 2 - 1 3
tricyclics, 1 6 - 1 9 in vocal manifestations o f anxiety, 251
trait, 1, 31, 156 AP7 (see 2-Amino-7-phosphonoheptanoic
vocal manifestations of acid)
animal models of, 2 5 1 , 252 Appetite-motivated conditioning, deficits in,
anxiogenic drugs in, 251 240-241
Index 263
Barbiturates, 117, 194, 2 1 3 , 2 1 4 - 2 1 5 in contrast effects, 2 2 1 - 2 2 2

Basal corticosterone concentrations, 4 1 - 4 3 in extinction, 2 1 3 - 2 1 4 , 2 1 7 - 2 1 8
Behavioral changes, 2 3 2 - 2 3 9 in panic disorder, 10, 12
Behavioral contrast, 2 2 3 - 2 2 4 in punished behavior, 141
Behavioral tests, 2 3 9 - 2 4 3 Chlorgyline, 100
Benzodiazepine (BZ) cues, 1 0 8 - 1 0 9 Chlorimipramine, 2 1 8 , 2 5 6
Benzodiazepines jö-Chlomaltrexamine, 2 5 3
and adrenocorticotropic hormone, 4 3 />-Chlorophenylalanine (/TCPA), 100, 101
antistress effect of, 4 3 C h l o φ r o m a z i n e , 141, 194
in anxiety provocation, 11 Cholinergic mechanisms, 2 5 8
in anxiety-provoked vocalization, 2 5 4 - 2 5 6 C L 2 1 8 8 7 2 , 4 3 - 4 4 , 112
in anxiety reduction, 10-11 Clinical studies o f hypothalamic-pituitary-
and basal corticosterone concentrations, 4 1 - adrenal hormones
43 in anxiety, 3 0 - 3 4
binding, 4 5 - 4 6 , 109 of exogenous administration of, 3 9 - 4 0
in conditioned emotional response, 1 9 2 - 1 9 4 Clomipramine, 1 2 - 1 3
in extinction, 2 1 3 , 2 1 4 - 2 1 5 Clonazepam, 10
and panic attacks, 2 Clonidine, 15, 159, 1 9 7 - 1 9 8 , 2 2 3 , 2 5 2 , 257
in panic disorder, 10, 12 Clorazepate, 14
in phobias, 10-11 Clovoxamine, 256
and plasma corticosterone concentrations, Clozapine, 141
43-44 jo-CMC [see 3-(methoxycarbonyl)-amino-iÖ-
and stress, 7 - 8 carboline]
withdrawal, 15, 1 2 0 - 1 2 1 Cocaine, 141, 195
Beta-carboline FG 7 1 4 2 (see FG 7142) Cognitive therapy, 2 0 (see also Psychological
Bulbectomy (see Olfactory bulbectomized rats) therapy)
Bupropion, 76 Conditioned emotional response (CER)
Buspirone animal models o f
in anxiety, 1 1 3 - 1 1 5 background of, 187
in anxiety disorder, 1 5 - 1 6 conclusions about, 1 9 5 - 1 9 6
and basal corticosterone concentrations, 4 2 and conditioned suppression, 1 8 7 - 1 8 9
in contrast effects, 2 2 2 drugs in, 189, 1 9 2 - 1 9 5
in fear-potentiated startle effect, 197, 199 benzodiazepines in, 1 9 2 - 1 9 4
and plasma corticosterone concentrations, c h l o φ r o m a z i n e in, 194
44 in operant responding, early models of, 1 3 3 -
in punished behavior, 136 134
in punished drinking, 142, 144 Conditioned fear, 2 0 6 - 2 0 7
Conditioned stimulus (CS), 187, 2 0 6
Caffeine, 141, 159 Conditioned suppression, 142, 1 8 7 - 1 8 9
c A M P (see Cyclic adenosine monophosphate) Conditioning, 2 4 0 - 2 4 1 (see also Conditioned
Carbamazepine, 9 emotional response; Fear-potentiated
i^-Carboline, 110 startle effect)
i^-Carboline-carboxylic acid ethyl ester (β- "Conflict," 133
CCE), 9 1 - 9 4 , 96, 1 0 9 - 1 1 0 , 118, 1 7 0 - Consummatory behavior, 2 1 7 , 2 2 1 - 2 2 3 , 2 2 3 -
171 224
Carbon dioxide, 159 Continuous reinforcement, 2 1 3 - 2 1 5 , 2 1 8 -
Cardiac disease, 30 219
Catecholamine systems, 100 Contragonists, 11
Causative factors, 7 - 8 Contrast effects
ß-CCE (see /?-Carboline-carboxylic acid ethyl antidepressants in, 2 2 3 - 2 2 4
ester) anxiolytics in, 2 2 1 - 2 2 3
C D P (see Chlordiazepoxide) buspirone in, 222
Central amine pathways, 4 4 chlordiazepoxide in, 221
Central cholinergic mechanisms, 258 conclusions about, 2 2 4 - 2 2 6
Central neurotransmitter systems imipramine in, 2 2 3 , 2 2 4
after olfactory bulbectomy, 2 4 4 - 2 4 5 term of, 2 2 0
and stressors, 5 7 - 6 5 "Controllability," 86
CER (see Conditioned emotional response) Corticosterone concentrations, 3 5 - 3 7 , 4 1 - 4 4
Cerebrospinal fluid (CSF), 7, 30 Corticotropin releasing factor (CRF), 8
C G S 19755, 116 Corticotropin releasing h o r m o n e (CRH)
C G S 9896, 110, 1 1 1 - 1 1 2 and adrenocorticotropic hormone, 29, 3 1 -
Chlordiazepoxide (CDP) 32, 4 0
antistress effect of, 4 3 in anxiety-provoked vocalization, 2 5 7 - 2 5 8
in anxiety disorder, 14 in depression, 34
in benzodiazepine cues, 1 1 1 - 1 1 3 and g a m m a - a m i n o butyric acid release, 45
264 Index
Corticotropin releasing hormone (cont.) in fear-potentiated startle effect, 197

and norepinephrine, 41 in panic disorder, 10, 1 2 - 1 3
and stress, 29 and plasma corticosterone concentrations,
Cortisol levels, 3 3 - 3 4 43
pCPA {see p-Chlorophenylalanine) in punished behavior, 141
C R F {see Corticotropin releasing factor) and Z K 9 5 9 6 2 , 110-111
C R H {see Corticotropin releasing hormone) Differential reinforcement for low-rate
CS {see Conditioned stimulus) responding (DRL), 2 1 9 - 2 2 0
CSF {see Cerebrospinal fluid) 3,4-Dihydroxyphenylacetic acid ( D O P A C ) ,
Cyclic adenosine monophosphate (cAMP), 61-63
60-61 (I)-1 -(2-,5-Dimethoxy-4-iodophenyl)-2-amino-
propane HCl (DOI), 257
DA {see Dopamine) Discrimination leaming, 2 1 7 - 2 1 8 , 220, 2 2 3 -
Defensive behaviors, 1 7 1 - 1 7 2 224
Depression DMI {see Desmethylimipramine)
and 5-hydroxytryptamine, 9 Dominance, 8 4 - 8 7
and anticonvulsants, 9 D O P A C {see 3,4-DihydroxyphenyIacetic acid)
with anxiety, 4 - 6 , 155 D o p a m i n e (DA), 6 1 - 6 2 , 65, 67, 244, 257
causative factors of, 7 - 8 Dothiepin, 13
conclusions about, 20 Drinking suppressed by punishment
and corticotropin releasing hormone, 34 conclusions about, 1 4 2 - 1 4 4
diagnostic classification of, 3 - 4 conditioned suppression, 142
diagnostic issues of, 3 - 4 definitions of, 1 4 1 - 1 4 2
drugs producing, 100-101 unconditioned suppression, 142
drug treatment of, 9 - 1 0 , 1 9 - 2 0 D R L {see Differential reinforcement for low-
epidemiological studies of, 1 rate responding)
ethanol in, 9 9 - 1 0 0 Drug discrimination models
genetic studies of, 5 - 6 antidepressant cues, 1 2 2 - 1 2 3
and hypothalamic-pituitary-adrenal anxiogenic cues, 118-121
hormones anxiolytic cues
animal studies of, 3 7 - 3 9 benzodiazepine cues, 1 0 8 - 1 0 9
clinical studies of, 3 2 - 3 4 excitatory amino acids, 1 1 6 - 1 1 8
exogenous administration of, 3 9 - 4 1 nonbenzodiazepine benzodiazepine
imipramine in, 20 receptor ligands, 1 0 9 - 1 1 3
monoamine oxidase inhibitors in, 9 serotonin cues, 1 1 3 - 1 1 6
neurochemistry of, 7 term of, 108
and nonhuman primate social behavior, 9 7 - background of, 1 0 7 - 1 0 8
99 conclusions about, 1 2 3 - 1 2 4
pharmacotherapy of, 9 - 1 0 Drug treatment, 9 - 1 0 , 1 9 - 2 0 {see also
psychological treatment of, 1 9 - 2 0 Pharmacology; Pharmacotherapy)
and stress, 7 - 8 DSM-III-R (see Diagnostic and Statistical
symptoms of, 4 Manual)
therapeutic issues of
antidepressants, 11-16 EAA {see Excitatory a m i n o acids)
benzodiazepines, 10-11 E n d o φ h i n levels, 64
drug or psychological treatment, or both, Energized behaviors, 17
19-20 Ethanol, 9 9 - 1 0 0 , 141, 189, 192, 2 1 4 - 2 1 5 , 2 2 2
pharmacotherapy of depression, 9 - 1 0 Excitatory a m i n o acids (EAA), 1 1 6 - 1 1 8
tricyclics, 1 6 - 1 9 Exploratory behavior tests
Desipramine, 13 background of, 160
Desmethylimipramine (DMI), 33, 76, 2 1 8 - holeboard test, 1 6 3 - 1 6 4
219 light-dark transitions, 1 6 6 - 1 6 7
Dexamethasone, 3 3 - 3 4 open field test, 1 6 0 - 1 6 2
Diagnostic and Statistical Manual (DSM-III-R) plus-maze test, 1 6 4 - 1 6 6
on anxiety, 1-2, 131, 155 staircase test, 1 6 7 - 1 6 8
on anxiety disorders, 2, 31 Extinction
on depression, 4 antidepressants in, 2 1 8 - 2 2 0
on generalized anxiety disorder, 155, 158 anxiolytics in
o n panic disorder, 155, 158 consummatory behavior, 217
Diagnostic classifications, 1-2, 3 - 4 , 31 {see continuous reinforcement, 2 1 3 - 2 1 5
also DSM-III-R; ICD-9; ICD-10) discrimination leaming, 2 1 7 - 2 1 8
Diagnostic issues, 1-3, 3 - 4 energized behaviors, 217
Diazepam intermittent reinforcement, 2 1 5 - 2 1 7
in anxiety disorder, 15 barbiturates in, 2 1 3 , 2 1 4 - 2 1 5
in extinction, 214 benzodiazepines in, 2 1 3 , 2 1 4 - 2 1 5
Index 265
chlordiazepoxide in, 2 1 3 , 214, 2 1 7 - 2 1 8 5-HIAA (see 5-Hydroxyindoleacetic acid)

chlorimipramine in, 218 Holeboard test, 1 6 3 - 1 6 4
conclusions about, 2 2 4 - 2 2 6 Homology, 156
diazepam in, 2 1 4 5-HTP (see 5-Hydroxytryptophan)
ethanol in, 2 1 4 - 2 1 5 H u m a n studies, 2 0 2 - 2 0 3 (see also Clinical
flumazenil in, 2 1 4 studies; Primate social behavior)
8-Hydroxy-2-(di-n-propyl-amino)-tetralin
FE {see Amsel frustration effect) ( 8 - O H D P A T ) , 1 1 4 - 1 1 5 , 199
Fear (see also Fear-potentiated startle effect) 6-Hydroxydopamine ( 6 - O H D A ) , 70, 244,
and amygdala, 2 0 4 - 2 0 7 256-257
and anxiety, 251 5-Hydroxyindoleacetic acid (5-HIAA), 6 3 , 87
conditioned, 2 0 6 - 2 0 7 5-Hydroxytryptamine (5-HT) systems, 1 0 0 -
pharmacology of, 197 101
Fear-potentiated startle effect 5-Hydroxytryptamine (5-HT) uptake blockers,
and amygdala, 2 0 0 - 2 0 2 9
animal models of 5-Hydroxytryptophan (5-HTP), 13, 6 1 , 6 3 ,
advantages of, for studying pharmacology 100, 1 1 4 - 1 1 5
o f fear or anxiety, 197 Hyperactivity, 241
background of, 1 9 6 - 1 9 7 Hypoglycemia, 43
conclusions about, 2 0 3 - 2 0 4 Hypothalamic-pituitary-adrenal (HPA)
drugs in, 1 9 7 - 1 9 9 hormones
as model of anticipatory anxiety, 2 0 2 - 2 0 3 animal studies of, 3 4 - 3 9
neural systems involved in, 1 9 9 - 2 0 2 and anxiety
buspirone in, 197, 199 animal studies of, 3 4 - 3 9
Clonidine in, 1 9 7 - 1 9 8 clinical studies of, 3 0 - 3 2
diazepam in, 197 exogenous administration of, 3 9 - 4 1
gepirone in, 199 and anxiety disorder, 32
8-hydroxy-2-(di-n-propyl-amino)-tetralin in, anxiolytics in, 4 1 - 4 4
199 clinical studies of, 3 9 - 4 0
ipsapirone in, 199 conclusions about, 4 6 - 4 7
m o φ h i n e i n , 197 and "controllability," 86
S C H - 2 3 3 9 0 i n , 199 and depression
FG 7142, 1 1 , 3 7 - 3 8 , 159 animal studies of, 3 7 - 3 9
FG 7142 cue, 1 2 1 - 1 2 2 clinical studies of, 3 2 - 3 4
Flumazenil, 1 1 , 4 3 , 4 6 , 2 1 4 exogenous administration of, 3 9 - 4 1
Fluvoxamine, 13, 76, 256 and separation, 171
social variables on, 89
G A B A (see G a m m a - a m i n o butyric acid) and stress, 2 9 - 3 0 , 4 4 - 4 6
GABA-benzodiazepine receptor complex (see Hypothalamic-pituitary-adrenal system (see
G a m m a - a m i n o butyric acid- Hypothalamic-pituitary-adrenal
benzodiazepine receptor complex) hormones)
G A D (see Generalized anxiety disorder)
G a m m a - a m i n o butyric acid (GABA), 214, 2 4 4
ICD-9 (International Classification of
G a m m a - a m i n o butyric acid-benzodiazepine
Diseases), 1-2, 4
receptor complex, 7, 4 3 - 4 6 , 1 1 2 - 1 1 3 ,
ICD-10 (International Classification of
117, 145
Diseases), 2, 4, 10
Generalized anxiety disorder ( G A D )
Idazoxan, 9
animal models o f
Imidazopyridine, 111
background of, 1 5 8 - 1 6 0
Imipramine
exploratory behavior tests, 1 6 0 - 1 6 8
in anxiety-provoked vocalization, 2 5 1 , 256
future, 1 7 2 - 1 7 5
in depression, 20, 99
other tests, 1 7 0 - 1 7 2
in contrast effects, 2 2 3 , 2 2 4
social behavior tests, 1 6 8 - 1 7 0
initial doses of, 16
antidepressants in, 1 4 - 1 5
in panic attacks, 2
benzodiazepines in, 2
in panic disorder, 12, 14, 1 9 - 2 0
DSM-III-R on, 155, 158
Instrumental behavior, 221
m o n o a m i n e oxidase inhibitors in, 2
Intermittent reinforcement, 2 1 5 - 2 1 7
and panic disorder, 2
International Classification of Diseases (see
Genetic studies, 5 - 6
ICD-9; ICD-10)
Gepirone, 16, 199
Inverse agonists, 11
Glucocorticoids, 30
Ipsapirone, 15, 4 4 , 115, 199
Gonadal hormones, 85
Irritability test, 239
Hamilton Anxiety and Depression scales, 5, 6
Head dipping, 1 6 3 - 1 6 4 Kainic acid lesions, 2 4 4
266 Index
Leamed helplessness, 3 7 - 3 8 , 46 N M D A antagonists (see 7V-methyl-D-aspartate

Levoprotiline, 243 antagonists)
Light-dark transitions, 166-167 7V-methyl-D-aspartate ( N M D A ) antagonists,
Lorazepam, 10 116-118
íZ-Lysergic acid diethylamide (LSD), 114, 115, Nonbenzodiazepine benzodiazepine receptor
195 ligands, 1 0 9 - 1 1 3
L-Tryptophan, 100 N o n h u m a n primate social behavior
and antidepressants, 9 9 - 1 0 0
MAOIs (see Monoamine oxidase inhibitors) and anxiety, 9 1 - 9 7
Maprotiline, 13 and anxiogenic drugs, 9 1 - 9 7
Maudsley reactive and nonreactive rat strains, and anxiolytics, 9 1 - 9 7
162 background of, 8 3 - 8 4 , 9 0 - 9 1
m-Chlorophenyl piperazine (m-CPP), 17, 118 and depression, 9 7 - 9 9
m-CPP (see m-Chlorophenyl piperazine) and drugs producing depression, 1(X)-101
M D M A (see 3,4 Methylenedioxymeth- Noradrenaline, 244
amphetamine) Norepinephrine (NE)
Medial forebrain bundle (MFB), 71 and anxiety, 7
Meprobamate, 194 and corticotropin releasing hormone, 41
3-(Methoxycarbonyl)-amino-i9-carboline and depression, 7
(ß'CMC), 112 opioids on, 65
3-Methoxy-4-hydroxyphenylethylene glycol and stressors, 67
(MHPG), 7 stressors on, 5 8 - 6 1
3,4 Methylenedioxymethamphetamine /^-Norepinephrine receptors, 6 0 - 6 1
( M D M A ) , 257 Nortriptyline, 13
a-Methyl-p-tyrosine ( a M P T ) , 100
MFB (see Medial forebrain bundle) Observer-rated anxiety, 170-171
M H P G (see 3-Methoxy-4-hydroxyphenyl- Obsessive compulsive disorder (OCD), 155,
ethylene glycol) 176
Mianserin, 218 O C D (see Obsessive compulsive disorder)
Milacemide, 256 6 - O H D A (see 6-Hydroxydopamine)
M K 8 0 1 , 116, 117 8 - O H D P A T [see 8-Hydroxy-2-(di-n-propyl-
"Model,'' 155 (see also Animal models; Drug amino)-tetralin]
discrimination models) Olfactory bulbectomized rats
Monoamine neurotransmitters, 256 (see also background of, 2 3 1 - 2 3 2
specific names oO behavioral changes following, 2 3 2 - 2 3 9
Monoamine-oxidase inhibitors (MAOIs) behavioral tests to evaluate, 2 3 9 - 2 4 3
in anxiety, 11, 1 3 - 1 4 conclusions about, 246
in depression, 9 neurochemical changes following, 2 4 3 - 2 4 6
and differential reinforcement for low-rate and neurochemical connection between
responding, 219 bulbs and limbic regions, relevant, 2 3 4 -
in generalized anxiety disorder, 2 236
in panic attacks, 2 and strain, effects of, 236
in panic disorder, 1 3 - 1 4 , 19, 159 surgical procedure in, 2 3 2 - 2 3 4
Monoamines, 2 5 6 - 2 5 7 Open field test, 1 6 0 - 1 6 2 , 241
M o φ h i n e , 138, 197, 222 Operant responding suppressed by punishment
M o φ h i n e sulfate, 2 5 2 - 2 5 3 and anxiety, early models of, 1 3 3 - 1 3 4
a M P T (see a-Methyl-p-tyrosine) conclusions about, 141
Muricide behavior test, 2 3 9 - 2 4 0 definition of, 1 3 2 - 1 3 3
with monkey, 138, 141
Naloxone, 2 5 3 - 2 5 4 with other species, 141
N E (see Norepinephrine) with pigeon, 138
Negative contrast (see Contrast effects) with rat, 1 3 4 - 1 3 8
Neural systems, 1 9 9 - 2 0 2 Opiates, 194, 2 5 2 - 2 5 4
Neurochemistry Opioids, 65
of anxiety, 1 4 4 - 1 4 5
and anxiety-provoked vocalization, 2 5 2 - Panic attacks, 2
258 Panic disorder
of anxiolysis, 144-145 and alcoholism, 6
after olfactory bulbectomy, 2 4 3 - 2 4 6 alprazolam in, 10, 12
Neurotoxins, 2 4 3 - 2 4 4 animal models of
Neurotransmitter systems background, 1 5 8 - 1 6 0
and stress, 4 4 - 4 6 exploratory behavior tests, 1 6 0 - 1 6 8
and stressors, 5 7 - 6 5 future, 1 7 2 - 1 7 5
Nialamide, 218 other tests, 1 7 0 - 1 7 2
Nicotine, 141, 159 social behavior tests, 1 6 8 - 1 7 0
Index 267
benzodiazepines in, 10, 12 Psychotropic drugs, 2 3 9 - 2 4 3

chlordiazepoxide in, 10, 12 P T Z {see Pentylenetetrazole)
clomipramine in, 1 2 - 1 3 Punished behavior
clonazepam in, 10 amphetamines in, 141
diazepam in, 10, 1 2 - 1 3 and animal models o f psychopathology, 132
drug treatment of, 1 9 - 2 0 and anxiety, 1 3 1 - 1 3 2
DSM-III-R on, 155, 158 and anxiolytics, 1 3 1 - 1 3 2
F G 7 1 4 2 in, 11 background of, 131
fluvoxamine in, 13 buspirone in, 136
and generalized anxiety disorder, 2 caffeine in, 141
imipramine in, 12, 14, 1 9 - 2 0 chlordiazepoxide in, 141
lorazepam in, 10 c h l o φ r o m a z i n e in, 141
m o n o a m i n e oxidase inhibitors in, 1 3 - 1 4 , clozapine in, 141
19, 159 cocaine in, 141
maprotiline in, 13 conclusions about, 1 4 5 - 1 4 6
pharmacotherapy for, 14 diazepam in, 141
phenelzine in, 12, 14, 15, 19 and drinking suppressed by punishment,
prevalence studies of, 4 141-144
psychological treatment of, 1 9 - 2 0 ethanol in, 141
tricyclics in, 159 and neurochemistry o f anxiolysis, 1 4 4 - 1 4 5
Pargyline, 2 2 4 nicotine in, 141
Partial reinforcement extinction effect (FREE), and operant responding suppressed by
215-216 punishment
Passive avoidance deficits, 2 4 0 and anxiety, early models of, 1 3 3 - 1 3 4
PCP {see Phencyclidine) conclusions about, 141
Pentobarbital, 1 4 1 , 2 1 4 definition o f term, 1 3 2 - 1 3 3
Pentylenetetrazole (PTZ), 159 with monkey, 138, 141
Pentylenetetrazole cue, 118-121 with other species, 141
Pharmacology, 197 {see also Drug treatment) with pigeon, 138
Pharmacotherapy, 9 - 1 0 , 14 {see also Drug with rat, 1 3 4 - 1 3 8
treatment) pentobarbital in, 141
Phencyclidine (PCP), 1 1 6 - 1 1 7 and term "punishment," 133
Phenelzine, 2, 12, 14, 15, 19 "Punishment," 133
Phobias, 1 0 - 1 1 , 1 4 - 1 5 , 175
Picrotoxin, 4 3 R e b o u n d anxiety, 10
Pituitary /9-endoφhin, 6 4 Relapse rates, 2 0
Placebo, 12, 1 9 - 2 0 Reproductive suppression, stress-induced, 8 9 -
Plasma corticosterone concentrations 90
and benzodiazepines, 4 3 - 4 4 R e s e φ i n e , 100, 195
buspirone on, 4 4 R H A {see R o m a n High Avoiders)
and diazepam, 4 3 Ritanserin, 4 2 - 4 3 , 4 4
increases in, 3 5 - 3 7 R L A {see R o m a n Low Avoiders)
ipsapirone on, 4 4 R o 1 5 - 1 7 8 8 , 111
after olfactory bulbectomy, 2 4 5 - 2 4 6 R o m a n high- and low-avoidance rat strains,
Plus-maze test, 35, 1 6 4 - 1 6 6 162
Post-traumatic stress disorder (PTSD), 1 7 6 - R o m a n High Avoiders ( R H A ) , 162
177 R o m a n Low Avoiders (RLA), 162
FREE {see Partial reinforcement extinction
effect) S C H - 2 3 3 9 0 , 199
Prevalence studies, 4 Schizophrenia, 30
Primate social behavior Separation vocalization, 170
animal studies of, 87.-89 Serotonin, 145, 2 4 4 - 2 4 5
background of, 8 3 - 8 4 , 9 0 - 9 1 Serotonin cues, 1 1 3 - 1 1 6
and dominance, 8 5 - 8 7 Serontonergic activity, 6 2 - 6 3
and observer-rated anxiety, 170-171 Serotoninergic drugs, 1 5 - 1 6 {see also specific
and social variables o n hypothalamic- names oO
pituitary-adrenal hormones, 89 Serotoniner^c function, 1 0 0 - 1 0 1
and stress-induced reproductive suppression, Social behavior {see N o n h u m a n primate social
89-90 behavior; Primate social behavior)
Propranolol, 15 Social behavior tests, 1 6 8 - 1 7 0
Protriptyline, 218 Social interaction test, 1 6 8 - 1 7 0
P T S D {see Post-traumatic stress disorder) Social phobia, 1 4 - 1 5
Psychological treatment, 1 9 - 2 0 Social variables, 89
Psychopathology, animal models of, 132 Sodium amobarbital, 218, 2 2 2
Psychotherapy {see Psychological treatment) Sodium lactate, 159
268 Index
Spatial leaming, 241 Therapeutic issues

Spoken language (see Vocal manifestations o f antidepressants, 1 1 - 1 6
anxiety) benzodiazepines, 10-11
Staircase test, 167-168 drug or psychological treatment, or both,
State anxiety, 3 1 , 156 19-20
Stress pharmacotherapy o f depression, 9 - 1 0
and adrenocorticotropic hormone, 2 9 - 3 0 tricyclics, 1 6 - 1 9
and anxiety, 7 - 8 Trait anxiety, 3 1 , 156
and benzodiazepines, 7 - 8 Trazodone, 13
and cardiac disease, 30 Tricyclics
and corticotropin releasing hormone, 29 in anxiety provocation, 1 6 - 1 7
and depression, 7 - 8 as anxiolytic agents, 1 2 - 1 3
endocrinological responses to, 2 9 - 3 0 effectiveness of, in both anxiety and
and hypothalamic-pituitary-adrenal depression, 17
hormones, 2 9 - 3 0 , 4 4 - 4 6 in panic disorder, 159
neurochemical responses to, 2 9 - 3 0 paradox of, 1 7 - 1 9
and neurotransmitter systems, 4 4 - 4 6 in punished drinking, 142, 144
and reproduction suppression, 8 9 - 9 0 1 ,-[3-(Trifluromethyl) phenylj-piperazine HCl
and schizophrenia, 30 (TFMPP), 257
Stressors
and 5-hydroxytryptamine, 6 1 , 6 3 Unconditioned suppression, 142
antidepressants in, 76
background of, 57 Vocal manifestations of anxiety
behavioral changes with, 6 5 - 7 3 animal models of, 2 5 1 , 2 5 2
central neurotransmitter changes with, 5 7 - anxiogenic drugs in, 251
65 anxiolytics in, 251
and dopamine, 67 background of, 2 5 1 - 2 5 2
and e n d o φ h i n levels, 6 4 Clonidine in, 252
genetic differences or responses to, 7 3 - 7 6 conclusions about, 258
individual differences or responses to, 7 3 - 7 6 imipramine in, 251
and norepinephrine, 5 8 - 6 1 , 67 and neurochemistry, 2 5 2 - 2 5 8
and neurotransmitter systems, 5 7 - 6 5
on serotoninergic activity, 6 2 - 6 3 Yohimbine, 115, 159
Suicide, 33
Symptoms, 2 - 3 , 4 Zimelidine, 13
Z K 9 1 296, 110-111
Taste aversion, conditioned, 241 Zolpidem, 111, 112
Tetrahydrodeoxycorticosterone, 45

Anxiolytics and Antidepressants

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Anxiolytics and Antidepressants

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EDITORIAL BOARD

Α. C. SARTORELLI, New Haven

Some Recent Volumes

SPECIALIST SUBJECT EDITOR

U.S.A. Pergamon Press, Inc., Maxwell House, Fairview Park,

U.K. Pergamon Press pic, Headington Hill Hall,

PEOPLE'S REPUBLIC Pergamon Press, Xizhimenwai Dajie, Beijing Exhibition Centre,

GERMANY Pergamon Press GmbH, Hammerweg 6,

BRAZIL Pergamon Editora Ltda, Rua Εςβ de Queiros, 346,

AUSTRALLi Pergamon Press Australia Pty Ltd., P.O. Box 544,

JAPAN Pergamon Press, 8th Floor, Matsuoka Central Building,

Copyright © 1991 Pergamon Press, Inc.

AU rights reserved. No pari of this publication may be reproduced, stored

Library of Congress Cataloging in PuMication DaU

Psychopharmacology of anxiolytics and antidepressants / specialist

Printed in the United States of America

The paper used in this publication meets the minimun) requirements of

therefore may be of limited use either in screening new compounds or in investigating

J. S. Andrews Richard G. Lister

CLINICAL PHARMACOLOGY OF ANXIOLYTICS

2.1.1. Diagnostic Classification

system is presently employed, soon to be replaced by the ICD-10, which is presently

2.1.2. Symptom Variables

TABLE 1.1. Diagnostic Classifications of Anxiety: Comparison o / D S M - I I I - R , I C D - 9 and I C D - 1 0

DSM-III-R diagnosis ICD-9 diagnosis ICD-10 diagnosis

Phobic Disorders Phobic States Phobic Disorders

TABLE 1.2. Symptom Subcomponents of Anxiety and Depression

Symptom Anxiety Depression

Mood Apprehension, worry, irritability Sadness, flatness, loss of feeling, anhedonia

Cognitions Thoughts of impending disaster or Thoughts of guilt, worthlessness, uselessness,

Physiology Tachycardia, sweating, Anorexia, constipation, sexual impotence,

Behavior Hand-wringing, pacing, scratching, Agitation or retardation, suicide attempts,

2.2.1. Diagnostic Classification

2.2.2. Symptom Variables

2.3. COEXISTENCE OF DEPRESSION AND ANXIETY

DSM-III-R diagnosis ICD-9 diagnosis ICD-10 diagnosis

Affective disorders Affective psychoses Mood disorders

Anxiety Shared Depression

Fears (specific) Depressed mood Guilt

severe ("neurotic") depression, family studies do not support a genetic vulnerability in

Anxiety/Panic Phobic Avoidance

Depression '4- ' Demoralization

FIG. 1.1. Relationship between anxiety and depression.

FIG. 1.2. Neurochemical aspects of stress and anxiety.

stress > panic

4.1. PHARMACOTHERAPY OF DEPRESSION

TABLE 1.5. Established Treatments for Anxiety and Depression

Acute Maintenance Acute Maintenance

Tricyclics Tricyclics E C T (severe) Tricyclics

MAOIs = monoamine-oxidase inhibitors; ECT = electroconvulsive treatment.

to interpersonal rejection, and reactivity of mood. Such patients responded better to

4.2. BENZODIAZEPINES IN ANXIETY REDUCTION

4.3. BENZODIAZEPINES A N D ANXIETY PROVOCATION

4.4. ANTIDEPRESSANT DRUGS FOR ANXIETY

In 1962, two influential reports were published on the effectiveness of antidepressants

4.4.1. Tricyclic Antidepressants as Anxiolytic Agents

TABLE 1.6. Trials of Imipramine in Panic Patients

Klein, 1964, 1967 Klein, 1964 Sheehan et al., 1980 (phenelzine)

TABLE 1.7. Trials of Clomipramine in Panic Patients

Escobar and Colgan, 1975 den Boer et al., 1987 (fluvoxamine)

1984) and 5-hydroxytryptophan (Kahn et al., 1987) have shown clomipramine to be

4.4.2. Other Antidepressants

TABLE 1.8. Trials of MAOIs in Panic Patients

Lipsedge et al., 1973 (iproniazid) King, 1962 Sheehan et al., 1980

Long-term treatment outcome is controversial. A recent review suggests that improve

1 9 8 7 ) , a triazolopyridazine that acts only at the type 1 benzodiazepine receptors, indi

CRAWLEY, J. N . , GLOWA, J. R . , MAJEWSKA, M . D . , and PAUL, S. M . (1986) Anxiolytic activity of an endog