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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

Brainstem areas activated by diazepam withdrawal as

measured by Fos-protein immunoreactivity in rats
Lucas Baptista Fontanesi, Renata Ferreira, Alicia Cabral, Vanessa Moreno Castilho,
Marcus Lira Brando, Manoel Jorge Nobre
Instituto de Neurocincias & ComportamentoINeC, Campus USP, Ribeiro Preto, 14040-901, SP, Brazil
Laboratrio de Psicobiologia, Faculdade de Filosofia Cincias e Letras de Ribeiro Preto, Universidade de So Paulo (USP), Ribeiro Preto,
14040-901, SP, Brazil

A R T I C LE I N FO

AB S T R A C T

Article history:

In the 1970s, chronic treatment with benzodiazepines was supposed not to cause

Accepted 5 July 2007

dependence. However, by the end of the decade several reports showed that the

Available online 14 July 2007

interruption of a prolonged treatment with diazepam leads to a withdrawal syndrome

characterized, among other symptoms, by an exaggerated level of anxiety. In laboratory

Keywords:

animals, signs that oscillate from irritability to extreme fear-like behaviors and convulsions

Withdrawal

have also been reported. In recent years many studies have attempted to disclose the neural

Diazepam

substrates responsible for the benzodiazepines withdrawal. However, they have focused on

Anxiety

telencephalic structures such as the prefrontal cortex, nucleus accumbens and amygdala. In

Fos

this study, we examined the Fos immunoreactivity in brain structures known to be

Brainstem

implicated in the neural substrates of aversion in rats under spontaneous diazepam-

dPAG

withdrawal. We found that the same group of structures that originally modulate the
defensive responses evoked by fear stimuli, including the dorso-medial hypothalamus, the
superior and inferior colliculus and the dorsal periaqueductal gray, were most labeled
following diazepam withdrawal. It is suggested that an enhanced neural activation of neural
substrates of fear in the midbrain tectum may underlie the aversive state elicited in
diazepam-withdrawn rats.
2007 Elsevier B.V. All rights reserved.

1.

Introduction

Benzodiazepines compounds are widely used for the treatment

of anxiety and sleep disorders and are also prescribed for their
sedative, muscle relaxant and anticonvulsant properties
(Woods et al., 1987; Bateson, 2002). Initially, chronic treatment
with benzodiazepines was supposed not to cause dependence.
However, it has been demonstrated that, on abrupt withdrawal
from benzodiazepine, chronic exposure patients can experience

a number of symptoms indicative of a dependent state. Among

the symptoms raised, an exaggerated level of anxiety (rebound
anxiety), more intense than that presented during the prescription of the drug, has been mentioned (Chouinard, 2004; Rynn
and Brawman-Mintzer, 2004). Additionally, clinical reports have
denounced the appearance of high levels of fear in patients
experiencing benzodiazepine withdrawal (Rosebush and
Mazurek, 1996). In the laboratory, similar aspects of the
withdrawal phenomena can be reproduced in animals. Actually,

Corresponding author. Fax: +55 1636024830.

E-mail address: mjnes@usp.br (M.J. Nobre).
0006-8993/$ see front matter 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.brainres.2007.07.007

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signs that oscillate from irritability to extreme fear behaviors

and convulsions have already been described (Petursson and
Lader, 1981a,b; Owen and Tyrer, 1983; Emmett-Oglesby et al.,
1983; Lacerra et al., 1999; Woods et al., 1987; Ashton, 2005).
The great majority of methods used in the investigation of the
neurobiological mechanisms implicated in the production of fear
and anxiety as, for example, chemical or electrical stimulation or
lesions of brain structures, can provide only indirect evidences
on the cerebral operation. In this case, the use of immunohistochemical techniques, such as the Fos protein detection, has
been employed because it allows a clear visualization of the
neurons activated during the presentation of a specific fear
stimulus (Dragunow and Faull, 1989; Reiman et al., 1989; Bullitt,
1990). In fact, Fos-like immunoreactivity has been used as a
marker of the neuronal activation that can be precipitated by a
wide range of stimuli as, for example, brain electrical stimulation
(Sandner et al., 1992; Vianna et al., 2003), local injection of
neurotransmitters (Stone et al., 1991; Richard et al., 1992;
Ferreira-Netto et al., 2005), stress induced by immobilization
(Imaki et al., 1992; Honkaniemi et al., 1992), pain (Bullitt, 1990) and
abstinence of several types of drugs of abuse such as opiates
(Hayward et al., 1990), psychostimulants (Persico et al., 1995),
alcohol (Moy et al., 2000) and benzodiazepines (Dunworth et al.,
2000). Using this technique, it has been shown that some
prosencephalic structures are activated by benzodiazepine
withdrawal, such as the shell region of the nucleus accumbens
(Dunworth et al., 2000), one of the most important areas involved
with the expression of motivational factors linked to drugs of
abuse. This type of activation is also produced by anxiogenic
stimuli that induce increase in dopamine release and induction
of Fos immunoreactivity in this structure (Abercrombie et al.,
1989; Smith et al., 1997). In the same context, a simple exposure
to the elevated plus-maze produces great Fos labeling in limbic
areas (Silveira et al., 1993; Sandner et al., 1993) related with the
production of the behavioral and autonomic responses associated with the expression of fear.
Drug withdrawal may function as an unconditioned stressor,
and as such could activate a particular set of structures involved
with the organization of fear states, particularly those belonging
to the well-known brain aversion system, such as the amygdala,
dorsal periaqueductal gray (dPAG) (the main effector pathway of
the defensive behavior responses), superior and inferior colliculi
(structures included in the processing of visual and auditory
information of aversive nature, respectively) and the medial
hypothalamus (mainly tied to the production of the somatic
responses of fear) (Gray, 1982; Graeff, 1990; Brando et al., 1999,
2005). To examine this possibility, in this study we analyzed
whether the expression of fear behaviors observed in rats
abruptly withdrawn from diazepam could lead to a consequent
activation of the same brain structures that modulate the expression of the defensive behavior of animals exposed to
dangerous situations such as, for example, exposure to the
open arms of the elevated plus-maze, among others. To this end,
we used the immunohistochemical technique for the detection
of Fos-protein expression in selected structures of diazepamwithdrawn rats.
The great majority of studies in the literature that investigate
the chronic effects of a drug of abuse has mainly used intraperitoneal injections as the method of delivery of the drug.
However, it was demonstrated that rats under chronic systemic

injections of placebo solutions tend to present an increase in the

anxiety levels when tested in the elevated-plus maze (Griebel
et al., 1994). In this case, it could be argued that the aversion
promoted by the exposure to chronic systemic injections could
lead to an increase in the number of Fos-like immunoreactive
neurons in structures that normally modulate this class of
emotional responses. To circumvent this problem, in this work,
we used a new model of oral drug intake, adapted by Schleimer
et al. (2005), in which the animals voluntarily drink the drug or
control solutions.

Fig. 1 Percentage of entries in the open arms (A), number of

enclosed arms entries (B) and percentage of time spent in the
open arms (C) of the elevated plus-maze of rats that did not
receive any treatment (No Treat) and rats treated chronically
p.o. with sucrose or diazepam. As ANOVA showed no
significant differences between the two control groups used
in this study, all the differences shown in the graphs are
relative to comparisons between the diazepam-treated rats
and those belonging to the sucrose group. Independent
groups of diazepam treated animals were tested at the
dependence (30 min after the last drug intakeDzp DEP) or
withdrawal (48 h after the last drug intakeDzp W). Data are
presented as mean S.E.M. One-way ANOVA followed by
post-hoc NewmanKeuls; p b 0.05.

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2.

Results

2.1.

Plus-maze

One-way ANOVA revealed that the chronic treatment with

sucrose, per se, did not promote any effect on the behavior of
rats after the interruption of the long-term treatment, as
there were no observed significant differences in any of the
registered measures on the behavior of rats of this group,
when compared with those that did not suffer any intervention (No Treat). On the other hand, significant group
differences in both the percentage of entries [F (3,72) = 5.35;
p b 0.005] and on the percentage of time spent in the open
arms [F (3,72) = 18.55; p b 0.001] were observed in animals
under diazepam effects (Dzp DEP) or during withdrawal (Dzp
W), when compared with those that received sucrose only.
NewmanKeuls post-hoc showed that the chronic oral intake
procedure was effective in producing a significant anxiolyticlike effect in rats tested 30 min after the last oral
administration of diazepam (Dzp DEP); as revealed by the

increase in the percentage of entries (Fig. 1A) and in the total

open arms time (Fig. 1C). An anxiogenic-like effect was
achieved in rats on 48-h withdrawal from diazepam (Dzp W),
as these animals stayed less time in the open arms of the
maze (Fig. 1C) than the animals of the sucrose group. The
number of closed arms entries [F (3,72) = 3.22; p N 0.05]
showed a trend towards significance (p = 0.055) to this
condition among the groups tested. As shown in Fig. 1B,
this effect was due to a tendency of rats under diazepam
effect (Dzp DEP) to increase motor activity, when compared
with the sucrose control group.

2.2.

Fos immunoreactivity

Statistical analysis of the effects of the diazepam withdrawal

on Fos-protein expression was conducted in brain areas of 22
animals (n = 11 for group) randomly chosen from each of the
sucrose- or diazepam-withdrawal groups (Dzp W) (see Table 1).
The mean number of Fos-protein immunoreactivity in neuronal nuclei is shown in Figs. 2, 4 and 6, for each brain region
studied.

Table 1 Mean (S.E.M.) number of Fos-positive neurons/0.1 mm2 in brain regions of 48-h diazepam-withdrawn rats
perfused 2 h after plus-maze exposure
Structures

Telencephalon

Diencephalon

Mesencephalon

PrL
CG1
CG2
CeA
MeA
BLA
CA1
CA2
CA3
AcbC
AcbSh
PV
PaV
AH
LH
DMH
DMPAG
DLPAG
LPAG
VLPAG
DR
MnR
SC
IC
LC
Cun

Sucrose

p b 0.05

Diazepam

Left

Right

Mean

Left

Right

Mean

6.18 1.72
21.40 4.43
2.86 0.87
2.01 0.72
8.16 1.60
5.15 1.26
0.61 0.17
0.65 0.25
1.22 0.33
1.55 0.48
2.01 0.55
19.74 5.41
16.18 2.55
13.77 2.02
7.84 1.48
3.75 0.75
1.35 0.33
1.25 0.24
2.36 0.31
5.04 0.77
6.26 0.70
7.79 0.87
1.37 0.12
1.91 0.33
2.67 0.41
3.72 0.76

7.20 1.27
21.45 4.71
2.66 0.79
1.15 0.60
6.98 1.56
4.97 1.11
1.23 0.39
0.46 0.18
1.36 0.23
1.92 0.88
1.94 0.85
19.51 5.52
17.11 3.05
15.17 1.87
8.92 1.11
3.57 0.90
1.23 0.31
1.47 0.32
1.58 0.30
6.58 0.73
8.68 0.86
8.55 0.95
1.21 0.13
2.10 0.40
3.37 0.37
3.40 0.45

6.69 1.46
21.43 4.04
2.76 0.79
1.58 0.63
7.57 1.41
5.06 1.06
0.92 0.27
0.56 0.20
1.29 0.25
1.74 0.67
1.98 0.67
19.63 5.43
16.64 2.60
14.47 1.67
7.38 1.23
3.66 0.75
1.29 0.32
1.36 0.23
1.97 0.27
5.81 0.56
7.47 0.78
8.17 0.91
1.29 0.11
2.01 0.35
3.02 0.36
3.56 0.34

14.70 2.39
24.67 2.04
3.23 1.39
1.81 0.60
9.54 1.62
5.36 1.36
1.67 0.32
1.17 0.24
1.36 0.34
3.04 1.16
3.68 1.32
29.50 6.21
27.57 5.77
21.77 1.97
11.97 1.60
11.10 1.99
11.14 1.25
7.50 1.37
9.28 1.50
11.52 1.32
13.45 1.71
9.37 0.58
6.65 1.01
13.70 1.78
1.88 0.28
9.21 1.42

18.57 2.51
22.44 2.53
4.29 1.71
1.45 0.42
9.49 1.92
5.76 1.16
2.27 0.69
1.19 0.25
1.79 0.43
3.75 1.61
3.38 1.00
27.74 5.85
31.07 6.68
22.10 2.65
13.41 1.53
9.99 1.13
12.70 1.43
8.07 0.98
10.14 1.59
13.27 2.13
13.79 1.75
10.93 0.68
6.87 0.57
13.62 1.83
1.3 0.21
10.11 1.16

16.64 2.23
23.56 2.14
3.76 1.55
1.63 0.41
9.51 1.71
5.56 1.21
1.97 0.48
1.18 0.18
1.57 037
3.40 1.35
3.53 1.14
28.62 5.85
29.32 6.15
21.93 2.00
12.69 1.49
10.54 1.14
11.92 1.34
7.79 1.03
9.71 1.42
12.39 1.61
13.62 1.73
10.15 0.63
6.76 0.72
13.66 1.36
1.60 0.20
9.66 1.15

Number of Fos-positive neurons per region on the left and right sides were counted using a computerized image analysis system (Image Pro Plus
4.0, Media Cybernetics). Asterisks indicate the regions in which withdrawn-rats displayed differential Fos-expression (p b 0.05) in relation to
control group (sucrose). Differences between groups were analyzed with the Student t-test for each region in study. PrL, prelimbic cortex; CG1
and CG2, cingulate areas 1 and 2, respectively; CeA, central nucleus of the amygdala; MeA, medial nucleus of the amygdala; BLA, basolateral
nucleus of the amygdala; CA1, CA2 and CA3, hippocampal areas 1, 2 and 3, respectively; AcbC, nucleus accumbens (core region); AcbSh, nucleus
accumbens (shell region); PV, paraventricular thalamic nucleus; PaV, paraventricular hypothalamic nucleus; AH, anterior hypothalamus; LH,
lateral hypothalamus; DMH, dorsomedial hypothalamus; DMPAG, dorsomedial periaqueductal gray; DLPAG, dorsolateral periaqueductal gray;
LPAG, lateral periaqueductal gray; VLPAG, ventrolateral periaqueductal gray; DR, dorsal raphe nucleus; MnR, median raphe nucleus; SC, superior
colliculus; IC, inferior colliculus; LC, locus ceruleus; Cun, cuneiform nucleus.

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Fig. 2 Mean number of Fos-immunoreactive neurons in the telencephalic structures of 48-h diazepam-withdrawn rats
submitted to the plus-maze. Data are expressed as mean S.E.M. of Fos-positive cells in 0.1 mm2 area of tissue. The number of
Fos-positive neurons per region was bilaterally counted using a computerized image analysis system (Image Pro-Plus 4.0,
Media Cybernetics). * Significant differences on the number of Fos-positive cells in each structure studied between diazepam
(Dzp W) and sucrose groups. The analysis was performed with the use of the Student t-test. The level of significance was set at
p b 0.05.

2.2.1.

Telencephalon

As revealed in Figs. 2 and 3 withdrawal from diazepam did not

cause important Fos expression in almost all telencephalic
regions. In fact, the Student t-test revealed significant difference in the Fos expression only in the pre-limbic cortex
(PrL) [t20 = 3.73; p b 0.05] (Fig. 3, upper right). Otherwise, many
other areas mainly involved in withdrawal of drugs of abuse,
as the core (AcbC) and shell (AcbSh) regions of the nucleus
accumbens, showed no alterations on Fos-protein immunoreactivity (Fig. 3, bottom right).

2.2.2.

Diencephalon

High neural activation was observed in diencephalic structures

(Figs. 4 and 5). Forty-eight hours of diazepam withdrawal was
effective in promoting a robust increase in Fos expression in
the anterior (AH) [t20 = 2.86; p b 0.05] and lateral hypothalamus
(LH) (Fig. 5, upper right) [t20 = 2.75] and also in the dorsomedial
(DMH) [t20 = 5.03; p b 0.05] nuclei of the hypothalamus of the
abstinent rats (Fig. 5, bottom right). Student t-test revealed that
the comparisons between the withdrawal and sucrose groups
reached marginal significance (p = 0.056) of the withdrawal on
the expression of Fos positive neuronal nuclei in the paraventricular region of the hypothalamus (PaV).

2.2.3.

Mesencephalon

Statistical analysis indicated significant differences in Fos expression in almost all mesencephalic areas studied (Figs. 6 and 7),
as the dorsomedial [DMPAG: t20 = 7.74; p b 0.05], dorsolateral
[DLPAG: t20 = 6.11; p b 0.05], lateral [LPAG: t20 =5.35; p b 0.05] and
ventrolateral [VLPAG: t20 = 3.87; p b 0.05] columns of the periaqueductal gray and dorsal raphe nucleus [DR: t20 = 3.24; pb 0.05]

(see Fig. 7, upper right panel). Student t-test also showed significant differences in the superior [SC: t20 = 7.51; p b 0.05] and
inferior [IC: t20 = 8.28; p b 0.05] colliculi (Fig. 7, bottom right) and
cuneiform nucleus [Cun: t20 = 5.07; p b 0.05]. Interestingly, of all
mesencephalic structures studied, only locus ceruleus showed a
significant decrease in Fos-protein expression, when compared
with the control group [LC: t20 =3.44; pb 0.05].

3.

Discussion

The interruption of a long period of treatment with benzodiazepines produces great probability of developing withdrawal
symptoms in humans or in laboratory animals (Lukas and
Griffiths, 1982; Ladewig, 1984). In fact, anxiety-like states have
been reported as a common symptom in benzodiazepinewithdrawn patients. Avoidance of these unpleasant symptoms
seems to negatively-reinforce continuous benzodiazepine use
and is one of the main components of craving for the drug (Busto
et al., 1986; Busto and Sellers, 1991). An increase in anxiety-like
states is also observed in rats under spontaneous diazepamwithdrawal and submitted to several types of animal models of
anxiety (Greenblatt and Shader, 1974; Emmett-Oglesby et al.,
1983; File, 1990). In our study, the interruption of the chronic
regimen of diazepam caused a significant decrease in the
percentage of time spent in the open arms of the plus-maze.
This anxiogenic effect promoted by diazepam withdrawal was
accompanied by significant Fos-positive immunoreactivity in
telencephalic, diencephalic and mesencephalic areas including
all columns of the periaqueductal gray, the deep layers of the
superior colliculus, the central nucleus of the inferior colliculus,

BR A IN RE S E A RCH 1 1 66 ( 20 0 7 ) 3 5 4 6

39

Fig. 3 Photomicrograph of Fos-like immunoreactive cells (dark dots) in coronal sections showing Fos expression through the
prelimbic cortex (PrL, above) and on the core (AcbC) and shell (AcbSh) regions of the nucleus accumbens (below) of rats submitted to
spontaneous diazepam-withdrawal (Dzp W, right) and tested 48 h after the interruption of the treatment, when compared with its
controls (Suc, left). Cg1 = cingulate cortex, area 1; fmi = forceps minor of the corpus callosum; Cl= claustrum; IL = infralimbic cortex;
LV= lateral ventricle; LacbSh= lateral accumbens shell; aca = anterior commissure, anterior part; ec= external capsule.

the medial aspects of the dorsal raphe nuclei, the anterior, lateral
and dorsomedial nuclei of the hypothalamus and the prelimbic
cortex, all of them mainly linked to the modulation of the
emotional, autonomic and motor expression of the fearmotivated behaviors. For example, the prelimbic cortex, amygdala and hypothalamus, in conjunction with other structures,
are part of many well-defined anxiety- and fear-related circuits
in the brain, so that anxiogenic drugs that promote high levels of
aversion also significantly activate these circuits (Charney et al.,
1998; Gorman et al., 2000; Gray and McNaughton, 2000; Rosen
and Schulkin, 1998). Parts of the prefrontal cortex, including the
prelimbic region, have been reported to be involved in the
production of fear- and anxiety-related behaviors (Espejo, 1997;
Jinks and McGregor, 1997; LeDoux, 1995). Additionally, many
studies on acute fear have proposed that brain areas such as the
amygdala, hippocampus, hypothalamus and periaqueductal
gray are putatively involved in fear processing (Fendt and
Fanselow, 1999; Graeff, 1994; Gray and McNaughton, 2000;
LeDoux, 1995). Others have implicated the periaqueductal gray,
amygdala, medial hypothalamus, raphe nuclei, superior and
inferior colliculus and locus ceruleus in the expression of the fear
behaviors (Graeff, 1990, 1994; Brando et al., 1999, 2003, 2005).
The activation of the amygdala results in behavioral and
physiological responses associated with anxiety and panic-

like behavior. It is intriguing that no changes in Fos-expression

in this region were observed in our study. However, a similar
result was also obtained by Borlikova et al. (2006), who showed
that a single alcohol-withdrawal experience does not induce
significant Fos expression in this area, contrasting with a
significant Fos expression following repeated withdrawal
procedures. The activation of the paraventricular hypothalamic nucleus (PaV) promotes release of a variety of hormones
that are involved in the neuroendocrine and autonomic
responses to fear and psychological stress, whereas the
activation of the lateral hypothalamus seems to be important
for the cardiovascular expressions of fear and anxiety. Also, in
laboratory animals, electrical stimulation of the anterior
hypothalamic nucleus elicits hissing and threat postures
characteristic of defensive behaviors. Electrical or chemical
stimulation of some mesencephalic structures belonging to
the well-known brain aversion system, such as the superior
and inferior colliculus and periaqueductal gray, also promote
defensive behaviors in rats (Brando et al., 1999, 2005). The
activation of brainstem structures was also observed in several
studies using Fos-protein expression as a neuronal marker of
anxiety-like states (Senba et al., 1993; Sandner et al., 1993;
Silveira et al., 1995; Beck and Fibiger, 1995; Beckett et al., 1997;
Canteras and Goto, 1999), and after intraperitoneal injection of

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Fig. 4 Number of Fos-immunoreactive neurons in the

diencephalic structures of 48-h diazepam-withdrawn rats
submitted to the plus-maze. Data are expressed as
mean S.E.M. of Fos-positive cells in 0.1 mm2 area of tissue.
The number of Fos-positive neurons per region was
bilaterally counted using a computerized image analysis
system (Image Pro-Plus 4.0, Media Cybernetics). * Significant
difference on the number of Fos-positive cells in each
structure studied between diazepam (Dzp W) and sucrose
groups. The analysis was performed with the use of the
Student t-test. The level of significance was set at p b 0.05.

drugs that elicit panic-like symptoms (Singewald and Sharp,

2000). All these common sets of structures involved in the
expression of the emotional, autonomic and motor components of fear-related behaviors were also strongly activated in
rats suffering withdrawal which were submitted to the plusmaze, showing that the same neuronal changes verified during
the presentation of fear stimuli seems to be occurring during
diazepam withdrawal.
The main neurobiological mechanism associated with the
effects of drugs of abuse has been the dopaminergic mesolimbic
system and its connections with the basal prosencephalon
(Koob, 1992; Koob and Le Moal, 1997). In this context, in a
previous experiment, Dunworth et al. (2000) investigated
whether some diazepam-withdrawal symptoms could be
lessened by prior withdrawal experience in mice undergoing a
single or repeated flumazenil-precipitated diazepam withdrawal. In the third experiment of this study, they evaluated the Fosprotein expression in brain areas that they speculated would be
important in either seizure sensitivity or the expression of an
aversive state. The immunohistochemistry results showed a
significant increase in Fos expression in the shell region of the
nucleus accumbens of mice submitted to a single withdrawal
experience; contrasting with our findings that showed no
significant alteration on this measure in the same area. On the
other hand, no differences were observed after flumazenil

injection in rats undergoing repeated diazepam-withdrawal

experience. Concerning this discrepancy, we must pay attention
to some methodological aspects that could clarify the differences obtained between the two studies. Most importantly, as
revealed above, on Dunworth's experiments, Fos immunohistochemistry assays were performed in mice through the
flumazenil-precipitated diazepam-withdrawal procedure, in
contrast with the spontaneous diazepam-withdrawal method
used in our experiments. This difference is seminal to understand the increase of Fos expression observed in the former
study. Concerning this point, the study of Motzo et al. (1997)
demonstrated that a challenge administration of diazepam
inhibits dopamine output in the nucleus accumbens. On the
other hand, the discontinuation of long-term treatment with
diazepam does not alter the basal dopamine release in this
region. However, a single intraperitoneal injection of flumazenil
(in a dose that has no effect on the basal dopamine levels) is
effective in enhancing the release of this neurotransmitter in
the nucleus accumbens of rats chronically treated with
diazepam and tested after either 6 h or 5 days of withdrawal.
In this case, we could argue that the enhanced Fos expression
observed in the Dunworth's study could be due to a hyperfunctioning of the dopaminergic neurons on the accumbens,
following flumazenil injection. Besides, the flumazenil concentration (20 mg/kg) used in the experiment of Dunworth et al. was
much more larger than that noted in the study of Motzo (4 mg/
kg). In this case, an increase on the basal activity of the
dopaminergic cells that could contribute to promote Fos
expression on the accumbens, could not be disregarded.
The absence of Fos expression in the nucleus accumbens in
the present study may be related to the notion that benzodiazepine withdrawal recruits neural substrates different from the
dopaminergic one (Lingford-Hughes and Nutt, 2003) to promote
its effects. Indeed, changes in the GABA system have been
postulated to underlie diazepam-withdrawal syndrome (Miller
et al., 1988; Galpern et al., 1991; Toki et al., 1996). Secondary
alterations in other neurotransmitter systems as, for example,
the glutamatergic and serotoninergic mechanisms have also
been considered (Allison and Pratt, 2003; Andrews et al., 1997)
Additionally, we have found that the inhibition of the glutamatergic neurotransmission through local injections of AMPAkainate or NMDA antagonists in the dPAG reduce the fear
behaviors in diazepam-withdrawn rats, implicating the excitatory amino acids of the dPAG in the modulation of the aversive
states induced by diazepam withdrawal (Souza-Pinto et al.,
2007). As the mechanisms that modulate fear are primarily
located in the brainstem, it is possible that these changes take
place initially in the mesencephalon. This is supported by the
pronounced Fos-positive immunoreactivity in structures basically involved in the modulation of fear behaviors, such as the
periaqueductal gray and the superior and inferior colliculi of rats
under diazepam withdrawal. Thus, it is likely that the fear
elicited by diazepam withdrawal is the result of the activation of
the neural substrates of aversion in the brainstem, such as the
defense reaction elicited by stimulation of the dPAG (Brando
et al., 1999, 2005). On the other hand, prosencephalic structures,
such as amygdala, are involved in the modulation of the anxiety
promoted by associative learning, such as that generated in
aversive conditioning. In support of the present results, we have
found that rats under ethanol withdrawal had an increase in

BR A IN RE S E A RCH 1 1 66 ( 20 0 7 ) 3 5 4 6

41

Fig. 5 Photomicrograph of Fos-like immunoreactive cells (dark dots) in coronal sections showing Fos expression through the
paraventricular (PvA), anterior (AH) and lateral (LH) regions of the hypothalamus (above), and the dorsomedial hypothalamic
nucleus (below) of rats submitted to spontaneous diazepam-withdrawal (Dzp W, right) and tested 48 h after the interruption of
the treatment, when compared with its control (Suc, left). f = fornix; 3V = third ventricle; VMH = ventromedial hypothalamus;
opt = optic tract; mt = mamillothalamic tract; ic = internal capsule.

Fig. 6 Number of Fos-immunoreactive neurons in the mesencephalic structures of 48-h diazepam-withdrawn rats submitted
to the plus-maze. Data are expressed as mean S.E.M. of Fos-positive cells in 0.1 mm2 area of tissue. The number of Fos-positive
neurons per region was bilaterally counted using a computerized image analysis system (Image Pro-Plus 4.0, Media
Cybernetics). * Significant difference on the number of Fos-positive cells in each structure studied between diazepam (Dzp W)
and sucrose groups. The analysis was performed with the use of the Student t-test. The level of significance was set at p b 0.05.

42

BR A IN RE S EA RCH 1 1 66 ( 20 0 7 ) 3 5 46

Fig. 7 Photomicrograph of Fos-like immunoreactive cells (dark dots) in coronal sections showing Fos expression through the
regions of the periaqueductal gray (above) and inferior collicullus (below) of rats submitted to spontaneous
diazepam-withdrawal (Dzp W, right) and tested 48 h after the interruption of the treatment, when compared with its control
(Suc, left). DMPAG = dorsomedial periaqueductal gray; DLPAG = dorsolateral periaqueductal gray; LPAG: lateral periaqueductal
gray; VLPAG: ventrolateral periaqueductal gray; Aq = Aqueduct of Sylvius; DR = dorsal raphe; DCIC = dorsal cortex of the
inferior colliculus; ECIC = external cortex of the inferior colliculus; CIC = central nucleus of the inferior colliculus.

reactivity to the electrical stimulation of the dPAG along with a

reduction in their ultrasonic vocalizations (a phenomenon
usually observed during exposure to an intense uncontrollable
stressor), showing that ethanol withdrawal sensitizes the
substrates of fear at the level of dPAG (Cabral et al., 2006).
In summary, neural substrates of fear seem to be recruited
by the withdrawal from diazepam intake in rats, as revealed by
the Fos-protein immunohistochemistry assay. In this context,
this is the first report showing that an increase of the activation
of the neural substrates of fear in the midbrain tectum may
underlie the aversive states elicited by diazepam withdrawal.

4.

Experimental procedures

4.1.

Animals

Wistar rats, weighing 100110 g at the beginning of the treatment, from the animal house of the campus of Ribeiro Preto,
University of So Paulo, were used. They were housed in groups
of four in Plexiglas-walled cages, lined with wood shavings
changed every 3 days, maintained in a 12:12 dark/light cycle
(lights on 07:00 h) at 24 1 C, and given free access to food and

water. Before the beginning of the treatments, the animals had a

three-day habituation period to the lodging conditions. The
experiments reported in this article were performed in compliance with the recommendations of SBNeC (Brazilian Society for
Neuroscience and Behavior), which are in accordance with the
rules of the National Institutes of Health Guide for Care and Use
of Laboratory Animals.

4.2.

Behavioral procedure

In the first part of our study, we tested the efficacy of the perorally
(p.o.) drug intake procedure in producing signs of abstinence 48 h
after the interruption of the diazepam regimen, which had lasted
for 18 days, by means of the elevated plus-maze test. On day 18,
the anxiolytic effects of diazepam were also tested. Two indices of
anxiety were used: the percentage of entries and time spent in the
open arms of the maze. We also recorded the number of entries in
the closed arms as a measure of the animal's motor activity.

4.3.

Administration of drugs by oral route

The self-administration of diazepam p.o. in rats suffers the bias

of the gustative and temporal factors in function of a delay in the

BR A IN RE S E A RCH 1 1 66 ( 20 0 7 ) 3 5 4 6

production of its reinforcing effects. In this case, water

deprivation and sucrose were two elements added to our
experimental design that helped us to circumvent this problem.
In fact, the use of deprivation for the establishment of
motivational drives has been very successful when emotional
aspects of the behavior are evaluated. On the other hand, it is
well known that sucrose, by itself, has high reinforcing effects
and it has been found that sucrose-withdrawn animals have
increased anxiety levels. In our study, as we shall see, the
maximum dose of 100 g of sucrose did not have any consequence on the motivational drive of the animals as neither the
percentage of entries nor the time spent in the open arms of the
plus maze were changed in these animals, in comparison with
the no-treatment group.
The oral voluntary ingestion of diazepam started 3 days after
the arrival of the animals at the laboratory's animal house. The
chronic administration of a placebo solution by daily injections
can, by itself, induce an increase in the anxiety levels in animals
tested in the elevated plus-maze (Griebel et al., 1994). To avoid
these caveats we used an oral procedure, adapted by Schleimer
et al. (2005), in which the animals were submitted daily to 14 h of
water deprivation (19:00 to 9:00), followed by 10 h of water ad
libitum (9:00 to 19:00). This procedure began 2 days before the
start of the 18 treatment days, as a period of habituation of the
animals to the drinking procedure. Diazepam was separately
dissolved in a concentration of 10 mg/ml of saline plus
propilenoglycol (5%) and offered, once daily, in a volume of
1 ml/kg diluted in a solution of 2 ml of tap water added to sucrose
(5%) plus propilenoglycol (5%). The solutions were available to
the animals, in glass pipettes of 5 ml, at the end of the daily
period of water deprivation, delivered during each of the 18 days
of treatment. Except for the first 2 days of treatment, in which
control and experimental solutions were available for 30 min,
the animals that did not drink for 10 min were discarded from
the experiments. This was necessary to avoid prolonging the
deprivation period. Still, to evaluate the possible aversive effects
of water deprivation or reinforcing effects of the sucrose
solution per se, a second control group was included in which
the animals had food and water ad libitum during the whole
period of the experiment. Thus, four groups of animals were
formed: a) No treatment group (No Treat, n = 20), b) Sucrose p.o.
intake (n = 21), c) diazepam p.o. intake dependence condition
(Dzp DEP, n = 16) and d) diazepam p.o. intake withdrawal
condition (Dzp W, n = 19).

4.4.

Elevated plus-maze test

The elevated plus-maze was made of wood and consisted of

four arms of equal dimensions (50 cm 12 cm). Two of the
arms were enclosed by 40-cm-high walls and were arranged
perpendicularly to two opposite open arms. The apparatus
was elevated 50 cm above the floor, with a 1 cm Plexiglas rim
surrounding the open arms to prevent falls (Pellow et al., 1985).
The apparatus was located inside an isolated room with 20 lx
of luminosity at the end the open arms. The recording of the
behaviors of the animals in the plus-maze was made through
a camera (Everfocus, USES) linked to a monitor and videocassette, external to the experimental room. The tests were
conducted 30 min (we named this situation the dependence
condition, in which the animals were tested during the effect

43

of the drug) or 48 h after the last oral drug intake (the withdrawal condition, in which the animals were tested free of the
drug). The tests lasted 5 min. The animals were placed in the
center of the plus-maze facing one of the closed arms. Each
animal was tested just once.

4.4.1.

Statistical analysis

The data are presented as mean S.E.M. The behavioral data

were analyzed by means of a one-way ANOVA. The dependent
factor was the number of entries in the closed arms and percentage of entries and time spent in the open arms; the independent factor was no treatment (No Treat), sucrose, diazepam
dependent (Dzp DEP) or diazepam withdrawal (Dzp W) groups.
NewmanKeuls's post-hoc comparisons were carried out whenever significant overall F-values were obtained. In all cases a
probability level of p b 0.05 was considered to be significant.

4.5.

Fos protein immunoreactivity

We evaluated the Fos-protein expression in rats under 48 h of

diazepam-withdrawal. Eleven animals from each of the
sucrose and diazepam oral regimens were randomly chosen
for immunohistochemical protocols. Two hours after the plusmaze test, these animals were deeply anaesthetized with
urethane (1.25 g/kg, i.p., Sigma, USA) and intracardially
perfused with 0.1 M phosphate-buffered saline followed by
4% paraformaldehyde in 0.1 M PBS (pH 7.4). The brains were
removed and immersed (4 C) for 2 h in paraformaldehyde and
then stored for at least for 48 h in 30% sucrose in 0.1 M PBS
cryoprotection. They were then quickly frozen in isopentane
( 40 C) and sliced by the use of a cryostat ( 19 C). To keep the
experimental conditions similar for all animals of both
sucrose and diazepam-withdrawal groups, all incubations
had brain slices of each structure analyzed in this study.
Two adjacent series of 40 m thick brain slices were obtained.
One series was Nissl stained and used for neuroanatomical
comparison purposes and the other series was collected for the
immunohistochemical studies. Tissue sections were collected
in 0.1 M PBS and subsequently processed free-floating according
to the avidinbiotin procedure, using the Vecstatin ABC Elite
peroxidase rabbit IgG kit (Vector, USA, ref. PK 6101). All reactions
were carried out under agitation at room temperature. The
slices were first incubated with 1% H2O2 for 10 min, washed four
times with 0.1 M PBS (5 min each) and then incubated overnight
at room temperature with the primary Fos rabbit polyclonal IgG
(Santa Cruz, USA, SC-52) at a concentration of 1:2000 in PBS+
(0.1 M PBS enriched with 0.2% Triton-X and 0.1% Bovine Serum
Albumin, BSA). Sections were again washed three times (5 min
each) with 0.1 M PBS and incubated for 1 h with secondary Fos
biotinylated anti-rabbit IgG (H+ L) (Vecstatin, Vector Laboratories) at concentration of 1:400 in PBS+. After another series of
three 5-min washings in 0.1 M PBS the sections were incubated
for 1 h with the avidinbiotinperoxidase complex in 0.1 M PBS
(A and B solution of the kit ABC, Vecstatin, Vector Laboratories)
at concentration of 1:250 in 0.1 M PBS, and then were again
washed three times in 0.1 M PBS (5 min per wash). Fos immunoreactivity was revealed by the addition of the chromogen
3,3-di-aminobenzidine (DAB, 0.02%, Sigma) to which hydrogen
peroxide (0.04%) was added just prior to use. Finally the tissue
sections were washed twice with 0.1 M PBS.

44
4.5.1.

BR A IN RE S EA RCH 1 1 66 ( 20 0 7 ) 3 5 46

Quantification of Fos-positive cells

Tissue sections were mounted on gelatin-coated slides, dehydrated for observation and cell counting under bright-field
microscopy. The nomenclature and nuclear boundaries utilized
were based on the atlas of Paxinos and Watson (2005). Cells
containing a nuclear brownblack reaction product with areas
between 10 and 80 m2 were identified and automatically
counted as Fos-positive neurons by a computerized image
analysis system (Image Pro Plus 4.0, Media Cybernetics, USA).
Sections of 26 different regions at different levels in the brain
were collected, according to a method used in previous studies
(Lamprea et al., 2002; Vianna et al., 2003; Ferreira-Netto et al.,
2005). Mounted sections of the tissue were observed using a light
microscope (Olympus BX-50) equipped with a video-camera
module (Hamatsu Photonics C2400) and coupled to a computerized image analysis system indicated above. Counting of Fospositive cells was performed under a 10 objective at a
magnification of 100 in one field per area encompassing the
entire brain region included in quantification. An area of the
same shape and size per brain region was used for each rat. The
system was calibrated to ignore background staining. All brain
regions were bilaterally counted for each rat. The analyzed
encephalic regions and their respective AP coordinates from
bregma (Paxinos and Watson, 2005) were as follows: the
prelimbic cortex (PrL) (AP: 3.72 mm to 3.24 mm), the cingulate
areas 1 (CG1) and 2 (CG2) (AP: 1.56 mm to 1.44 mm), the central
(CeA), medial (MeA) and basolateral nuclei of the amygdala
(BLA) (AP: 2.40 mm to 2.52 mm), the CA1, CA2 and CA3
hippocampal areas (AP: 2.64 mm to 2.92 mm), the dorsal
aspects of the nucleus accumbens core (AcbC) and shell (AcbSh)
(AP: 1.68 mm to 1.44 mm), the paraventricular thalamic nucleus
(PV) (AP: 2.16 mm to 2.40 mm), the paraventricular (PaV, all
subnuclei), anterior (AHC, central and posterior regions) and the
lateral hypothalamic nuclei (LH, peduncular part) (AP: 1.80 to
1.92 mm), the dorsomedial hypothalamus (DMH, all subnuclei)
(AP: 3.00 mm to 3.24 mm), the dorsomedial (DMPAG), dorsolateral (DLPAG), lateral (LPAG) and ventrolateral (VLPAG) parts
of the intermediate portions of the periaqueductal gray
(AP: 6.48 mm to 6.96 mm), the dorsal (DR) and median
raphe nucleus (MnR) (AP: 7.68 mm to 7.80 mm), the deep
layers of the superior colliculus (SC) (AP: 7.44 mm to 7.68 mm),
the ventral part of the central nucleus of the caudal inferior
colliculus (IC) (AP: 8.16 mm to 8.52 mm), the locus ceruleus
(LC) (9.48 to 9.96 mm) and the cuneiform nucleus (CnF)
(AP: 7.80 mm to 8.04 mm). Nuclei were counted individually
and expressed as number of Fos-positive cells per 0.1 mm2
(Lamprea et al., 2002; Vianna et al., 2003; Ferreira-Netto et al.,
2005), as shown in Table 1. The choice of the areas sampled in
this study took into consideration the importance of these
regions in the modulation of the emotional, autonomic and
motor expression of the fear-motivated behaviors (Azuma
and Chiba, 1996; Beck and Fibiger, 1995; Brando et al., 1999,
2003, 2005; Campeau et al., 1997; Canteras et al., 1997;
Canteras and Swanson, 1992; Charney et al., 1998; Conde
et al., 1990; Emmert and Herman, 1999; Espejo, 1997; Fendt
and Fanselow, 1999; Graeff, 1990, 1994; Gorman et al., 2000;
Gray and McNaughton, 2000; Jay et al., 1989; Jinks and
McGregor, 1997; LeDoux, 1995; Li and Sawchenko, 1998; Risold
and Swanson, 1996; Rosen and Schulkin, 1998; Silveira et al.,
1993), although we are aware that many other brain regions,

not specified here, also contribute to the modulation/expression of behaviors elicited by fear stimuli.

4.5.2.

Statistical analysis

Fos-protein expression was analyzed only in withdrawal

animals (sucrose and Dzp W) through Student t-test for each
region in study. The significance level was set at p b 0.05.

Acknowledgment
This work was supported by grants from FAPESP (04/02859-0).

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