Behavioral Neuroscience Copyright 1995 by the American Psychological Association, Inc.

1995, Vol. 109, No. 4, 648-662 0735-7044/95/53.00

Pavlovian Conditional Vocalizations of the Rat: A Model System for

Analyzing the Fear of Pain
George S. Borszcz
Dartmouth College

Presentation of a 6-s light conditional stimulus (CS) that overlapped with a 1-s tailshock
unconditional stimulus (US) generated audible conditional vocalization responses (VCRs) during
the CS period. The rate of conditioning was observed to be directly related to the intensity of the
tailshock US (0.15 mA-0.80 mA). The amplitude, duration, and number of VCRs was also directly
related to US intensity, whereas the latency of VCRs from CS onset was inversely related to US
intensity. VCRs were not observed in rats given explicitly unpaired presentations of CS and US
(0.80 mA). The capacity of tailshock to support development of VCRs was found to depend on its
capacity to elicit vocalization afterdischarges (VADs). Sonographic analysis of vocalizations
revealed that VCRs and VADs share spectrographic characteristics. Results are discussed in terms
of VCRs' providing a model system for analyzing the fear of pain and its suppression.

Studies of chronic pain behaviors in humans have empha-
sized the contribution of the development of fear-avoidance
beliefs in the etiology of these syndromes (Fordyce, Shelton, &
Dundore, 1982; Lethem, Slade, Troup, & Bentley, 1983;
McCracken, Zayfert, & Gross, 1992; Philips, 1987; Waddell,
Newton, Henderson, Somerville, & Main, 1993). These studies
indicate that the negative affective-motivational state associ-
ated with the pain of acute injury (Melzack & Casey, 1968)
supports the conditioning of a central fear state through
Pavlovian contingencies. The fear state in turn generates
avoidance of the cues, and activities that were associated with
the injury and thereby initially promotes recuperation and
healing. However, for some individuals the avoidance re-
sponses remain although tissue damage decreases. The mainte-
nance of these chronic pain behaviors is believed to be
sustained by the Pavlovian conditional anticipation of pain
(i.e., fear of pain) rather than the pain itself. Consequently, as
tissue damage remits, the fear of pain may remain but is no
longer supported by noxious sensory input; rather, it is
maintained by the reduction of emotional distress that derives
from avoidance of the cues and activities that were associated
with the initial injury. For example, a strong correlation has
been reported between the development of the fear of pain
and both work-loss and disability in activities of daily living
(Waddell et al., 1993). But it is important to note that little
relationship was found between these chronic pain behaviors
and biomedical measures of the current intensity of pain.
In recognition of the seminal involvement of Pavlovian
conditional fear in the development and maintenance of
This research was supported by Grant NS-27668 from the National
Institute of Neurological Disorders and Stroke.
I wish to acknowledge the excellent technical assistance of Christine
P. Johnson.
Correspondence concerning this article should be addressed to
George S. Borszcz, Department of Psychology, 6207 Gerry Hall,
Dartmouth College, Hanover, New Hampshire 03755. Electronic mail
may be sent via Internet to George.S.Borszcz@Dartmouth.EDU.
chronic pain behaviors, the present study was designed to
evaluate an animal model of the fear of pain. Audible
vocalizations of the rat that were generated by the pairing of a
discrete conditional stimulus (CS; light) with a noxious uncon-
ditional stimulus (US; tailshock) were examined. Two features
of conditional vocalization responses (VCRs) recommend
them as a model of the fear of pain. First, considerable
progress has been made in delineating the mammalian vocaliza-
tion circuit (for reviews see Sutton & Jiirgens, 1988; Vogt &
Barbas, 1988). This anatomical specificity indicates that the
neural circuit(s) responsible for generating VCRs may eventu-
ally be identified and encourages the prospect of identifying
the neural mechanisms that suppress their generation. Second,
the mammalian vocalization circuit comprises rhinencephalic-
diencephalic structures that have been shown to mediate the
fear and anxiety associated with chronic pain in humans. The
amygdala, anterior cingulate cortex, midline thalamic nuclei,
and dorsolateral periaqueductal gray (dlPAG) are compo-
nents of the mammalian vocalization circuit (Jiirgens, 1982;
Jurgens & Miiller-Preuss, 1977; Jiirgens & Ploog, 1970; Jur-
gens & Pratt, 1979a, 1979b; MacLean, 1985; Magoun, Atlas,
Ingersoll, & Ranson, 1937; Robinson, 1967). Lesions of these
structures have been reported to alleviate the emotional
distress associated with chronic pain in humans, and they are
especially effective when fear and anxiety are components of
the pain state (Ballentine, Cassidy, Flanagan, & Marino, 1967;
Bouckoms, 1984; Brown, 1977; Corkin & Hebben, 1981; Foltz
& White, 1962; Hassenbusch, Pillay, & Barnett, 1990; Jelasic,
1966; Mark, Ervin, & Yakovlev, 1963; Schvarcz, 1977; White &
Sweet, 1969). Correspondingly, stimulation of the amygdala,
anterior cingulate cortex, anterior cingulum, and dlPAG gener-
ates reports of fear and anxiety in human subjects (W. P.
Chapman et al., 1954; Gloor, Olivier, & Quesney, 1981; Meyer,
McElhaney, Martin, & McGraw, 1973; Nashold, Wilson, &
Slaughter, 1974; Schvarcz, 1977). Therefore, VCRs may reflect
activation of the rhinencephalic-diencephalic structures that
are known to be critical to the production of fear and anxiety
associated with chronic pain in humans.

648
 PAVLOVIAN CONDITIONAL VOCALIZATIONS
The present study is a parametric analysis of Pavlovian
conditional audible vocalizations in the rat and thereby pro-
vides a foundation for subsequent analysis of the neural
mechanisms underlying the generation and suppression of
these responses. The rate of conditioning was evaluated as a
function of the intensity of the tailshock US. Changes in the
form (i.e., latency, duration, amplitude, and number) of VCRs
were examined both during the course of conditioning and as a
function of US intensity. The relation between conditioning
and the generation of various unconditional responses (URs)
was also investigated. Three URs that are organized at
different levels of the neuraxis were monitored during condi-
tioning (Borszcz, Johnson, Anderson, & Young, 1992; Carroll
& Lim, 1960; Hoffmeister, 1968). Spinal motor reflexes (SMRs)
consist of tail flexion and hindlimb withdrawal and are orga-
nized at spinal levels. Vocalizations elicited during tailshock
(VDSs) are organized below the junction of the medulla and
pons, and vocalizations that extend beyond tailshock offset
(VADs, or vocalization afterdischarges) are integrated within
the rhinencephalon-diencephalon. I have previously reported
that a Pavlovian conditional avoidance response in rats relies
on the capacity of tailshock to generate VADs (Borszcz, 1993).
I therefore hypothesized that the capacity of tailshock to
support the acquisition of VCRs would also rely on its
generation of VADs. Finally, I performed spectrographic
analysis on VCRs, VADs, and VDSs to determine whether
these vocalizations are distinguishable by their sonographic
characteristics.
Figure 1. Apparatus used for conditioning audible vocalizations in the rat. See text for details.
649
Method
Subjects
Forty-eight male Long-Evans-derived rats, 120-180 days old at the
beginning of the experiment, were used. The rats were individually
housed under a 14:10-hr light-dark cycle and given ad-lib access to
food and water. They were handled 2-3 times per day for at least 1
week prior to training. All training was conducted during the light
portion of the light-dark cycle.
Apparatus
The apparatus in which the rats were trained is shown in Figure 1.
The rat was restrained in a torso sling suit (Harvard Apparatus, South
Natick, MA) that was modified with Velcro strapping. The rat was
restrained to a Plexiglas pedestal by additional strapping that ran
underneath its torso through loops in the suit. This design maintained
the rat in a crouching posture throughout training. The apparatus was
contained within a sound-attenuating isolation chamber.
Unconditional stimulus. Shock electrodes were constructed of two
0-gauge stainless steel insect pins. The pins were held in place by the
chucks of insect pin holders that were embedded in plastic clothespins.
The tension of the clothespins on the tail was adjusted to ensure that
circulation to the tail was not restricted. The electrodes were placed
intracutaneously on opposite sides of the tail 7.0 cm (cathode) and 8.5
cm (anode) from the base. Current (20-ms pulses at 25 Hz for 1,000
ms) was delivered to the tail by means of a computer-controlled
shocker. The intensity, duration, and timing of tailshocks were
 650 GEORGE S. BORSZCZ
controlled by a microcomputer. Different current intensities were
generated by applying different input voltages to the shocker by means
of a digital-to-analog converter. Current intensity was monitored by an
analog-to-digital converter that digitized (500-Hz sampling rate) an
output voltage of the shocker that was proportional to the current
delivered. It is important to emphasize that this form of tailshock does
not produce the sensation of vibration that is generated by the
superficially applied 60-Hz AC current commonly used in studies of
aversive conditioning. Bromm and Meier (1984) reported that intracu-
taneously applied pulsed DC current predominately activates primary
afferent nociceptors (A-delta and C fibers) and generates a sharp,
stabbing or burning pain in humans rather than the diffuse paresthesia
produced by superficially applied AC current.
Unconditional responses. The rat's tail distal to the shock elec-
trodes was attached with cotton thread to a semi-isotonic displacement
transducer (Lafayette Model 76614, Lafeyette, IN). The arm of the
transducer was positioned behind and perpendicular to the tail such
that the thread extended in a straight line directly behind the rat.
Movement of the transducer arm beginning with shock onset was used
to measure SMRs. The output voltage of the transducer was amplified
(x50) and then digitized (500-Hz sampling rate) by a second analog-to-
digital converter. I calibrated this system by determining the relation
between digital conversions of voltage outputs from the transducer/
amplifier and millimeter movements of the transducer arm. The
computer used this derived function to convert digitized voltages to
millimeters of tail movement. SMR was defined as movement of the
transducer arm by at least 0.5 mm. Once SMR criterion was exceeded,
the output voltage of the transducer was monitored for 2,000 ms. The
microcomputer recorded the latency (in milliseconds), peak amplitude
(in millimeters), and magnitude (integrated voltage output expressed
as centimeters x milliseconds) of tail movement on each trial. Displace-
ments up to 100 mm could be detected, and latencies in 2-ms
increments could be measured.
Vocalizations were measured by a pressure-zone microphone (Real-
istic Model 33-1090, Tandy, Ft. Worth, TX) placed approximately 16
cm in front of the rat. The microphone was attached to an audio
amplifier (Technics Model SA-160, Tandy, Ft. Worth, TX) and a
10-band frequency equalizer. The frequency equalizer was adjusted to
selectively amplify frequencies above 1500 Hz. At 80 dB, frequencies
below 1500 Hz were attenuated by approximately 12 dB. The response
function of the system was relatively flat (±0.5 dB) from 1500 to 18000
Hz. The filtering of low frequencies prevented extraneous noise (i.e.,
rats' respiration and movement artifacts) from contaminating vocaliza-
tion records. The output of the amplifier was integrated by a
Coulbourn Instruments (Allentown, PA) contour following integrator
(2-ms time base) and then digitized (500-Hz sampling rate) by a third
analog-to-digital converter of the microcomputer.
I calibrated the audio system by determining the relation between
the peak digitized output of the converter and the amplitude (SPL, B
Scale) of a 2.5-kHz pure tone—the approximate fundamental fre-
quency of pain-induced vocalizations of the rat (Levine, Feldmesser,
Tecott, Gordon, & Izdebski, 1984). The derived function was used to
convert analog-to-digital inputs to decibels. Sound intensities up to
103.0 dB could be measured. The most intense vocalization measured
during any sampling period was 97.1 dB. The microcomputer recorded
the peak intensity (in decibels), latency (in milliseconds), and duration
(in milliseconds) of vocalizations during the shock epoch (i.e., VDS)
and for the 2,000-ms interval following shock termination (i.e., VAD).
The ambient background noise level in the isolation chamber was 54.0
dB. Sounds above 57.0 dB were considered to be vocalizations.
Conditional stimulus. A discrete 6-s light CS was provided by a
150-W lightbulb in the otherwise dark isolation chamber. The light-
bulb was placed above the microphone such that it was approximately
4 cm in front and 12 cm above the rat's head. The timing of CS
presentations was controlled by the microcomputer. The tailshock US
was presented during the last second of CS presentation.
Conditional responses. Conditional responses (CRs) were moni-
tored during the first 5 s of CS presentation. VCRs were measured
with the audio system used to measure VDSs and VADs. On each trial
the microcomputer recorded three features of VCRs: (a) latency (in
milliseconds) from CS onset to the first VCR, (b) peak amplitude (in
decibels) of the most intense VCR, and (c) total duration (in
milliseconds) of VCRs. The experimenter recorded the total number
of VCRs generated on each trial by monitoring the output of the audio
system on a digital storage oscilloscope.
Tail movements during the first 5 s of CS presentation were
monitored by the displacement transducer used to measure SMRs.
The microcomputer recorded the latency, peak amplitude, and magni-
tude of the first tail movement that exceeded the 0.5-mm criterion.
Spectrographic analysis. For all rats on each trial, vocalizations
during the first 5 s of the CS period, during US presentation, and for
the 2,000-ms interval following US offset were recorded on stereo
cassette tape (Optimus SCT-36 high-speed stereo cassette deck,
Tandy, Ft. Worth, TX) for subsequent spectrographic analysis. The
unfiltered vocalizations were recorded on one track of the cassette
tape. The other track recorded three different computer-generated
tones that corresponded to the three intervals described above and
thereby permitted identification of VCRs, VDSs, and VADs. For
sonographic analysis the audiotapes were digitized (44.1-kHz sampling
rate) by an Audiomedia card (Digidesign Inc., Menlo Park, CA)
installed in a NuBus slot of a Macintosh Ilfx computer. Digital
recording was controlled by the Tape Deck module of Sound Designer
II software (Digidesign Inc.). This software was also used to display
both channels (vocalizations and tones) of the audiotapes and to select
the vocalizations to be analyzed. Selected vocalizations were trans-
ferred to Soundscope/8 software (GW Instruments Inc., Somerville,
MA) for sonographic analysis. Spectrograms were produced using the
Spectrogram module with the display range set at 0-20 kHz and the
number of data points used by the Fast Fourier Transform (FFT)
algorithm set at 2,048. Power spectra were generated using the
Average Spectrum module with the display range and number of FFT
points set as for spectrogram production.
Procedure
Eight rats were randomly assigned to each of five different groups
based on the intensity of tailshock used during training (i.e., 0.15 mA,
0.20 mA, 0.40 mA, 0.60 mA, or 0.80 mA—paired groups). An
additional group of eight rats received explicitly unpaired presenta-
tions of the CS and US (0.80 mA—unpaired controls). Rats were
adapted to the apparatus for 30 min on the day prior to the beginning
of training. Training was conducted on the next 5 consecutive days.
On each training day, rats were placed in the apparatus and given 10
min of adaptation. Paired groups received 15 trials per day of paired
presentations of the CS and US on a variable interval (VI) 2.5-min
schedule (range = 2-3 min). Presentation of the US overlapped with
the last 1 s of the 6-s light CS. The unpaired control group received 15
unpaired presentations per day of the CS and US on a VI 1.25-min
schedule (range = 1-1.5 min). Stimuli were presented in a pseudoran-
dom order with the restriction that the CS or US could not be
presented on four consecutive trials and that the order of presentation
had to differ on each day. Vocalizations and tail movements were
recorded in the same manner as for the paired groups. For both
US-alone and CS-alone trials, responses were measured during the CS
period, US period, and post-US period irrespective of whether the CS
or US was presented.
 PAVLOVIAN CONDITIONAL VOCALIZATIONS 651

VDS
Data Analysis
D1T1
Each rat was evaluated to determine whether a criterion level of
conditioning had developed. Rats that generated VCRs on at least 8 of
the 15 trials on Day 5 were judged to have met the criterion for
conditioning, and their data were analyzed separately from those of
rats that failed to meet the criterion. Increases in the percentage of
VCRs generated by each group over the 75 conditioning trials
(CS-alone trials for the unpaired controls) were analyzed with one-
factor repeated measures analysis of variance (ANOVA). Differences
between paired and unpaired groups in the percentages of rats
generating VCRs were assessed by two-factor repeated measures
ANOVA. Changes in the latency, amplitude, duration, and number of
VCRs generated during the course of conditioning were also assessed
with one-factor repeated measures ANOVA. On trials during which
VCRs were not generated, latency was arbitrarily assigned as 5,000 ms.
Differences between groups at the end of training were assessed over
the last block of three trials via Student's t tests for independent groups
with the alpha level divided by the number of comparisons (Bonferroni
correction).
URs generated only on the first trial (first US-alone trial for
unpaired controls) were analyzed because VCRs contaminated UR
assessment on subsequent trials. Group differences in UR perfor-
mance were analyzed with one-factor ANOVA. Three rats from the
0.15-mA group did not generate VADs, and the latency of the VAD
was arbitrarily assigned as 2,000 ms. Pairwise comparisons between
groups were made via Student's t test for independent groups with
Bonferroni corrections of the alpha level.
For each rat that exhibited the criterion level of conditioning, three
VCRs were subjected to spectrographic analysis. The total number of
trials in which VCRs were generated was counted, and the first,
middle, and last trials were analyzed. When multiple VCRs were
generated on a trial, a middle response was selected for analysis. For
all rats, VDSs and VADs on the first trial were subjected to Figure 2. Oscilloscope (MacScope, Hanover, NH) records from a rat
spectrographic analysis. The first VDS and first VAD were generally in the 0.80-mA group during the first 2 days of training. Each day (D)
selected when multiple responses were present. The frequencies of the consisted of 15 trials (T). Integrated vocalizations are represented in
fundamental and the harmonics of each vocalization were calculated the upper trace and spinal motor reflexes (SMRs) in the bottom trace
from the peaks of the power spectra by an experimenter unaware of of each record. The bars between the traces indicate the 5-s condi-
the type of vocalization being analyzed. tional stimulus (CS) period (open bar), the 1-s unconditional stimulus
(US) period (stippled bar), and the 2-s post-US period, (solid bar).
Conditional vocalization responses (VCRs) were assessed during the
Results CS period, vocalizations during shock (VDSs) were assessed during
Pairs of oscilloscope traces over the first 2 days of training the US period, and vocalization afterdischarges (VADs) were assessed
during the post-US period.
from a rat in the 0.80-mA group are shown in Figure 2. In each
pair the top trace represents VCRs, VDSs, and VADs, and the
bottom trace represents SMRs. For the rat depicted in Figure the 0.15-mA-COND (met criterion) and the 0.15-mA-NC
2, VCRs developed during the 1st day of training (D1T1- (failed to meet criterion) groups.
D1T15; D = day, T = trial) and increased in number, ampli-
tude, and duration while decreasing in latency from CS onset. Percentage VCR
VCRs were maintained over the 24-hr intersession interval as
reflected by their generation on the first trial of Day 2 (D2T1). Increases in the percentage of rats generating VCRs during
Over the course of Day 2, the number, amplitude, and conditioning are shown in Figure 3. For the sake of clarity,
duration of VCRs continued to increase and the latency percentage responding is presented as the means of blocks of
continued to decrease. By the conclusion of Day 2, the three consecutive trials, and symbols are presented for every
amplitude of VCRs was similar to the amplitude of VDSs and second block. Percentage responding on the first trial (FT) of
VADs (compare VCRs on D2T15 with VDSs and VADs on each day is also depicted. Two-factor repeated measures
D1T1). ANOVA of all paired groups that met the criterion level of
The course of conditioning depicted in Figure 2 is represen- conditioning and the unpaired controls over the course of the
tative of all rats that met the criterion level of conditioning. All 75 conditioning trials revealed significant main effects of
8 rats in the 0.20-mA, 0.40-mA, 0.60-mA, and 0.80-mA groups groups, F(5, 38) = 27.19, p < .001, and trials, F(74, 2,812) =
demonstrated the criterion level of conditioning. However, U.76,p < .001, and a significant Groups x Trials interaction,
only 4 of 8 rats from the 0.15-mA group met the criterion, and F(370, 2,812) = 1.76, p < .001. The interaction reflects the
this group was divided accordingly into two separate groups: finding that whereas all paired groups demonstrated a progres-
 652 GEORGE S. BORSZCZ
---o— 0.80mA
0.60mA
---a-- o.40mA
0.20mA
0.15mA-COND
0.1SmA-NC
UNP
Day 4
Figure 3. Percentage of rats in each group generating conditional
vocalization responses (VCRs) over the 5 days of training. Each day
consisted of 15 trials. Separate groups of 8 rats received paired
presentations of the conditional stimulus (CS) with different intensi-
ties of the tailshock unconditional stimulus (US; 0.80 mA, 0.60 mA,
0.40 mA, and 0.20 mA). The 0.15-mA-NC group (n = 4) failed to
reach the conditioning criterion, and its data were analyzed separately
from those of the 0.15-mA-COND group (n = 4) that met the
criterion. All rats from all other paired groups met the conditioning
criterion. Unpaired controls (UNP; n = 8) received explicitly unpaired
presentations of the CS and US (0.80 mA). Percentage responding
during CS-alone trials is depicted for the UNP group. For the sake of
clarity, percentage responding is presented as the means of blocks of
three consecutive trials, and symbols are presented for every second
block. Percentage responding on the first trial (FT) of each day is also
represented.
sive increase in percentage responding [for the 0.20-mA,
0.40-mA, 0.60-mA, and 0.80-mA groups, allFs(74,518) > 4.0,
slips < .001; for the 0.15-mA-COND group, F(74,222) = 2.83,
p < .001], the unpaired controls did not demonstrate a signifi-
cant change in percentage responding during training, F(74,
518) = 1.03, p > .40. Individual comparisons of paired groups
with unpaired controls also revealed significant Groups x
Trials interactions (all Fs > 2.0, allps < .001). On the other
hand, the 0.15-mA-NC group did not exhibit an increase in
percentage responding during training, F(74, 222) = 1.23,p >
.10, and did not differ from the unpaired controls (F < 1).
The results observed over all 75 trials were also observed
when only the first trials of each day were analyzed. These
results demonstrate that the conditioning that develops during
each daily session is maintained during the 24-hr intersession
intervals. Overall two-factor repeated measures ANOVA over
the first trials of each day revealed significant main effects of
groups, F(5, 38) = 9.69,p < .001, and trials, F(4,152) = 33.35,
p < .001, and a significant Groups x Trials interaction, F(20,
152) = 1.91, p < .05. As with the analysis of all trials, the
interaction reflects the observation that only paired groups
that met the conditioning criterion exhibited a significant
increase in percentage responding (all Fs > 2.6, all ps < .05).
The unpaired controls and the 0.15-mA-NC group did not
demonstrate an increase in first-trial responding during train-
ing (Fs < 1).
As depicted in Figure 3, the rate of conditioning was found
to be directly related to the intensity of the tailshock US.
Tailshock intensity was found to be inversely related to the
number of trials required by groups to reach at least 80%
responding (Pearson's r = -.93). Nevertheless, no differences
were observed between paired groups in percentage respond-
ing at the end of training. One-way ANOVA of percentage
responding over the last block of three trials on Day 5 revealed
no significant differences between paired groups that had met
the conditioning criterion, F(4, 31) = 1.07, p > .35.
No group demonstrated an increase in tail movements
during CS presentations. Occasionally tail movements were
recorded during the CS period, but examination of oscillo-
scope records indicated that these tail movements were small
(less than 4.7 mm) and coincided with VCRs. When multiple
VCRs were produced, the SMR trace revealed small tail
movements that lagged slightly behind the VCRs. These tail
movements were judged to be movement artifacts associated
with vocalizing and were not subjected to further analysis. The
intermittent occurrence of these tail movements presumably
reflects slight postural adjustments made by the animal.
VCR Performance
Changes in the number, amplitude, duration, and latency of
VCRs in paired groups during the course of conditioning and
differences in these performance variables as a function of US
intensity are depicted, respectively, in Figures 4, 5, 6, and 7.
Over the course of the 75 conditioning trials all paired groups
demonstrated a progressive increase in the number (all
Fs > 2.4, allps < .001), amplitude (all Fs > 2.6, allps < .001),
and duration (all Fs > 2.2, all ps < .001) of VCRs and a
progressive decrease in the latency of VCRs from CS onset (all
Fs > 2.4, allps < .001). Identical results were obtained when
performance was analyzed across first trials (for number, all
Fs > 3.9, allps < .05; for amplitude, all Fs > 3.8, allps < .05;
for duration, all Fs > 3.3, all ps < .05; and for latency, all
Fs > 3.1, allps < .05).
Like the rate of conditioning, the performance of VCRs was
also related to US intensity. The mean number, mean ampli-
tude, and mean duration of VCRs on the last block of three
trials on Day 5 were directly related to US intensity: Pearson's
« = .98, .95, and .94, respectively. The mean latency of VCRs
over these trials was inversely related to US intensity (Pear-
son's r = —.79). Unlike percentage responding, VCR perfor-
mance differed among paired groups at the end of training.
One-way ANOVA over the last block of three trials on Day 5
revealed significant group differences on all four performance
variables: number, F(4, 31) = 7.47, p < .001; amplitude, F(4,
31) = 18.71,p < .001; duration, F(4, 31) = 7.19,p < .001; and
latency, F(4, 31) = 12.22, p < .001. Pairwise group compari-
sons with the 0.80-mA group revealed that VCRs were
significantly reduced in number, amplitude, and duration and
significantly elevated in latency in the 0.15-mA-COND and
 PAVLOVIAN CONDITIONAL VOCALIZATIONS 653

on all three performance variables, all Js(6) > 4.7, all ps <
.001. Moreover, the VAD performance of the rat in the
0.15-mA-NC group that generated the response did not
overlap with the VAD performance of the 0.15-mA-COND
group. The VADs generated by the rat in the 0.15-mA-NC
group were reduced compared with the smallest VADs gener-
ated by the 0.15-mA-COND group: for the 0.15-mA-COND
group, latency = 2,089 ms, amplitude = 9.2 dB above criterion,
and duration = 254 ms; for the 0.15-mA-NC rat, latency =
FT Day 2 FT 3
Day
2,102 ms, amplitude = 4.2 dB above criterion, and duration =
104 ms. These results indicate that the tailshock US must
generate VADs of a minimum intensity for it to support VCR
conditioning.
—O— 0.80mA
—•— 0.60mA
—o— 0.40mA
Spectrographic Analysis of Vocalizations
• 0.20mA
tr-- O.ISmA-COND Representative spectrograms of VDSs, VADs, and VCRs
from rats in the 0.60-mA group are shown in Figure 11. For all
three types of vocalizations the power in each frequency band
FT Day 4 FT Day 5 increased with US intensity, but the frequencies of the funda-
mental and the harmonics remained constant across US
Figure 4. Mean number of conditional vocalization responses (VCRs)
intensity (all Fs < 1). Similarly, whereas the power in each
generated by paired groups in which all rats met the conditioning
criterion (COND). Rats received 5 days of training with 15 trials per
frequency band of VCRs increased during the course of
day. For the sake of clarity, data are presented as the means of blocks conditioning, the frequencies of the fundamental and the
of three consecutive trials, and symbols are presented for every second harmonics did not change (all Fs < 1). These results indicate
block. Mean numbers of VCRs on the first trial (FT) of each day are
also represented. All groups demonstrated a significant increase in the
number of VCRs across all trials and across FTs. Asterisks indicate
significantly fewer VCRs on the last block of trials when compared
with the 0.80-mA group (Student's t test for independent groups,
alpha = .0125).

0.20-mA groups (all ts > 3.4, attps < .01). The amplitude of
VCRs was also significantly reduced in the 0.40-mA group
compared with the 0.80-mA group, t(U) = 4.16,p < .001.

Unconditional Responses FT Day 1 FT Day 2 FT Day 3

The performance of SMRs, VDSs, and VADs of all groups

on the first training trial are shown in Figures 8, 9, and 10,
respectively. All performance variables for all three URs were
---O— 0.80mA
related to US intensity. One-way ANOVA across paired -» 0.60mA
groups that met the conditioning criterion revealed a signifi- —-D— 0.40mA
••— 0.20mA
cant increase in the amplitude of all three URs, all Fs(4,31) > --•A—- 0.15mA-COND
42.4, all/7S < .001, a significant increase in the magnitude of
SMRs, F(4, 31) = 41.4,^ < .001, a significant increase in the
16 18 20 21 23 25
duration of VDSs and VADs, all Fs(4, 31) > 21.5, all ps < FT Day 4 FT Day 5
.001, and a significant decrease in the latency of all three URs,
allFs(4, 31) > 10.0, all ps < .001. The 0.80-mA group did not Figure 5. Mean peak amplitude of conditional vocalization responses
differ from the unpaired controls on any performance variable (VCRs) depicted as decibels (dB) above the criterion level of back-
for any response, all ts( 14) < 1.96, all ps > .05. ground noise (57.0 dB) in the isolation chamber. Data are from paired
It is important to note that only the performance of VADs groups in which all rats met the conditioning criterion (COND). Rats
distinguished the 0.15-mA-COND and the 0.15-mA-NC groups. received 5 days of training with 15 trials per day. For the sake of clarity,
The tailshock US elicited SMRs and VDSs in all rats of both data are presented as the means of blocks of three consecutive trials,
and symbols are presented for every second block. Mean amplitudes of
groups, and the performance of these URs did not differ
VCRs on the first trial (FT) of each day are also represented. All
between groups, all rs(6) < .50. However, whereas all 4 rats in groups demonstrated a significant increase in the amplitude of VCRs
the 0.15-mA-COND group generated VADs on the first trial, across all trials and across FTs. Asterisks indicate significantly less
only 1 of 4 rats in the 0.15-mA-NC group generated VADs. intense VCRs on the last block of trials when compared with the
Consequently, the groups differed in their production of VADs 0.80-mA group (Student's t test for independent groups, alpha = .0125).
 654 GEORGE S. BORSZCZ

that the sonographic differences among vocalizations are not 5000

dependent on the intensity of the vocalization. 4000-

The mean frequencies of the fundamental and the harmon-
ics of VDSs, VADs, and VCRs are shown in Table 1. These 3000
means are collapsed across US intensity. The most striking
2000-
observation was the similarity between VADs and VCRs and
their differences with VDSs. VADs and VCRs did not differ in 1000-

their fundamental frequency or in the frequencies and number

of their harmonics, all ft (70) < 1.26, all ps > .20. Alterna- 1 3 5 6 8 10 11 13 15

tively, the frequencies of the fundamental and the first and FT Day 1 FT Day 2 FT Day 3

second harmonics of VDSs were significantly lower than those

of either VADs or VCRs, all ft (70) > 22.9, allps < .001. In
addition, whereas VADs and VCRs exhibited only two harmon- £ 4000-

ics, VDSs were composed of five or six harmonics. The second <J 3000- —It-- 0.15H1A-COND
harmonic of VADs and VCRs did not exceed 11.5 kHz, • 0.20mA
whereas the frequency of the highest harmonic of VDSs was 2000- — -0-- 0.40mA
never less than 14.0 kHz. These results demonstrate that 1000-
•— 0.60mA
VADs and VCRs are discriminable from VDSs on the basis of o— 0.80mA

their sonographic characteristics. 16 18 20 21 23 25

Discussion
The development of audible vocalizations in the rat that is
described in the present study is consistent with established
principles of Pavlovian conditioning (Gormezano, 1966; Ka-
FT Day 4 FT Day 5
Figure 7. Mean latency (in milliseconds) from conditional stimulus
(CS) onset to the first conditional vocalization response (VCR). Data
are from paired groups in which all rats met the conditioning criterion
(COND). Rats received 5 days of training with 15 trials per day. For
the sake of clarity, data are presented as the means of blocks of three
consecutive trials, and symbols are presented for every second block.
Mean VCR latencies on the first trial (FT) of each day are also
represented. All groups demonstrated a significant decrease in VCR
latency across all trials and across FTs. Asterisks indicate significantly
longer VCR latency on the last block of trials when compared with the
0.80-mA group (Student's nest for independent groups, alpha = .0125).

min & Brimer, 1963; Pavlov, 1927), and therefore I conclude

that these vocalizations are CRs. The failure of VCRs to
develop in the group that received explicitly unpaired presen-
FT Day 1 FT Day 2 FT Day 3 tations of the CS and US demonstrates that VCRs arise as a
result of contingent presentation of the two stimuli rather than
from sensitization or pseudoconditioning. The maintenance of
VCRs over the 24-hr intersession intervals also demonstrates
that the within-session increases in percentage VCR and VCR
o— 0.80mA
-•— 0.60mA
performance are not the result of sensitization generated by
---D-- 0.40mA repeated presentation of the tailshock US. The direct relation-
-•— 0.20mA ship between US intensity and both the rate of conditioning
A— 0.15mA-COND
and the performance of VCRs is also consistent with estab-
lished parametric features of Pavlovian CRs.
The VCR also satisfies the requirements of a suitable model
system for analyzing the neuronal basis of learning in verte-
Figure 6. Mean duration (in milliseconds) of conditional vocalization brates (Kapp & Pascoe, 1986; Thompson, 1976). A Pavlovian
responses (VCRs) during the 5-s conditional stimulus (CS) period. CR is recommended because the paradigm permits control
Data are from paired groups in which all rats met the conditioning over the stimuli (CS and US) that produce the response and
criterion (COND). Rats received 5 days of training with 15 trials per allows precise measurement of the CR. These features permit
day. For the sake of clarity, data are presented as the means of blocks development of the CR to be characterized under a variety of
of three consecutive trials, and symbols are presented for every second stimulus conditions, and differences in CR development can
block. Mean durations of VCRs on the first trial (FT) of each day are
also represented. All groups demonstrated a significant increase in the
then be correlated with electrophysiological activity in poten-
duration of VCRs across all trials and across FTs. Asterisks indicate tial neural substrates. For example, activity in the neural
significantly shorter duration of VCRs on the last block of trials when substrates responsible for generating VCRs should reflect the
compared with the 0.80-mA group (Student's t test for independent VCR's sensitivity to US intensity with respect to the rate of
groups, alpha = .0125). conditioning and performance (latency, duration, and ampli-
 PAVLOVIAN CONDITIONAL VOCALIZATIONS 655

SMR Because these neurophysiological techniques are limited by

200 -i the length of time neural activity can be recorded, it is
necessary for the CR to develop rapidly. Maintenance of the
•" 150 CR between sessions permits evaluation of the effects of
various manipulations on the retention versus acquisition of
the CR. Finally, it is recommended that the efferent motor
» 100-
pathways responsible for generating the CR be identifiable.
Localization of the motor neurons that produce the CR
50 H provides the basis for experiments designed to identify the
VDS
400n

.15mA .15mA .20mA .40mA ,60mA .80mA UNP

NC COND — 300-

60-
» 200
50-

0)
•o 40- 100-

30-

20- ,15mA ,15mA ,20mA ,40mA ,60mA ,80mA UNP

NC COND
c
CO 10-
o 40-1

.15mA .15mA ,20mA .40mA .60mA .80mA UNP

400-
NC COND
l«
< « 20-

0
•o 300- 10-
'«' CD
•o
x
200-
E .15mA ,15mA ,20mA ,40mA ,60mA ,80mA UNP
NC COND
IB
0) 100-
800-

(A
E
.15mA .15mA ,20mA ,60mA ,80mA UNP ^ 600-
NC COND
o
Groups
« 400-
3
Figure 8. Mean performance of the spinal motor reflex (SMR) on the Q
first conditioning trial for paired groups (n = 8) and on the first 200-
unconditional stimulus (US)-alone trial for unpaired controls (UNP;
n = 8). Paired groups are designated by the intensity of the tailshock
US; the UNP group received 0.80-mA tailshock. The performance ,15mA ,15mA ,20mA ,40mA ,60mA ,80mA UNP
variables measured were latency (in milliseconds) from US onset, peak NC COND
amplitude (in millimeters), and magnitude (in centimeters x millisec- Groups
onds). Peak amplitude and magnitude increased, and latency de-
creased, across paired groups that received increasing levels of Figure 9. Mean performance of vocalizations during shock (VDSs) on
tailshock. The UNP group did not differ from the 0.80-mA group on the first conditioning trial for paired groups (n = 8) and on the first
any measure. The 0.15-mA-NC group (n = 4) failed to reach the unconditional stimulus (US)-alone trial for unpaired controls (UNP;
conditioning criterion, and its data were analyzed separately from n = 8). Paired groups are designated by the intensity of the tailshock
those of the 0.15-mA-COND group (« = 4), which met the criterion. US; the UNP group received 0.80-mA tailshock. The performance
These groups did not differ on any measure. variables measured were latency (in milliseconds) from US onset, peak
amplitude (in decibels above criterion background noise), and dura-
tion (in milliseconds). Peak amplitude and duration increased, and
latency decreased, across paired groups that received increasing levels
tude). Another recommended characteristic of a CR, which is
of tailshock. The UNP group did not differ from the 0.80-mA group on
met by the VCR, is that it develop to a significant degree within any measure. The 0.15-mA-NC group (n = 4) failed to reach the
one session and endure between sessions. Single-session acqui- conditioning criterion, and its data were analyzed separately from
sition is largely a practical requirement of studies designed to those of the 0.15-mA-COND group (n = 4), which met the criterion.
correlate electrical activity of a neuron with CR development. These groups did not differ on any measure.
 656 GEORGE S. BORSZCZ

2000
VAO nucleus, facial nucleus and hypoglossal nucleus, and expiratory
motor neurons located in the ventral horn of the lower thoracic
and upper lumbar spinal cord. These motor neurons generate
the laryngeal, articulatory, and respiratory activity that consti-
tute a vocalization. Sources of afferent projections to these
motor neurons that coordinate their activity to produce vocaliza-
tions and possibly VCRs are discussed in the following section.

VCRs as a Model of the Fear of Pain

The "two-process" theory of Pavlovian conditioning pro-
posed by Konorski (1967) and adapted by others (Borszcz,
1993; Thompson et al, 1986; Wagner & Brandon, 1989;
Weinberger, 1982) posits that an aversive US is composed of
two separate but interrelated sensory attributes. Within this
framework, the VCR is a CR that reflects the association of the
CS with the affective-motivational (protopathic-emotive) at-
tributes of an aversive US. These attributes of the US are
believed to generate an unconditional fear state in the animal,
and therefore VCRs are indicative of the conditioning of a
central fear state. In addition, the sensory-discriminative
(epicritic-sensory) attributes of an aversive US support the
.15mA .60mA ,80mA conditioning of CRs that mimic the withdrawal reflexes elic-
COND
ited by the US (i.e., SMRs in the present study). These
attributes of the US code its particular sensory dimensions
-~ 1200- (location, timing, and modality), and therefore these CRs are
ooo- believed to be adaptive to the particular US that was used.
Furthermore, the conditioning of adaptive CRs is proposed to
800-
be supported by the conditional fear state. This interrelation-
3 600- ship is thought to account for the observation that develop-
o ment of adaptive CRs requires substantially more conditioning
400-
a trials than do CRs that represent the conditional fear state.
a> 200-
S # Consequently, the failure to observe conditional SMRs in the
present study may have been the result of insufficient training.
.]5mA .ISmA .20mA .40mA .60mA ,80mA UNP The development of Pavlovian conditional tail flexion has been
NC COND
Groups reported to require 200-500 conditioning trials (Miller, Greco,
& Vigorito, 1981), compared with the 75 trials used in the
present study.
Figure 10. Mean performance of vocalization afterdischarges (VADs) Although VCRs generated in the present study reflect a
on the first conditioning trial for paired groups (n = 8) and on the first conditional fear state, it is necessary to establish that these
unconditional stimulus (US)-alone trial for unpaired controls (UNP;
responses represent the anticipation of pain if they are to be
n = 8). Paired groups are designated by the intensity of the tailshock
US; the UNP group received 0.80-mA tailshock. The performance used as a model system of analyzing the fear of pain. This issue
variables measured were latency (in milliseconds) from US offset, peak turns on the determination of whether the US is noxious.
amplitude (in decibels above criterion background noise), and dura- When evaluating whether or not a stimulus to be used in
tion (in milliseconds). Peak amplitude and duration increased, and animal studies is noxious it is necessary to determine whether
latency decreased, across paired groups that received increasing levels it generates pain in humans and selectively activates primary
of tailshock. The UNP group did not differ from the 0.80-mA group on nociceptive afferents (Hammond, 1989). As discussed above,
any measure. The 0.15-mA-NC group (n = 4) failed to reach the the tailshock US used in the present study was modeled after
conditioning criterion, and its data were analyzed separately from the stimulus described by Bromm and Meier (1984) and used
those of the 0.15-mA-COND group (« = 4), which met the criterion. extensively in the study of pain and analgesia in humans (see
These groups differed on all measures of VAD performance. Asterisk review by Bromm, 1989). The intracutaneously pulsed DC
indicates Student's Mest for independent groups, p < .001.
current predominately activates peripheral nociceptors (A-
delta and C fibers) and generates reports of sharp pain and
burning in humans. I have confirmed from personal observa-
afferent links in the neural circuit responsible for its produc- tion that these are the sensations generated by the stimulus
tion. As previously noted, the mammalian vocalization circuit used as the US in the present study, and therefore VCRs may
has been described in considerable detail (see review by Sutton reasonably be viewed as reflecting the anticipation of pain.
& Jurgens, 1988). Its most efferent level consists of phonotory On the other hand, the superficially administered 60-Hz AC
motor neurons located in nucleus ambiguus, trigeminal motor current that is often used in studies of aversive conditioning is
 PAVLOVIAN CONDITIONAL VOCALIZATIONS 657

A. Calc
El T
20
KHz
F2 •18
F3
F4
16

14

12

10

0
0.0 118-4 36.8 355.2 473.7 592.1 7J&, 828.9 947.3 msec

B. Calc 20
PI KHz
F2
18
P3
F4
16

14

12

^HI****"^ r*. 10

Htiate v^Miitr.r.

0 110 220 330 440 551 661 771 881 991 1101 1211 1321 1432 1542 1652 1762 1872

Figure 11. Spectrograms from rats in the 0.60-mA group. Time (milliseconds) is represented on the
abscissa and frequency (kilohertz) on the ordinate. A: Vocalization during shock (VDS; left) and
vocalization afterdischarge (VAD; right) from the same rat on the first conditioning trial. These
vocalizations were selected from the trial record, and therefore the time scale does not reflect their actual
temporal association. B: Conditional vocalization responses (VCRs) from three different rats. These
vocalizations were taken from the middle trial of all the trials in which the rats generated VCRs. The left
spectrogram is from the animal whose VDS and VAD are depicted in A. Note the different time scales in
AandB.

likely not to be noxious. This form of stimulation generates
sensations of vibration and pressure without pain in human
participants at intensities up to 4 mA. At higher intensities, an
increasingly aversive, but not painful, paresthesia is produced
with a steady transition to painful sensation from 4.6 mA to 5.4
mA (Bromm, 1989; Bromm & Meier, 1984; Price & Tursky,
1975). Studies that use this stimulus as a US typically do not
use intensities in the painful range. Consequently, the fear
conditioning supported by this form of stimulation likely
reflects the anticipation of the aversive but non-noxious
attributes of the US.
The reliance of VCR acquisition on the capacity of the US to
generate a minimum intensity of VAD also supports the
proposition that VCRs reflect the fear of pain. I have previ-
658 GEORGE S. BORSZCZ
Table 1
Mean Frequencies (in kilohertz) of the Fundamental and the
Harmonics of Vocalizations During Shock (YDS), Vocalization
Afterdischarges (VAD), and Conditional
Vocalization Responses (VCR)
Vocalization
YDS VAD VCR
Harmonics M SE M SE M SE
Fundamental 2.61 ±.022 3.66* ±.027 3.56* ±.028
1 5.02 ±.043 7.27* ±.047 7.14* ±.042
2 7.75 ±.060 10.26* ±.055 10.16* ±.065
3 10.04 ±.070
4 12.85 ±.102
5 14.63 ±.123
6 16.47 ±.060
Note. VAD and VCR did not differ on any measure. Means are
collapsed across unconditional stimulus intensity. *Comparison of
VDS with either VAD or VCR on the frequencies of the fundamental
or the corresponding harmonics, independent Student's t test, p <
.001.
ously reported that the capacity of noxious tailshock to support
the conditioning of an avoidance response in rats also relies on
its generation of VADs, and I argued that VADs reflect
processing of the affective-motivational attributes of this
noxious US that are critical to the conditioning of a fear state
that reflects the anticipation of pain (Borszcz, 1993, 1995).
That VADs reflect processing of the emotional attributes of
the pain experience is supported by pharmacological and
neuroanatomical evidence. Drug treatments (i.e., systemically
administered morphine, fentanyl, benzodiazepines, or neuro-
leptics) that have been shown to preferentially suppress the
emotional component of human pain (C. R. Chapman &
Feathers, 1973; Gracely, McGrath, & Dubner, 1978; Price,
Harkins, Rani, & Price, 1986; Price, Von der Gruen, Miller,
Rani, & Price, 1985) have been shown to preferentially raise
the threshold of VADs (Borszcz, Johnson, & Fahey, 1994;
Carroll & Lim, 1960; Hoffmeister, 1968; Levine et al, 1984). In
addition, VADs rely on rhinencephalic-diencephalic struc-
tures for their production (Carroll & Lim, 1960; Hoffmeister,
1968). Although the neural circuit responsible for the genera-
tion of VADs has not yet been described, the mammalian
vocalization circuit is composed of rhinencephalic (anterior
cingulate cortex, amygdala), thalamic (medial and intralami-
nar nuclei), and midbrain (dlPAG) structures that underlie the
negative emotional state associated with pain in humans
(Ballentine et al., 1967; Brown, 1977; Bouckoms, 1984; W. P.
Chapman et al., 1954; Corkin & Hebben, 1981; Foltz & White,
1962; Gloor et al., 1981; Hassenbusch et al., 1990; Jelasic, 1966;
Mark et al., 1963; Meyer et al., 1973; Nashold et al., 1974;
Schvarcz, 1977; White & Sweet, 1969). I have postulated,
therefore, that the development of CRs that reflect the fear of
pain may depend on the capacity of a noxious US to generate
neural activity in the rhinencephalic-diencephalic circuit that
is responsible for the production of VADs.
VCRs and the Mammalian Vocalization Circuit
It is tempting to speculate that because VADs and VCRs
share sonographic characteristics they may also share underly-
ing neural substrates. An examination of the mammalian
vocalization circuit may therefore provide insight into the
forebrain structures responsible for production of these re-
sponses. The motor neurons that generate the phonotory and
respiratory activity that constitute a vocalization require facili-
tatory input from the dlPAG and laterally boarding tegmen-
tum in order for a vocalization to be initiated. The dlPAG
projects directly and indirectly (via the lateral reticular forma-
tion of the pons and medulla) to nucleus ambiguus, trigeminal
motor nucleus, and nucleus retroambiguus, which contains
respiratory premotor neurons (Holstege, 1989; Jiirgens &
Ploog, 1988). These projections of the dlPAG coordinate the
activity of motor neurons to produce a vocalization. Lesions of
the dlPAG lead to total or partial mutism without paresis of
the vocal folds (Adametz & O'Leary, 1959; Blanchard, Wil-
liams, Lee, & Blanchard, 1981; Chaurand, Vergnes, & Karli,
1972; Jiirgens & Pratt, 1979b). Correspondingly, electrical or
chemical stimulation of the dlPAG yields natural-sounding
vocalizations, whereas stimulation of nucleus ambiguus is
either ineffective or produces vocalizations that are acousti-
cally degraded and not within the animal's repertoire (Jiirgens,
1991; Jiirgens & Ploog, 1970, 1988; Magoun et al., 1937). In
addition, the intensity of electrical stimulation of the dlPAG is
directly related to the loudness of the elicited vocalization and
to the magnitude of laryngeal and respiratory muscle contrac-
tions (P. J. Davis & Zhang, 1991; Larson, 1991b; Larson,
Ortega, & DeRosier, 1988). The coordinating function of the
dlPAG is also supported by the results of studies that have
recorded single-unit activity in this structure from monkeys
trained in an operant paradigm to vocalize in order to obtain a
reward (Larson, 1991a, 1991b; Larson & Kistler, 1986). Unit
activity in the dlPAG was shown to anticipate the operant
conditioned vocalizations, and discharges were correlated with
the intensity and duration of both the vocalization and EMG
recordings of laryngeal and respiratory muscles.
The type of vocalization that is generated is believed to be
determined by inputs from the limbic forebrain (amygdala,
anterior cingulate cortex, substantia innominata, anterior per-
forated substance, stria terminalis, septum, preoptic area,
nucleus accumbens, zona incerta, ventral tegmental area,
midline thalamus, lateral hypothalamus) to various rostro-
caudal subdivisions of the dlPAG. Stimulation of different
regions along the rostro-caudal extent of the dlPAG has been
reported to elicit many different call types in cats and monkeys
(Handler & Carrive, 1988; Jiirgens & Ploog, 1970; Larson,
1990; Zhang, Bandler, & Carrie, 1990) and sonic versus
ultrasonic vocalizations in rats (Bandler & Depaulis, 1991;
Jensen & Yaksh, 1992; Keay, Depaulis, Breakspear, & Ban-
dler, 1990). For the monkey, these calls are known to accom-
pany different social interactions in a natural setting that
reflect disparate affective states (Grimm, 1967; Jiirgens, 1979).
Alternately, stimulation of limbic sites that project to the
dlPAG typically produce only one or a few call types in monkeys
(Jiirgens & Ploog, 1970; Robinson, 1967).
Accordingly, Jiirgens and Pratt (1979b) described one func-
tion of the dlPAG as integrating specific motivational states
(via limbic forebrain input) with their corresponding vocal
expressions (via outputs to nucleus ambiguus and nucleus
retroambiguus) by coordinating the internal and external
 PAVLOVIAN CONDITIONAL VOCALIZATIONS
stimuli that induce an animal to generate a vocalization.
Within this framework, a particular motivational state may be
characterized by the pattern of limbic input(s) to specific
subdivisions of the dlPAG. One behavioral manifestation of
this pattern of dlPAG activation is the production of a specific
call type. VADs may therefore be viewed as providing a
"readout" (cf. M. Davis, 1989) of the neural activity within the
limbic-midbrain circuit responsible for processing the affective-
motivational attributes of a noxious US that yields an uncondi-
tional fear reaction. VCRs, in turn, may reflect activation of a
similar limbic-midbrain circuit by a CS that has become
associated with the unconditional fear state. This interpreta-
tion is supported by the observation that lesions of the central
nucleus of the amygdala prior to training suppressed both
VADs and the acquisition of VCRs (Borszcz & Leaton, 1994).
The involvement of the amygdala in processing the affective-
motivational attributes of noxious stimuli has been previously
discussed (Borszcz, 1995).
The anterior cingulate cortex may permit the discrimination
of limbic sites that contribute to the production of VADs
versus VCRs. Lesion studies have demonstrated that monkeys
trained on a discriminative operant conditioning task to
vocalize either to obtain food reward or to postpone an electric
shock lose the ability to perform following ablation of the
anterior cingulate cortex (Aitken, 1981; Sutton, Larson, &
Lindeman, 1974; Sutton, Samson, & Larson, 1978). A similarly
conditioned operant lever-press response was retained by
these animals following the lesion. Correspondingly, single-
unit activity in the anterior cingulate cortex was found to
change prior to the utterance of a conditioned vocalization
(Sutton et al., 1978). On the other hand, vocalizations re-
corded between conspecifics were not altered in their rate of
occurrence, variety, or acoustic structure following ablation of
this limbic site (Franzen & Myers, 1973; Kirzinger & Jiirgens,
1982; Trachy, Sutton, & Lindeman, 1981).
These findings, coupled with the observations that the
anterior cingulate cortex is the only limbic site at which
stimulation generates a variety of call types and that the calls
are not accompanied by an emotional reaction, led to the
proposition that the anterior cingulate cortex mediates direct
control over "volitional" (conditional) vocalizations but not
unconditional species-specific vocalizations (Jiirgens, 1976;
Kirzinger & Jiirgens, 1982). The conditional emotional state
that generates the conditional vocalization is proposed to be
transmitted to the anterior cingulate cortex via direct projec-
tions from the other limbic sites at which stimulation-elicited
vocalizations are accompanied by emotional reactions (Jiir-
gens, 1976; Jiirgens & Muller-Preuss, 1977). Production of the
conditional vocalization is coordinated by the dlPAG, which
receives direct projections from the anterior cingulate cortex
(Jurgens & Muller-Preuss, 1977).
The Ethological Basis of VCRs
An evaluation of the circumstances under which rats in a
natural setting emit audible vocalizations may provide insight
into the particular features of the paradigm that support VCR
acquisition and thereby provide additional insight into the
neuronal substrates of VCRs. When rodents are approached
659
by a predator, they will display various defensive behaviors
depending on the proximity of the predator and the availability
of escape routes (Blanchard & Blanchard, 1987; Chance, 1962;
Fanselow, 1989). When contact with the predator is imminent
(distance less than 0.5 m) and an escape route is not available,
rodents will engage in defensive threat-attack behaviors. The
rat will rise up to face the predator, display its teeth, and emit
audible vocalizations. Continued approach by the predator will
elicit a jump-attack (Blanchard, Flannelly, & Blanchard,
1986). A good illustration of the former behaviors is shown in a
photograph provided by Flynn (1967, Figure 1) that depicts the
"affective" attack of a cat (elicited by hypothalamic stimula-
tion) on a rat.
I propose that VCRs represent a conditional defensive
reaction that mimics a component of the defensive threat-
attack response. Fanselow (1989) previously demonstrated
that freezing, a defensive behavior of a rodent to a distant
predator, can come under the associative control of contextual
cues when freely moving rats receive footshock in an observa-
tion chamber. In the present study, the use of a discrete visual
CS presented near the rat in its upper visual field coupled with
restraint provides an approximation of a cat's attack on a
cornered rat. That these features are critical to determining
the type of conditional defensive behavior that is elicited is
supported by my observation of rats that were trained in a
passive avoidance paradigm using tailshock (Borszcz, 1993).
When tested, they did not vocalize but rather engaged predomi-
nately in freezing and the stretched attention posture (a
defensive reaction to low levels of threat).
Conceptualization of VCRs as a conditional defensive
reaction suggests a substrate that may be responsible for
processing the CS. A class of neurons in the superior colliculus
of rats has been identified that responds preferentially to
looming stimuli presented to the upper visual field (Westby,
Keay, Redgrave, Dean, & Bannister, 1990). Redgrave and
Dean (1991) postulated that these neurons encode an ap-
proaching predator and in turn activate the dlPAG, which
coordinates the execution of defensive behaviors (also see
Redgrave, Westby, & Dean, 1993). In support of this interpre-
tation is the report that lesions of the superior colliculus
reduced a rat's defensive threat-attack reaction to an approach-
ing experimenter, but the response was initiated once contact
was made with the animal (Blanchard et al., 1981). Lesions of
the dlPAG completely eliminated the response. Correspond-
ingly, stimulation of the superior colliculus has been shown to
elicit defensive responding (Dean, Mitchell, & Redgrave,
1988; Dean, Redgrave, & Westby, 1989). Although the CS in
the present study did not loom, it was presented to the upper
visual field and it is reasonable to speculate that it may,
through its association with the US, also be processed by
neurons in superior colliculus that have been implicated in the
initiation of defensive threat-attack reactions. I am currently
investigating the efficacy of various CSs in supporting the
acquisition of VCRs.
Conclusion
The VCR is a Pavlovian conditional response that reflects
the fear of pain. Its ease of measurement and sensitivity to
 660 GEORGE S. BORSZCZ
parametric manipulations of US intensity recommend the
VCR as a model system for analyzing the fear of pain. This
recommendation is further supported by the feasibility of
identifying the neural substrates responsible for its acquisition.
Current studies are being directed toward delineating the
experimental variables that are critical to the acquisition of the
VCR, the neural circuit(s) responsible for its generation, and
the mechanisms by which it can be suppressed.
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Received October 25. 1994
Revision received January 17. 1995
Accepted January 18. 1995