Physiology & Behavior 147 (2015) 183–192
Contents lists available at ScienceDirect
Physiology & Behavior
journal homepage: www.elsevier.com/locate/phb
Psychosocial predator-based animal model of PTSD produces
physiological and behavioral sequelae and a traumatic memory
four months following stress onset
Phillip R. Zoladz a,⁎, Collin R. Park c,d, Monika Fleshner b, David M. Diamond c,d,e,f
a
Department of Psychology, Sociology & Criminal Justice, Ohio Northern University, Ada, OH 45810, USA
Department of Integrative Physiology & Center for Neuroscience, University of Colorado, Boulder, CO 80309, USA
c
Medical Research Service, VA Hospital, Tampa, FL 33612, USA
d
Department of Psychology, University of South Florida, Tampa, FL 33620, USA
e
Department of Molecular Pharmacology & Physiology, University of South Florida, Tampa, FL 33620, USA
f
Department of Center for Preclinical & Clinical Research on PTSD, University of South Florida, Tampa, FL 33620, USA
b
H I G H L I G H T S
•
•
•
•
PTSD-like sequelae induced by psychosocial stress persisted for at least 4 months.
Predator exposure generated a persistent fear-conditioned “traumatic” memory.
Psychosocial stress resulted in a long-term increase in anxiety and impaired cognition.
Predator-based psychosocial stress resulted in persistent physiological changes comparable to PTSD-like symptoms.
a r t i c l e
i n f o
Article history:
Received 27 January 2015
Received in revised form 17 April 2015
Accepted 18 April 2015
Available online 22 April 2015
Keywords:
PTSD
Stress
Animal model
Trauma
Fear
Memory
a b s t r a c t
We have a well-established animal model of PTSD composed of predator exposure administered in conjunction
with social instability that produces PTSD-like behavioral and physiological abnormalities one month after stress
initiation. Here, we assessed whether the PTSD-like effects would persist for at least 4 months after the initiation
of the psychosocial stress regimen. Adult male Sprague–Dawley rats were exposed to either 2 or 3 predatorbased fear conditioning sessions. During each session, rats were placed in a chamber for a 3-min period that terminated with a 30-s tone, followed by 1 h of immobilization of the rats during cat exposure (Day 1). All rats in the
stress groups received a second fear conditioning session 10 days later (Day 11). Half of the stress rats received a
third fear conditioning session 3 weeks later (Day 32). The two cat-exposed groups were also exposed to daily
unstable housing conditions for the entire duration of the psychosocial stress regimen. The control group received stable (conventional) housing conditions and an equivalent amount of chamber exposure on Days 1, 11
and 32, without cat exposure. Behavioral testing commenced for all groups on Day 116. The stress groups demonstrated increased anxiety on the elevated plus maze, impaired object recognition memory and robust contextual and cued fear conditioned memory 3 months after the last conditioning session. Combined data from the two
stress groups revealed lower post-stress corticosterone levels and greater diastolic blood pressure relative to the
control group. These findings indicate that predator-based psychosocial stress produces persistent PTSD-like
physiological and behavioral abnormalities that may provide insight into the neurobiological and endocrine sequelae in traumatized people with PTSD.
© 2015 Elsevier Inc. All rights reserved.
1. Introduction
⁎ Corresponding author at: Ohio Northern University, Department of Psychology,
Sociology, & Criminal Justice, 525 S. Main St., Ada, OH 45810, USA.
E-mail address: p-zoladz@onu.edu (P.R. Zoladz).
http://dx.doi.org/10.1016/j.physbeh.2015.04.032
0031-9384/© 2015 Elsevier Inc. All rights reserved.
Post-traumatic stress disorder (PTSD) is a unique psychiatric condition in that its diagnosis requires a distinct etiological event, specifically,
one or more intense and horrific traumatic experiences. Individuals who
develop PTSD following trauma exposure experience significant psychological distress by repeatedly reliving the trauma through intrusive,
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flashback memories [1–4]. This aspect of the disorder causes PTSD patients to avoid situations that remind them of their trauma and can also
facilitate the development of an array of other debilitating symptoms,
such as persistent anxiety, an exaggerated startle response, cognitive impairments and an impaired ability to extinguish conditioned fear [5–7].
Trauma exposure is a necessary, but not sufficient, component for
both the development and persistence of PTSD. Only a subset (10–
50%) of traumatized individuals develops PTSD, depending on a multitude of interacting risk factors, including the nature of the trauma, genetics, gender, social support and early life experiences [7,8]. Our
group has published a series of studies based on a predator-exposure
animal model of PTSD that we developed [9]. The primary components
of this model were based on trauma-induction features that are known
to be associated with a greater susceptibility of a subset of traumatized
people developing PTSD. Specifically, in our model, rats are immobilized
and placed in close proximity to a cat on two separate occasions. The
stressed rats are also exposed to daily social instability (randomized
cage mates) throughout the duration of the experiment to increase
the likelihood of producing long-lasting physiological and behavioral
changes in the rats. Thus, our PTSD model incorporates key elements
of trauma, such as uncontrollability, unpredictability, lack of social support and a re-experiencing of trauma that, in people, significantly increases the risk for the development and persistence of PTSD.
We have reported that exposing rats to our psychosocial predatorbased animal model of PTSD results in a number of physiological and
behavioral abnormalities, all of which are remarkably similar to those
observed in people with PTSD. For instance, three weeks after the second predator exposure, rats administered psychosocial stress exhibited
reduced growth rate, reduced thymus weight, greater adrenal gland
weight, increased anxiety, an exaggerated startle response, impaired
memory for new information, greater cardiovascular and hormonal reactivity to an acute stressor and an exaggerated physiological and behavioral response to the α2-adrenergic receptor antagonist, yohimbine
[9]. Importantly, these effects depend on the combination of both cat exposures and daily social instability.
In more recent studies, we demonstrated that three weeks after the
second predator exposure, psychosocially stressed rats exhibited a robust fear conditioned memory for the predator stress exposures, enhanced negative feedback of the hypothalamus–pituitary–adrenal
(HPA) axis and increased methylation of BDNF DNA in the hippocampus
[10–12]. Others have replicated and extended the findings from our
model, revealing effects of predator-based psychosocial stress on neurotransmitters and neuromodulators that resemble those observed in
PTSD patients [13,14]. Thus, extensive assessments fortify the conclusion that our predator-based psychosocial stress model of PTSD results
in physiological and behavioral changes that are comparable to those
seen in people with PTSD.
One aspect of our animal model of PTSD that has yet to be addressed
is how long the physiological and behavioral changes persist. This is
clearly a relevant issue for any animal model of PTSD since traumatized
individuals commonly experience debilitating symptoms that can
persist for years after the original trauma occurred. Thus, the purpose
of the present study was to examine if the PTSD-like symptoms would
persist for 4 months following the initiation of stress and whether additional predator exposure was necessary to maintain the presence of the
PTSD-like effects.
2. Material and methods
2.1. Animals
Experimentally naïve adult male Sprague–Dawley rats (225–250 g
upon delivery) obtained from Charles River laboratories (Wilmington,
MA) were used in the present experiment. The rats were housed on a
12-h light/dark schedule (lights on at 0700) in standard Plexiglas
cages (two per cage) with free access to food and water. Upon arrival,
all rats were given 1 week to acclimate to the housing room environment and cage changing procedures before any experimental manipulations took place. All procedures were approved by the Institutional
Animal Care and Use Committee at the University of South Florida.
2.2. Psychosocial stress procedure
The experimental groups and timeline of procedures are illustrated
in Fig. 1. Following the 1-week acclimation phase, rats were brought
to the laboratory and assigned to one of two psychosocial stress groups
or a no stress control group (N = 10 rats/group). On Day 1, rats from
these groups were exposed to a chamber for 3 min. During the last
30 s of the 3-min chamber exposure, a 74-dB, 2500 Hz tone was presented to the rats. The chamber (26 × 30 × 29 cm; Coulbourn Instruments; Allentown, PA) consisted of two aluminum sides, an aluminum
ceiling, and a Plexiglas front and back. The floor of the chamber
consisted of 18 stainless steel rods, spaced 1 cm apart. The purpose of
exposing rats to the chamber was to enable rats in the psychosocial
stress groups to associate the chamber (contextual fear conditioning)
and tone [auditory (cue) fear conditioning] with the acute stress experiences (i.e., immobilization plus cat exposure, as described below).
We then measured their fear conditioned memory by assessing immobility of the rats in the chamber when they were returned to the chamber
during behavioral testing, as previously described [10,12]. Locomotor activity in the chamber was measured during the fear conditioning sessions
and fear memory testing by a 24-cell infrared activity monitor (Coulbourn
Instruments; Allentown, PA) mounted on the top of the chamber, which
used emitted infrared body heat image (1300 nm) from the animals
to detect their movement. Immobility was defined as periods of inactivity lasting at least 7 s, based on previously employed methodology [10,12].
Following the 3-min chamber exposure, rats in the psychosocial
stress groups were removed from the chamber and immediately
immobilized in plastic DecapiCones (Braintree Scientific; Braintree,
MA), and then they were placed in a perforated wedge-shaped Plexiglas
enclosure (Braintree Scientific; Braintree, MA; 20 × 20 × 8 cm). The rats,
immobilized in the plastic DecapiCones within the Plexiglas enclosure,
were taken to the cat housing room where they were placed in a metal
cage (61 × 53 × 51 cm) with an adult female cat for 1 h. The Plexiglas enclosure prevented any contact between the cat and rats, but the rats were
still exposed to all non-tactile sensory stimuli associated with the cat.
Canned cat food was smeared on top of the Plexiglas enclosure to direct
the cat's attention toward the rats. An hour later, the rats were brought
to the laboratory, returned to their home cages and then were returned
to the vivarium. Rats in the no stress group were not immobilized or exposed to the cat; after the chamber exposure, they were placed in their
home cages in the laboratory where they remained for 1 h, and then
they were returned to the vivarium.
The first and second stress sessions were separated by a period of
10 days (i.e., Day 1 and Day 11), as per our previously employed methodology [9–12]. The second and third stress sessions were separated by
a period of 21 days (i.e., Day 11 and Day 32). Thus, the third stress
session occurred when, in previous work, we had initiated behavioral
testing. Rats that had been assigned to the first psychosocial stress
group (Stress × 2) were immobilized and exposed to the cat during
the first two, but not the third, stress sessions. During the third stress
session, rats in the “Stress × 2” group were given chamber exposure
only. Rats in the second psychosocial stress group (Stress × 3) were
immobilized and exposed to the cat during all three stress sessions.
Rats in the control group (No Stress) were given chamber exposure,
alone, during all three sessions.
The first and third acute stress sessions took place during the light
cycle, between 0800 and 1300 h, and the second acute stress session
took place during the dark cycle, between 1900 and 2100 h. The acute
stress sessions took place during different times of the day to add an
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Fig. 1. Experimental timelines and procedures. Rats in the two psychosocial stress groups were administered predator-based fear conditioning on Days 1 and 11 (Stress × 2 group) or on
Days 1, 11 and 33 (Stress × 3 group); see Material and methods for fear conditioning details. The control group (no stress) was placed in the chamber on Days 1, 11 and 33 without
subsequent cat exposure. Throughout the entire stress period (Days 1–115). Until the commencement of behavioral testing on Day 116, rats in both psychosocial stress groups were
given daily social instability. Twelve weeks following the third chamber exposure, beginning on Day 116, all rats were given a battery of behavioral tests to assess conditioned fear memory,
anxiety, startle response and novel object memory.
element of unpredictability as to when the rats could re-experience the
traumatic event.
2.3. Daily social stress
Beginning on the day of the first acute stress session (Day 1), rats in
the two psychosocial stress groups were exposed to unstable housing
conditions, as described previously, until behavioral testing (Day 116).
Rats in the psychosocial stress groups were housed two per cage, but
every day, their cohort pair combinations were changed. Therefore, no
rats in the psychosocial stress groups had the same cage mate on two
consecutive days during the 115-day stress period. Rats in the no stress
group were housed with the same cohort for the entire duration of the
experiment.
2.4. Behavioral testing
Twelve weeks following the third acute stress session (on Day 116),
all rats were given tests to measure their fear memory, anxiety, startle,
learning and memory, cardiovascular activity and corticosterone activity. On each day of behavioral testing (Days 116–120), rats were taken to
the laboratory and left undisturbed for 30 min before testing began. All
behavioral testing took place during the light cycle, between 0800 and
1500 h.
2.4.1. Contextual and cued fear memory
On Day 116, immobilization of the rats in the fear conditioning
chamber (context test) and in response to a tone (cue test) was
assessed. First, rats were placed for 5 min in the same chamber that
they were exposed to during each of the three acute stress sessions.
An hour after the 5-min context test, the rats were placed in a novel
chamber that had different lighting, walls and flooring texture. The
rats were placed in the novel chamber for a total of 6 min for a cuespecific test. After a 3-min baseline period, a 74-dB, 2500 Hz tone was
presented for the next 3 min. Immobility during the baseline period
provided a measure of general fear of a novel place, and immobility during the 3-min tone provided a measure of the fear response to the cue
that was, in the psychosocial stress groups, temporally associated with
the cat exposures.
2.4.2. Elevated plus maze
The elevated plus maze (EPM), a routine test of anxiety in rodents,
consisted of two open arms (11 × 50 cm) and two closed arms
(11 × 50 cm) that intersect each other to form the shape of a plus
sign. On Day 117, the rats were placed on the EPM for 5 min, and
their behavior was monitored by 48 infrared photobeams located
along the perimeter of the open and closed arms input to computer
software (Motor Monitor; Hamilton-Kinder; San Diego, CA). The dependent measures of interest were the percent of time rats spent in
the open arms and the number of ambulations (i.e., an indication of
overall movement) made by each rat.
2.4.3. Startle response
One hour after the EPM assessment, acoustic startle testing was
administered. The rats were placed inside a small Plexiglas box
(19 × 10 × 10 cm), which was inside a larger startle monitor cabinet
(Hamilton-Kinder; San Diego, CA; 36 × 28 × 50 cm). The small Plexiglas
box within this cabinet contained a sensory transducer on which the
rats were placed at the beginning of the trial. The sensory transducer
was connected to a computer (Startle Monitor; Hamilton-Kinder; San
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Diego, CA), which recorded the startle responses by measuring the maximum amount of force (Newtons) that rats exerted on the sensory
transducer for a period of 250 ms after the presentation of each auditory
stimulus. To control for any differences in body weight, the sensitivity of
the sensory transducer was adjusted prior to each trial via a Vernier adjustment with a sensitivity range of 0–7 arbitrary units. The startle trial
began with a 5-min acclimation period, followed by the presentation of
24 bursts of white noise (50 ms each), eight from each of three auditory
stimulus intensities (90, 100, and 110 dB). The noise bursts were presented in sequential order, and the time between each noise burst varied pseudorandomly between 25 and 55 s. Upon the commencement of
the first noise burst, the startle apparatus provided uninterrupted background white noise (57 dB).
2.4.6. Body weights
All rats were weighed before each acute stress session, as well as on
the first day of behavioral testing.
2.4.4. Novel object recognition
The novel object recognition (NOR) task was a modified version of
that which was employed by Baker and Kim [15]. On Day 118, the rats
were placed in an open field (Hamilton-Kinder; San Diego, CA;
40 × 47 × 70 cm) for 5 min to acclimate to the environment. A Logitech
camera that was mounted on the ceiling overlooking the open field
monitored rat behavior. This camera was connected to computer software (ANY-Maze; Stoelting; Wood Dale, IL) which scored rat behavior.
Twenty-four hours later (Day 119), the rats were placed in the same
open field with two identical (plastic/metal) objects for 5 min. The
objects were in opposite corners of the open field and secured to the
flooring to prevent the rats from displacing them. The objects were
counterbalanced across rats, as were the corners in which the objects
were placed. Three hours later, the rats were returned to the open
field for a final 5-min test trial, but this time, the open field contained
a replica of the object that had been there before and a novel object.
During this testing session, greater time spent by the rats in proximity
to the novel versus familiar object was an indication of intact memory
for the familiar object. The time that each rat spent with the objects during training and testing was quantified by specifying a zone around the
objects for the ANY-Maze software to score the duration of investigatory
behavior. These data were then used to calculate a discrimination index
[novel object investigatory time / (novel object investigator time +
familiar object investigatory time]. This index was then converted into
a percentage score by multiplying the result by 100. Chance performance (i.e., spending an equivalent amount of time with the novel
and familiar objects) was thus 50%.
3. Results
2.4.5. Blood sampling, cardiovascular activity and post-mortem dissections
On the final day of behavioral testing (Day 120), rats were brought,
one cage at a time, to a nearby procedure room for blood sampling.
The saphenous vein of each rat was punctured with a sterile, 27-gauge
syringe needle. A 0.2 cm3 sample of blood was then collected from
each rat. This first blood sample was collected within 2 min after the
rats were removed from the housing room. After obtaining this sample,
the rats were immobilized in plastic DecapiCones for 20 min, and then
another 0.2 cm3 sample of blood was collected via saphenous vein venipuncture. Immediately after collecting the second blood sample, the rats
were placed in Plexiglas tubes within a warming test chamber (~32 °C)
for 5 min to increase their body temperature, which enhanced blood
flow to their tails. Heart rate (HR) and blood pressure (BP) were
assessed using a tail cuff fitted with photoelectric sensors (IITC Life Science; Woodland Hills, CA). Three HR and BP recordings were obtained
from each rat (these three recordings were averaged to create single
HR and BP data points for each rat). Following the HR and BP measurement, the rats were returned to their home cages. An hour later, the
third and final blood sample (trunk blood) was collected following
rapid decapitation. Then, the adrenal glands and thymuses were removed and weighed. Once all of the blood has clotted at room temperature, it was centrifuged (3000 rpm for 8 min), and the serum was
extracted and stored at −80 °C until assayed by M.F. at the University
of Colorado at Boulder.
2.5. Statistical analyses
In most cases, one-way ANOVAs were used to analyze the data, with
group serving as the between-subjects factor. When repeated measures
variables were involved, mixed-model ANOVAs were utilized. Planned
comparisons (t-tests) were conducted between groups that were predicted to differ a priori. For all analyses, alpha was set at 0.05, and Holm–Sidak
post hoc tests were employed when necessary.
3.1. Fear conditioning and memory testing (see Fig. 2)
3.1.1. Fear conditioning sessions
Analysis of immobility during the first [F(2,27) = 1.00, p N 0.05] and
second [F(2,27) = 2.87, p N 0.05] 3-min chamber exposures revealed no
significant effect of group. Analysis of immobility during the third fear
conditioning trial revealed a significant effect of group [F(2,25) =
3.64, p b 0.05]. Both psychosocial stress groups exhibited greater immobility than the no stress group.
3.1.2. Fear conditioned contextual memory test
Analysis of immobility during the 5-min context test on Day 115
revealed a significant effect of group, F(2,26) = 16.65, p b 0.001. Both
psychosocial stress groups exhibited greater immobility than the no
stress group, and the “Stress × 3” group exhibited greater immobility
than the “Stress × 2” group.
3.1.3. Fear conditioned cue memory test
Analysis of immobility during the 6-min cue test revealed significant
effects of group, F(2,25) = 3.73, and tone, F(1,25) = 47.87, and a significant Group × Tone interaction, F(2,25) = 4.13 (p's b 0.05). There were
no group differences in immobility during the first 3 min of the cue test,
which was prior to tone delivery. During the 3-min tone delivery period,
both psychosocial stress groups exhibited greater immobility than the
no stress group.
3.2. Elevated plus maze (see Fig. 3)
3.2.1. Open arm time
Analysis of open arm time for the entire 5-min trial revealed no significant effect of group, F(2,26) = 0.81, p N 0.05. More specific analyses
revealed that, during the first minute of the 5-min trial, there was a significant effect of group, F(2,26) = 9.13, p b 0.001, indicating that rats in
both psychosocial stress groups spent less time in the open arms than
the no stress group.
3.2.2. Ambulations
Analysis of overall movement on the EPM (i.e., ambulations) during
the entire 5-min trial, F(2,27) = 0.63, and the first minute of the 5-min
trial, F(2,27) = 1.90, revealed no significant effects of group (p's N 0.05).
3.3. Startle response (see Fig. 4)
Analysis of startle responses revealed a significant effect of dB,
F(2,50) = 87.15, p b 0.001, indicating that rats exhibited greater startle
responses as the intensity of the white noise bursts increased. However,
the was no significant effect of group, F(2,25) = 2.85, and the Group ×
dB interaction was not significant, F(4,50) = 0.88 (p's N 0.05).
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Fig. 2. Immobility during the three stress sessions and the context and cue tests. There were no significant group differences during the first two sessions (a and b). By the third session (c),
both psychosocial stress groups exhibited greater immobility with chamber exposure than the no stress group. During the 4-month context memory test (d), both psychosocial stress
groups exhibited greater immobility than the no stress group, and the Stress × 3 group exhibited greater immobility than the Stress × 2 group. In the cue test (e), the psychosocial stress
groups displayed greater immobility than the no stress group during the tone (diagonal lines) compared to the pre-tone period (solid bars). Data are means ± SEM *p b 0.05 vs. the no
stress group; **p b 0.05 vs. all other groups.
Fig. 3. Behavior on the elevated plus maze (EPM). Both psychosocial stress groups spent less time in the open arms than the no stress group (left). This effect was not attributable to a
difference in overall activity level, as there were no significant group differences in ambulations (right). Data are means ± SEM. *p b 0.05 vs. the no stress group.
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effect of group, F(2,27) = 1.74, p N 0.05. The analysis comparing the discrimination indices of all groups during the first minute of the testing
trial revealed a significant effect of group, F(2,27) = 5.24, p b 0.05, indicating that both psychosocial stress groups exhibited lower discrimination indices than the no stress group. Further exploration of the time
that rats spent with the novel and familiar objects indicated that the
“Stress × 2” group spent significantly less time with the novel object
than the familiar object during the first minute of testing, t(9) = 2.55,
p b 0.05.
3.5. Body weights (see Fig. 6)
Fig. 4. Startle responses to the three auditory stimulus intensities. Although startle
responses increased as sound intensity increased, there were no significant group differences at any one intensity. Data are means ± SEM.
3.4. Novel object recognition (see Fig. 5)
3.4.1. Novel object recognition: habituation
The analysis of locomotor activity in the open field during the 5-min
habituation phase revealed no significant effect of group, F(2,27) =
0.13, p N 0.05. The analysis of time that the rats spent in each area of
the open field revealed no significant effect of group, F(2,27) = 0.22,
or quadrant, F(3,81) = 0.60, and the Group × Quadrant interaction
was not significant, F(6,81) = 0.28 (p's N 0.05). These findings indicate
that the rats did not display a preference for one area of the open field
over another.
3.4.2. Novel object recognition: training
Within-group comparisons showed that the no stress group, t(9) =
0.12, “Stress × 2” group, t(9) = 0.72, and “Stress × 3” group, t(9) = 1.03,
spent a comparable amount of time with each of the object replicas that
were placed in the open field during object recognition training
(p's N 0.05), indicating that no object preference effects were present.
Moreover, a between-groups comparison of the total amount of time
spent with both objects during training revealed no significant effect
of group, F(2,27) = 1.92, p N 0.05, indicating that all groups spent a comparable amount of time with both objects during training.
3.4.3. Novel object recognition: memory test
The analysis comparing the discrimination indices of all groups during the 5-min object recognition testing session revealed no significant
The analysis of weight gained over the course of the experiment
revealed a significant effect of time, F(2,52) = 614.76, p b 0.001, and a
significant Group × Time interaction, F(4,52) = 2.79, p b 0.05. Both psychosocial stress groups gained less weight than the no psychosocial
stress group between the first stress session and the second and third
stress sessions. However, the psychosocial stress groups both recovered
and gained the same amount of weight as the no stress group by the first
day of behavioral testing. Overall, there was no significant effect of
group, F(2,26) = 1.54, p N 0.05.
3.6. Organ weights (see Fig. 6)
The analysis of adrenal gland weights revealed no significant effect
of group, F(2,27) = 1.04, p N 0.05. However, the effect of group on thymus weight was significant, F(2,26) = 5.23, p b 0.05. This effect indicated
that the “Stress × 3” group exhibited reduced thymus weight relative to
the no stress group.
3.7. Cardiovascular activity (see Fig. 7)
The analysis of cardiovascular activity revealed no significant effects
of group for HR, F(2,16) = 0.12, systolic BP, F(2,16) = 1.43, or diastolic
BP, F(2,16) = 1.79 (p's N 0.05). In order to more thoroughly examine the
possibility that our animal model of PTSD resulted in long-term cardiovascular changes, we conducted additional statistical analyses by combining HR and BP data from both psychosocial stress groups and comparing
these data to the no stress group. The two psychosocial stress groups were
not significantly different from one another on any cardiovascular measure; thus, this analysis allowed us to increase statistical power and address whether 2 or 3 cat exposures, in general, exerted any long-term
effects on cardiovascular activity. These analyses revealed no significant
differences for HR and systolic BP; however, the effect for diastolic BP
was borderline significant and revealed that the psychosocial stress
group (“Stress × 2” + “Stress × 3”) exhibited greater diastolic BP than
the no stress group, t(17) = −1.948, p = 0.068.
Fig. 5. Novel object preference (expressed as a discrimination index) during novel object recognition testing. Over the entire 5-min testing trial (right), there were no significant group
differences. Upon examination of the first minute of the trial (left), both psychosocial stress groups exhibited significantly less preference for the novel object than the no stress group.
The dashed line at 50% is indicative of chance performance. Data are means ± SEM. *p b 0.05 vs. the no stress group.
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Fig. 6. Body weight gained (left) throughout the duration of the experiment and thymus weight (right) upon conclusion of behavioral testing. Both psychosocial stress groups gained less
body weight than the no stress group from the first stress session to the second and third stress sessions. However, this reduction in body weight gain recovered between the third stress
session and the commencement of behavioral testing. The Stress × 3 group, but not the Stress × 2 group, exhibited reduced thymus weight relative to the no stress group. Data are
means ± SEM. *p b 0.05 vs. the no stress group.
3.8. Corticosterone levels (see Fig. 7)
The analysis of corticosterone levels revealed a significant effect of
time point, F(2,54) = 104.22, p b 0.001, indicating that all groups
displayed elevated serum corticosterone levels, relative to baseline, following 20 min of acute immobilization stress and that these levels
remained elevated an hour later. There was no significant effect of
group, F(2,27) = 0.77, and the Group × Time interaction was not significant, F(4,54) = 0.64 (p's N 0.05).
To more thoroughly examine the possibility that our animal model
of PTSD resulted in long-term hormonal changes, we conducted additional statistical analyses by combining the corticosterone data from
both psychosocial stress groups and comparing these data to the
no stress group. These analyses revealed a significant main effect
of group, indicating that the two psychosocial stress groups exhibited lower corticosterone levels than the no stress group, overall,
F(1,26) = 4.19, p = 0.05, and a reliable Group × Time interaction,
F(2,52) = 3.08, p = 0.05. Post hoc comparisons revealed that 1 h following the cessation of acute immobilization stress, the psychosocial groups exhibited significantly lower corticosterone levels than
the no stress group (p's b 0.05).
4. Discussion
We have reported that this psychosocial predator-based animal
model of PTSD produces PTSD-like physiological and behavioral changes
that are present 4 months following the initiation of stress. In addition
to testing the persistence of our original paradigm (i.e., 2 cat exposures
combined with daily social instability), we also examined whether a
third cat exposure was necessary to permit the effects to be observed
so long following stress onset. Although we found that 2 cat exposures
were sufficient to produce a long-lasting contextual and cue-based fear
memory, as well as heightened anxiety on the elevated plus maze and
impaired object recognition memory, 3 cat exposures significantly increased the strength of the contextual fear memory and led to a significant reduction of thymus weight, which was not observed following 2
cat exposures. The combined data from both psychosocial stress groups
revealed lower corticosterone levels 1 h following acute immobilization
stress, relative to the no stress group. Moreover, psychosocial stress
produced a trend for an increase in diastolic BP following 20 min of
acute immobilization stress. We did not observe any significant effects
of psychosocial stress on startle response, and the body weight reduction that was observed in the psychosocial stress groups between the first and third stress sessions normalized by the time of
behavioral testing. Combined, these findings suggest that over the
course of the 4 months, some of the effects decreased in magnitude.
Most importantly, our experimental paradigm produced contextual
and cue-based fear memory, detectable changes in anxiety-like behavior and cognition and alterations in cardiovascular and hormonal
activity that were observable more than 4 months following stress onset.
This great durability of a large subset of effects indicates that our PTSD
model is useful for testing the neurobiological mechanisms of, and
novel treatments for, long-term traumatic memories and chronic
PTSD symptomatology.
Fig. 7. Effects of psychosocial stress on diastolic blood pressure (left) and corticosterone levels (right) when combining psychosocially stressed animals given 2 or 3 cat exposures. The
psychosocial stress group (Stress × 2 + Stress × 3) displayed borderline significant elevations of diastolic BP relative to the no stress group. The psychosocial stress group also exhibited
a greater recovery of corticosterone levels one hour following cessation of acute immobilization stress, as evidenced by significantly lower corticosterone levels than the no stress group at
the 80 min time point. Data are means ± SEM. β p = 0.068 vs. the no stress group; *p b 0.05 vs. the no stress group.
190
P.R. Zoladz et al. / Physiology & Behavior 147 (2015) 183–192
4.1. Collective effects of the psychosocial predator-based animal model
of PTSD
In previous work, we have found that our psychosocial predatorbased animal model of PTSD resulted in a number of physiological and
behavioral abnormalities, all of which are similar to those observed in
people with PTSD. Three weeks following the second predator exposure, psychosocially stressed rats exhibit reduced growth rate, reduced
thymus weight, greater adrenal gland weight, increased anxiety, an exaggerated startle response, impaired memory for new information,
greater cardiovascular and hormonal reactivity to an acute stressor
and an exaggerated physiological and behavioral response to the α2adrenergic receptor antagonist, yohimbine [9]. We have also shown
that, three weeks after the second predator exposure, psychosocially
stressed rats exhibit a robust fear memory for the predator stress exposures, enhanced negative feedback of the HPA axis and increased methylation of BDNF DNA in the hippocampus [10–12]. These findings highlight
a unique aspect of our animal model of PTSD — it manifests evidence of
associative and non-associative fear in rats. That is, psychosocially
stressed rats exhibit a memory for the initial “traumatic” experience, as
evidenced by classically-conditioned fear expression, and they demonstrate hyperarousal, cognitive symptoms and physiological symptoms
that appear to be independent of this memory.
Other investigators have replicated some of these findings and have
extended our animal model to characterize its effects on neurotransmitter levels and markers of inflammation. Wilson and colleagues reported
that our psychosocial predator-based model of PTSD resulted in decreased serotonin and increased norepinephrine and dopaminergic activity in the prefrontal cortex and hippocampus [13]. These investigators
also demonstrated that psychosocially stressed rats displayed increased
measures of inflammation and oxidative stress in the hippocampus, prefrontal cortex, adrenal glands and systemic circulation [14], effects that
have been observed 70 days following the initiation of stress in a paradigm of 3 cat exposures similar to the one used in the present study
[16]. Importantly, most of the effects of this animal model on neurotransmitter levels and markers of inflammation were reversed by administration of valproic acid or sertraline [16,17].
One empirical question that had yet to be addressed in our animal
model was how long the effects induced by the psychosocial stress
paradigm would last. In the present study, we have shown that although
some of the effects appeared to have diminished over time, a novel and
important finding is that psychosocially stressed rats displayed intact
conditioned fear memory for the cat exposures, heightened anxiety and
impaired cognition 4 months following the initiation of the stress experience. To our knowledge, the present study is the first to report PTSD-like
physiological and behavioral changes in rats this long after the initiation
of stress, potentially making it uniquely relevant to understanding the
mechanisms underlying long-term, or chronic, forms of PTSD.
Another question that was addressed in this study was whether a
third cat exposure was necessary to produce physiological and behavioral alterations so long after the initiation of stress. The only differences
that were produced by the additional cat exposure were a greater contextual fear memory and a greater reduction in thymus weight, relative
to rats given only 2 cat exposures. A reduction in thymus weight is
significant because it is suggestive of compromised immune system
function. That we observed a significant decrease in thymus weight in
the Stress × 3 group only suggests that a third cat exposure may
be necessary for this model to produce longer-lasting immune system alterations. We did observe additional effects upon combining
the cardiovascular and endocrinological data from both psychosocial stress groups and comparing this combined group to the no
stress group. These analyses revealed a statistical trend indicative
of elevated diastolic BP in the combined psychosocial stress group,
which is consistent with our previous findings [9,12] and cardiovascular
abnormalities observed in PTSD patients [7]. It is important to note,
though, that tail cuff measures of BP are notoriously less sensitive to
small changes in BP, relative to in vivo biotelemetry. Thus, the use of
more sensitive measures could have revealed a greater effect of psychosocial stress on cardiovascular measures. We also observed a greater reduction of corticosterone levels in the combined psychosocial stress group
one hour following exposure to acute immobilization stress. This finding
is consistent with our previous work showing significantly lower
baseline levels of corticosterone and a greater recovery of stressinduced corticosterone levels following dexamethasone administration in psychosocially stressed rats [10]. Psychosocially stressed animals may have demonstrated lower corticosterone levels one hour
after the immobilization stress because they had enhanced negative
feedback of the HPA axis, a finding that would be consistent with a
majority of the PTSD literature [18,19].
Despite the positive outcomes observed in the present study, we did
not observe an exaggerated startle response in psychosocially stressed
animals. This would appear to contradict our previous work, as well as
the PTSD literature. However, as we have reviewed previously [7], the
literature regarding startle response in PTSD patients is inconsistent
and has failed to conclusively provide evidence that an exaggerated
startle response is a cardinal symptom of the disorder. One alternative
explanation for the exaggerated startle response observed in some studies involving PTSD patients is that an exaggerated startle response is a
pre-existing risk factor for a subset of traumatized individuals to develop the disorder and is therefore not present in every person with PTSD
[20]. Another explanation is that when tested in a laboratory setting,
PTSD patients exhibit a context-dependent enhancement of startle
due to heightened reaction to novelty stress or anticipatory anxiety,
rather than exhibiting startle enhancement under true baseline conditions, such as in the safety of their home [21]. Thus, a failure to detect
an exaggerated startle response in our psychosocially stressed rats is
not inconsistent with the broader PTSD literature and may reflect the inherent variability in startle response data that is observed in such studies. It might also suggest that, over time, greater variability in the startle
response develops, which means an exaggerated startle response would
be more difficult to detect in individuals having PTSD for a longer period
of time [22,23].
We also did not observe an impairment of object recognition memory across the entire 5-min testing trial. Indeed, data suggestive of impaired object recognition memory in psychosocially stressed animals
was observed only during the first minute of testing. Perhaps more important is the finding that the “Stress × 2” group spent significantly less
time with the novel object than the familiar object during the first minute of testing. This finding raises the issue of whether the “Stress × 2”
group had an intact memory for the familiar object and chose to avoid
the novel object as a result of neophobia. Although the “Stress × 3”
group did not spend significantly less time with the novel object than
with the familiar object during the first minute of testing, the data
from this group were in the same direction as that of the “Stress × 2”
group. These findings could indicate that over time, rats exposed to
our predator-based psychosocial stress regimen develop PTSD-related
fear of novelty, especially because we have not observed neophobic
behaviors in psychosocially stressed animals in our previous work.
However, because we did not observe increased immobility of psychosocially stressed animals during the first 3 min of the cue test,
which took place in a novel environment, additional work is necessary to explore further this possibility.
4.2. Essential components of animal models of PTSD
Animal models of PTSD are essential to understand the disorder
because they provide control over environmental factors and stressinducing stimuli, thus affording investigators a greater opportunity to
make cause–effect inferences than can be accomplished in clinical
research. They also enable investigators to examine neurochemical
mechanisms that potentially underlie PTSD and test the efficacy of
novel therapeutic agents in reversing any stress-induced alterations of
P.R. Zoladz et al. / Physiology & Behavior 147 (2015) 183–192
physiology and behavior, which could lead to more treatment options
for PTSD patients in the future. However, PTSD is a complex psychological disorder that consists of several symptom clusters. The ability to
reproduce all symptoms of the disorder in a non-human animal model
is likely unrealistic, but a valid animal model of PTSD should model as
many aspects of the disorder as is possible to achieve in the species
being studied. Furthermore, these models should evidence phenomena that are known to exist in the human condition, such as a dosedependent relationship between the stressor and PTSD-like symptoms,
a long-term manifestation of the PTSD-like sequelae and associative and
non-associative fear-related behaviors [24].
Numerous stress paradigms have been employed to model PTSD in
rodents, such as repeated exposure to conditioned fear [25,26], electric
shock [27–38], underwater trauma [39–41], stress–restress and single
prolonged stress paradigms [42–46], and exposure to predators or
predator-related cues [47–57]. These paradigms have resulted in
heightened anxiety, exaggerated startle, cognitive impairments, enhanced fear conditioning, reduced social interaction and hormonal
changes that correlate with PTSD. Still, many animal models of PTSD
have modeled limited aspects of the human condition, and the effects
that have been produced last a short period of time. In addition, some
models provide evidence for a “traumatic” memory without demonstrating changes in anxiety levels, symptoms of hyperarousal and physiological alterations that are comparable to those observed in PTSD.
Other models have provided the opposite — that is, numerous behavioral and physiological changes without evidence for a memory component of PTSD. What is unique about our psychosocial predator-based
animal model of PTSD is that it produces a wide array of PTSD-like physiological and behavioral alterations, in addition to a memory for the
“traumatic” cat exposures. Furthermore, based on the present findings,
at least some of the effects produced by our model persist for 4 months
following stress onset. Thus, our animal model of PTSD can be used to
study the mechanisms underlying multiple facets of PTSD, as well as
its chronic nature.
5. Conclusions
We have found that our well-validated animal model of PTSD produces physiological and behavioral disturbances that can be observed
4 months following stress onset. Two cat exposures, as employed in
our original animal model of PTSD [9], resulted in an intact fear memory
for the predator stress experiences, heightened anxiety and impaired
cognition. A third cat exposure enhanced the contextual fear memory
for the predator stress and resulted in a significant reduction of thymus
weight. Data from the two psychosocial stress groups revealed greater
diastolic BP and lower corticosterone levels in these groups. Collectively,
our findings provide further validation of our predator-based psychosocial model of PTSD and suggest that it is useful for testing the mechanisms
and novel therapeutic strategies for persistent PTSD symptoms.
Acknowledgments
This research was supported by Award Number I01BX007080 from
the Biomedical Laboratory Research & Development Service of the VA
Office of Research and Development to DMD. The opinions expressed
in this paper are those of the authors and not of the Department of Veterans Affairs or the US government.
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