Progress in Neuro-Psychopharmacology & Biological Psychiatry 44 (2013) 1–16
Contents lists available at SciVerse ScienceDirect
Progress in Neuro-Psychopharmacology & Biological
Psychiatry
journal homepage: www.elsevier.com/locate/pnp
Differential effectiveness of tianeptine, clonidine and amitriptyline in blocking
traumatic memory expression, anxiety and hypertension in an animal model of PTSD
Phillip R. Zoladz a, Monika Fleshner b, David M. Diamond c, d, e, f,⁎
a
Department of Psychology, Sociology & Criminal Justice, Ohio Northern University, Ada, OH, USA
Department of Integrative Physiology & Center for Neuroscience, University of Colorado, Boulder, CO, USA
Medical Research Service, VA Hospital, Tampa, FL, USA
d
Department of Psychology, University of South Florida, Tampa, FL, USA
e
Department of Molecular Pharmacology & Physiology, University of South Florida, Tampa, FL, USA
f
Center for Preclinical & Clinical Research on PTSD, University of South Florida, Tampa, FL, USA
b
c
a r t i c l e
i n f o
Article history:
Received 10 October 2012
Received in revised form 22 December 2012
Accepted 4 January 2013
Available online 12 January 2013
Keywords:
Animal model
Cardiovascular activity
Corticosterone
Psychophysiology
PTSD
Stress
a b s t r a c t
Individuals exposed to life-threatening trauma are at risk for developing post-traumatic stress disorder
(PTSD), a debilitating condition that involves persistent anxiety, intrusive memories and several physiological disturbances. Current pharmacotherapies for PTSD manage only a subset of these symptoms and typically
have adverse side effects which limit their overall effectiveness. We evaluated the effectiveness of three different pharmacological agents to ameliorate a broad range of PTSD-like symptoms in our established
predator-based animal model of PTSD. Adult male Sprague–Dawley rats were given 1-h cat exposures on
two occasions that were separated by 10 days, in conjunction with chronic social instability. Beginning
24 h after the first cat exposure, rats received daily injections of amitriptyline, clonidine, tianeptine or vehicle. Three weeks after the second cat exposure, all rats underwent a battery of behavioral and physiological
tests. The vehicle-treated, psychosocially stressed rats demonstrated a robust fear memory for the two cat exposures, as well as increased anxiety expressed on the elevated plus maze, an exaggerated startle response,
elevated heart rate and blood pressure, reduced growth rate and increased adrenal gland weight, relative to
the vehicle-treated, non-stressed (control) rats. Neither amitriptyline nor clonidine was effective at blocking
the entire cluster of stress-induced sequelae, and each agent produced adverse side effects in control subjects. Only the antidepressant tianeptine completely blocked the effects of psychosocial stress on all of the
physiological and behavioral measures that were examined. These findings illustrate the differential effectiveness of these three treatments to block components of PTSD-like symptoms in rats, and in particular, reveal the profile of tianeptine as the most effective of all three agents.
Published by Elsevier Inc.
1. Introduction
Individuals who are exposed to intense trauma that threatens
physical injury or death are at significant risk for developing posttraumatic stress disorder (PTSD). People who develop PTSD respond
to a traumatic experience with intense fear, helplessness or horror
(American Psychiatric Association, 1994) and may endure chronic
psychological distress by repeatedly reliving their trauma through
intrusive, flashback memories (Ehlers et al., 2004; Hackmann et al.,
2004; Reynolds and Brewin, 1998, 1999; Speckens et al., 2006,
Abbreviations: ACTH, adrenocorticotropic hormone; BP, blood pressure; EPM, elevated plus maze; HPA, hypothalamus-pituitary-adrenal; HR, heart rate; MAOI, monoamine oxidase inhibitor; NOR, novel object recognition; PTSD, post-traumatic stress
disorder; SSRI, selective serotonin reuptake inhibitor; TCA, tricyclic antidepressant.
⁎ Corresponding author at: University of South Florida, Department of Psychology, 4202
E. Fowler Ave. PCD 4118G, Tampa, FL, 33620, USA. Tel.: +1 813 974 0480; fax: +1 813 974
4617.
E-mail address: ddiamond@mail.usf.edu (D.M. Diamond).
0278-5846/$ – see front matter Published by Elsevier Inc.
http://dx.doi.org/10.1016/j.pnpbp.2013.01.001
2007). The re-experiencing and avoidance symptoms of the disorder
may hinder everyday functioning in PTSD patients and can foster
the development of debilitating symptoms, such as persistent anxiety, exaggerated startle and cognitive impairments (Brewin et al.,
2000; Elzinga and Bremner, 2002; Nemeroff et al., 2006; Newport
and Nemeroff, 2000; Stam, 2007).
The finding that a subset of people with PTSD exhibit an improvement in their symptoms following treatment with selective serotonin
reuptake inhibitors (SSRIs) suggests a role of the serotonergic system
in the disorder (Asnis et al., 2004; Davis et al., 2006; Hidalgo and
Davidson, 2000; Ipser et al., 2006; Stein et al., 2006). Indeed, two
SSRIs, paroxetine and sertraline, are the only FDA-approved pharmacological treatments for the disorder (Albucher and Liberzon, 2002;
Van der Kolk, 2001; Vaswani et al., 2003). However, response rates
to SSRIs in PTSD patients rarely exceed 60% and full remission from
the disorder following SSRI treatment is achieved only 20–30% of
the time (Stein et al., 2002). In addition, some forms of PTSD, such
as combat-related PTSD, are resistant to SSRI treatment (Jakovljevic
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et al., 2003; Rothbaum et al., 2008; Stein et al., 2002). Moreover, SSRIs
tend to blunt the depressive components of PTSD, while having little
effect on the memory- and anxiety-related symptoms of the disorder
(Asnis et al., 2004; Boehnlein and Kinzie, 2007; Brady et al., 2000). It
is important to note that SSRIs tend to exert anxiogenic effects on
individuals early in the treatment phase (Browning et al., 2007;
Burghardt et al., 2004; Humble and Wistedt, 1992). Given the caveats
to the efficacy of SSRIs in treating PTSD, there is a need for additional
research in people with PTSD and in animal models of PTSD to facilitate the discovery of more effective treatments for the disorder.
Other work has examined the efficacy of first-generation antidepressants, the tricyclic antidepressants (TCAs) and monoamine oxidase inhibitors (MAOIs), in treating PTSD. TCAs inhibit the reuptake
of serotonin and norepinephrine. Research has shown that one such
antidepressant, amitriptyline, ameliorates a subset of the symptoms
of PTSD and, in some cases, may be more effective than SSRIs (Celik
et al., 2011; Davidson et al., 1990, 1993). Thus, in the present study,
we included amitriptyline as a positive control, as we expected it to
block anxiety-like symptoms in rats. MAOIs, on the other hand, interfere with the catabolism of monoamine transmitters. Overall, studies
that have been conducted on TCAs and MAOIs have generally shown
that they can reduce a subset of symptoms associated with PTSD
(Baker et al., 1995; Bisson, 2007; Burstein, 1984; Davidson et al.,
1990, 1993; Frank et al., 1988; Katz et al., 1994; Kosten et al., 1991;
Neal et al., 1997; Reist et al., 1989; Shestatzky et al., 1988). However,
both classes of drugs are rarely used as the first line of treatment for
PTSD and, instead, are typically employed only when SSRIs are ineffective (Albucher and Liberzon, 2002). Due to the numerous side effects of both drug classes, the dropout rates for these agents are
high (e.g., 30–50%). Additionally, patients who take MAOIs adhere
to a special low tyramine diet to avoid a potential life-threatening hypertensive crisis. Thus, despite some beneficial features of TCAs and
MAOIs in the treatment of PTSD symptoms, overall, these drugs are
not ideal for the treatment of people with anxiety disorders.
An alternative pharmacological approach is to target the elevated
baseline and stress-induced elevations of sympathetic activity which
are known to occur in PTSD (Buckley and Kaloupek, 2001; Pole,
2007). One indication of the accentuated sympathetic activity in
PTSD patients is the hyper-responsivity they exhibit to the administration of yohimbine, an α2-adrenergic receptor antagonist that inhibits noradrenergic autoreceptors and leads to increased central
norepinephrine activity (Rasmusson et al., 2000; Southwick et al.,
1993, 1999a, 1999b, 1999c). These findings, along with those of greater baseline norepinephrine levels in PTSD patients (Strawn and
Geracioti, 2008), have implicated a major role of the noradrenergic
system in the hyperarousal component of PTSD.
Based on observed hyperactivity of the noradrenergic system in
PTSD, recent work has examined the efficacy of drugs that reduce
central noradrenergic activity in treating the disorder. Studies have
found some support for the use of propranolol, a β-adrenergic receptor antagonist, to reduce a subset of symptoms of PTSD when administered after a traumatic event or with re-experiencing a traumatic
memory (Brunet et al., 2011; Hoge et al., 2012; Pitman et al., 2002;
Taylor and Raskind, 2002; Vaiva et al., 2003). Related work has
shown that clonidine, an α2-adrenergic receptor (noradrenergic
autoreceptor) agonist, and prazosin, an α1-adrenergic receptor antagonist, ameliorated symptoms of heightened anxiety and hyperarousal
in people with PTSD (Boehnlein and Kinzie, 2007). Though many
studies have examined the effects of clonidine on PTSD and found it
to be effective at reducing intrusive memories and hyperarousal
(Harmon and Riggs, 1996; Porter and Bell, 1999; Viola et al., 1997),
no randomized, placebo-controlled studies of clonidine's effects on
PTSD have been performed (Boehnlein and Kinzie, 2007). Recent
work has shown that prazosin is an effective treatment for hyperarousal
symptoms, intrusive thoughts, recurrent distressing dreams and
sleep disturbances in PTSD (Brkanac et al., 2003; Peskind et al., 2003;
Raskind et al., 2002, 2003; Taylor and Raskind, 2002; Taylor et al.,
2006). Collectively, these studies suggest that pharmacological reduction of adrenergic activity may be incorporated into the treatment of
PTSD patients.
Whereas the antidepressant tianeptine is commonly known for its
effectiveness in ameliorating symptoms of major depression, one
pilot study provided evidence that it has beneficial effects in the
treatment of PTSD (Onder et al., 2006). Tianeptine's primary mechanism of action involves the stabilization of glutamatergic neurotransmission and the enhancement of synaptic plasticity, particularly
under stress conditions (Brink et al., 2006; Kasper and McEwen,
2008; Kole et al., 2002; McEwen et al., 2010; Zoladz et al., 2008b).
Preclinical studies have shown that stress significantly increases glutamate levels (Bagley and Moghaddam, 1997; Lowy et al., 1993, 1995;
Moghaddam, 1993; Reznikov et al., 2007), inhibits glutamate uptake
(Yang et al., 2005), increases the expression and binding of glutamate
receptors (Bartanusz et al., 1995; Krugers et al., 1993; McEwen et al.,
2002) and increases calcium currents (Joels et al., 2003). Tianeptine
prevents the deleterious effects of stress on brain and behavior by
normalizing these alterations in glutamatergic neurotransmission.
Extensive preclinical research has shown that tianeptine reverses
stress-induced alterations of neuronal morphology and synaptic
plasticity and blocks the effects of stress on learning and memory
(Campbell et al., 2008; Diamond et al., 2004; Kasper and McEwen,
2008; McEwen et al., 2010; Shakesby et al., 2002; Vouimba et al.,
2006; Zoladz et al., 2008b). Overall, the clinical and preclinical findings suggest that tianeptine could be an effective pharmacological
treatment for individuals diagnosed with PTSD.
Our understanding of the mechanisms underlying PTSD may be
enhanced by animal models that mimic the experiences of traumatized individuals. To this end, we have developed an animal model
that includes conditions known to maximize the likelihood of PTSD
developing in people, including a threat to survival, a lack of control,
an intrusive reminder of the traumatic experience and a lack of social
support (Roth et al., 2011; Zoladz et al., 2008a, 2012). Specifically, we
have produced PTSD-like effects in rats by exposing them to two 1-h
periods of inescapable confinement in close proximity to a cat, in
conjunction with daily social stress, over a period of 1 month. We
demonstrated that rats that were administered this psychosocial
stress paradigm exhibited heightened anxiety, exaggerated startle, impaired cognition, increased cardiovascular reactivity and an exaggerated response to yohimbine administration, all of which are commonly
observed in people with PTSD. The purpose of the present experiment
was to investigate the influence of chronic administration of amitriptyline, clonidine or tianeptine on physiological and behavioral sequelae
induced by this predator-based psychosocial stress regimen. To emphasize that the design of this experiment has clinical relevance, administration of the pharmacological agents did not begin until 24 h
post-trauma. Our findings, therefore, are potentially relevant toward
optimizing clinically relevant outcomes based on a person seeking
treatment soon after an intense traumatic experience occurs.
2. Method
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.
P.R. Zoladz et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 44 (2013) 1–16
2.2. Psychosocial stress procedure
2.2.1. Acute stress sessions
Following the 1-week acclimation phase, rats were brought to the
laboratory and randomly assigned to “psychosocial stress” or “no psychosocial stress” groups (N = 10 rats/group). Rats from these groups
were first 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 sole purpose of exposing rats to the chamber was to allow rats in the psychosocial stress
groups to associate the chamber (contextual fear conditioning) and
tone [auditory (cue) fear conditioning] with the acute stress experience (i.e., immobilization plus cat exposure, as described below)
and measure their memory for the experience (via an assessment of
immobility in the chamber) during behavioral testing (see Zoladz et
al., 2012 for comparable methodology). Locomotor activity in the
chamber was measured during the acute stress sessions and behavioral testing by a 24-cell infrared activity monitor (Coulbourn Instruments; Allentown, PA) mounted on the top of the chamber, which
used the 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. Pilot data from our lab indicated
that the 7-s cut-off most closely represented off-line scoring of freezing behavior in rats.
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 returned to the laboratory. Rats in the no psychosocial stress
groups were not immobilized or exposed to the cat; rather, they
remained in their home cages in the laboratory during the 1-h period
the stressed group was exposed to the cat.
Psychosocial stressed rats were exposed to 2 acute stress sessions
which were separated by 10 days. The first acute stress session 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 two acute stress sessions took place during different times of the day to add an element of unpredictability as to
when the rats might re-experience the traumatic event (Zoladz et
al., 2008a). A lack of predictability in one's environment is a major
factor in the development and expression of PTSD, at least in a subset
of susceptible people (Orr et al., 1990; Regehr et al., 2000; Solomon et
al., 1988, 1989).
2.2.2. Daily social stress
Beginning on the day of the first acute stress session, and continuing for the next 31 days, rats in the psychosocial stress groups were
exposed to daily unstable housing conditions, as described previously
(Roth et al., 2011; Zoladz et al., 2008a, 2012). Rats in the psychosocial
stress groups were housed two per cage, but every day, their cohort
pair combinations were changed. Therefore, no rat in the psychosocial stress groups had the same cage mate on two consecutive days
during the 31-day stress period. All rats in the stress groups were administered the two cat exposures in conjunction with daily social instability because we found previously that the complete PTSD-like
3
profile was generated only in rats that were exposed to the combination of predator exposure and social stress (Zoladz et al., 2008a). Rats
in the control groups were housed with the same cohort pair for the
duration of the experiment.
2.3. Pharmacological agents
Twenty-four hours after the first acute stress session (i.e., Day 2),
all rats began receiving daily intraperitoneal (i.p.) injections of amitriptyline (5 or 10 mg/kg), clonidine (0.01 or 0.05 mg/kg), tianeptine
(10 mg/kg) or vehicle (0.9% saline). The injections occurred every
day throughout the 31-day period of psychosocial stress and also
throughout behavioral testing. Drug administration continued during
behavioral testing to prevent possible drug withdrawal effects
from influencing rat behavior. The injections were always administered in the morning (between 0900 and 1200 h) at a volume of
1 ml/kg. Ten rats from each of the psychosocial stress and no psychosocial stress groups were randomly assigned to each of the drug
conditions. Amitriptyline and clonidine were obtained from SigmaAldrich (St. Louis, MO), and tianeptine was provided by Servier Pharmaceuticals (France). The doses of amitriptyline and clonidine were
chosen based on the findings of preclinical studies demonstrating
the efficacy of similar doses in preventing stress-induced sequelae
in rats (Blanc et al., 1991; Ferretti et al., 1995; Katz and Hersh,
1981; Murphy et al., 1996; Soblosky and Thurmond, 1986). The single
dose of tianeptine was based on extensive findings from our laboratory and others which have demonstrated the effectiveness of this
dose in preventing acute and chronic stress effects on the brain and
behavior (Campbell et al., 2008; Conrad et al., 1999; Diamond et al.,
2004; Reagan et al., 2004; Vouimba et al., 2006; Zoladz et al., 2010).
2.4. Behavioral testing
Three weeks after the second acute stress session (Day 32), behavioral testing began; all rats were given tests to measure their fear
memory, anxiety, startle, learning and memory, cardiovascular activity and corticosterone levels. The three-week delay from the second
acute stress session to behavioral testing was based on comparable
time periods employed in other studies on the effects of stress on
brain and behavior (Adamec and Shallow, 1993; Cook and Wellman,
2004; Magarinos et al., 1996; McLaughlin et al., 2007; Park et al.,
2001; Watanabe et al., 1992a, 1992b, 1992c; Zoladz et al., 2008a).
On the first 4 days of behavioral testing (Days 32–35), rats were
taken to the procedure room across from the rat housing rooms,
where they received i.p. injections of the drug appropriate to the condition to which they had been assigned. Then, they 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 cue fear memory
On Day 32, rat behavior in response to the chamber (context test)
and tone (cue test) that had been previously paired with the acute
stress sessions was examined. Rats were placed in the same chamber
that they were exposed to during each of the two acute stress sessions for 5 min, and their immobility was recorded. One hour after
the 5-min context test, following methods and timing in our previous
work (Zoladz et al., 2008a, 2012), the rats were placed in a novel
chamber that had different lighting, walls and flooring from the
chamber in which they were placed during each of the two acute
stress sessions. The rats were placed in the novel chamber for a
total of 6 min (cue test). Three minutes into the cue test, the rats
were presented with a 74-dB, 2500 Hz tone that continuously played
for the remainder of the 6-minute testing period. The amount of
immobility recorded during the first 3 min of the cue test (i.e., no
tone) provided a measure of the general fear of a novel place, while
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the amount of immobility recorded during the last 3 min of the cue
test (i.e., tone) provided a measure of the fear response to the cue
that was, in the psychosocial stress group, specifically associated
with the two acute cat exposures.
2.4.2. Elevated plus maze
The elevated plus maze (EPM) is a routine test of anxiety in rodents
and 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 33, the rats were placed on the EPM for 5 min, and their behavior was scored by 48 infrared photobeams located along the perimeter
of the open and closed arms, which were connected to a computer program (Motor Monitor, Hamilton-Kinder, San Diego, CA). The primary
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
Acoustic startle testing was administered one hour after the EPM assessment following the methodology from our previous work (Zoladz
et al., 2008a, 2012). 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
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 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).
2.4.4. Novel object recognition
The novel object recognition (NOR) task was based on methods
described previously (Baker and Kim, 2002). On Day 34, 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. Their behavior was monitored by a Logitech camera that was mounted on the
ceiling overlooking the open field. This camera was connected to a
computer program known as ANY-Maze (Stoelting; Wood Dale, IL),
which scored rat behavior. Twenty-four hours later (Day 35), 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.
2.4.5. Blood sampling, cardiovascular activity & post-mortem dissections
On the final day of behavioral testing (Day 36), 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 cm 3 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. Then, the rats were removed from the DecapiCones, and another 0.2 cm 3 sample of blood was collected via saphenous vein venipuncture. Blood sampling at the 20 min time point was based on our
previous work (Zoladz et al., 2008a, 2012), and the well-established
finding that corticosterone reaches its maximum level approximately
20 min after stress onset (e.g., Ferland and Schrader, 2011). Immediately after collecting this blood sample, the rats were placed in Plexiglas tubes within a warming test chamber (~ 32 °C) for 5 min to
increase their body temperature. This enhanced blood flow to their
tails and allowed heart rate (HR) and blood pressure (BP) to be
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 HR and BP
measurements, the rats were returned to their home cages. An hour
later, one last blood sample (trunk blood) was collected following
rapid decapitation. Then, the adrenal glands and thymuses were removed and weighed. Once all of the blood had 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
The present study utilized a between-subjects, 2 × 6 factorial design.
The independent variables were psychosocial stress and drug. In most
cases, two-way, between-subjects ANOVAs were used to analyze the
data from the physiological and behavioral assessments, with psychosocial stress and drug serving as the between-subjects factors. When repeated measures variables were involved, mixed-model ANOVAs were
utilized to analyze the data. Planned comparisons (independent samples 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. Results
In all figures, the specific drug groups are identified by the following abbreviations: vehicle (VEH), amitriptyline — 5 mg/kg (AMI-5),
amitriptyline — 10 mg/kg (AMI-10), clonidine — 0.01 mg/kg
(C-0.01), clonidine — 0.05 mg/kg (C-0.05), tianeptine — 10 mg/kg
(TIA-10).
3.1. Fear memory expression
3.1.1. Acute stress sessions (Days 1 and 10)
During the first session, there was little evidence of freezing
(immobility) in any group, and no significant group differences
(ps > 0.05). This absence of freezing was evident because all rats
were naïve to the chamber, which had not yet been paired with an
adverse stimulus (cat exposure) in the psychosocial stress group.
During the second acute stress session, which took place 10 days
after the first, there was a significant main effect of psychosocial
stress, indicating that the psychosocial stress groups spent significantly more time immobile than the no psychosocial stress groups,
F(1,103) = 7.55, p b 0.01, (Fig. 1).
3.1.2. Fear conditioning context memory test (Day 32)
There were significant main effects of psychosocial stress,
F(1,97) = 11.96, and drug, F(5,97) = 3.90, and the Psychosocial
Stress × Drug interaction was significant, F(5,97) = 2.56 (ps b 0.05).
P.R. Zoladz et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 44 (2013) 1–16
Stress Session 2
Stress Session 1
80
80
No Psychosocial Stress
Psychosocial Stress
% Immobility
% Immobility
No Psychosocial Stress
Psychosocial Stress
60
60
40
40
20
20
0
0
VEH
AMI-5
AMI-10 C-0.01 C-0.05
TIA-10
VEH
AMI-5
Context Test
C-0.05 TIA-10
τ
No Psychosocial Stress
Psychosocial Stress
80
*
*
60
40
β
β
β
20
VEH
% Immobility
60
% Immobility
AMI-10 C-0.01
Cue Test - No Tone
No Psychosocial Stress
Psychosocial Stress
80
0
5
40
#
20
AMI-5 AMI-10 C-0.01 C-0.05
TIA-10
0
VEH
AMI-5 AMI-10 C-0.01 C-0.05 TIA-10
Cue Test - Tone
No Psychosocial Stress
Psychosocial Stress
80
% Immobility
60
τ
*
τ
*
*
*
*
40
20
0
VEH
AMI-5 AMI-10 C-0.01
C-0.05 TIA-10
Fig. 1. Immobility expressed by rats during various assessments of fear conditioning and memory testing. The vehicle-treated psychosocial stress group exhibited significant fear, as
indicated by an increase in immobility, to the context and cue that were paired with each of the two cat exposures during training. Amitriptyline and tianeptine were the most
effective agents in preventing the expression of fear memory. The data are expressed as the mean ± SEM. * pb 0.05 relative to the vehicle-treated no psychosocial stress group;
β = p b 0.05 relative to the vehicle-treated psychosocial stress group; τ= p b 0.05 relative to the respective drug-treated no psychosocial stress group; # = p b 0.05 relative to all
other groups.
The vehicle-treated psychosocial stress group spent significantly
more time immobile than the vehicle-treated no psychosocial stress
group (Fig. 1). Chronic treatment with 5 or 10 mg/kg of amitriptyline
or 10 mg/kg of tianeptine in groups that were psychosocially stressed
prevented the chronic stress-induced increase in immobility, as
evidenced by significantly less immobility than the vehicle-treated
psychosocial stress group and a lack of statistical significance relative
to each of the group's respective drug-treated no psychosocial
stress group. The group of psychosocially stressed rats treated with
0.01 mg/kg of clonidine also did not exhibit significantly greater immobility than its respective drug-treated control group; however,
the amount of immobility displayed by this group was not statistically
different from that of the vehicle-treated psychosocial stress group.
3.1.3. Fear conditioning pre-cue (novel environment) test (Day 32)
There were significant main effects of psychosocial stress,
F(1,100) = 9.40, and drug, F(5,100) = 2.72, and the Psychosocial
Stress × Drug interaction was significant, F(5,100) = 5.73 (ps b 0.05).
Chronic treatment with 0.05 mg/kg of clonidine in rats that were psychosocially stressed led to significantly greater immobility than all
other groups.
3.1.4. Fear conditioning cue memory test (Day 32)
There was a significant main effect of psychosocial stress,
F(1,100) = 12.04, and the Psychosocial Stress × Drug interaction was
significant, F(5,100) = 2.71 (ps b 0.05). The vehicle-treated psychosocial stress group spent significantly more time immobile than the
6
P.R. Zoladz et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 44 (2013) 1–16
vehicle-treated no psychosocial stress group. Additionally, chronic
treatment with 10 mg/kg of amitriptyline in groups that were not
psychosocially stressed led to significantly greater immobility than the
vehicle-treated no psychosocial stress group. Chronic treatment with
5 or 10 mg/kg of amitriptyline or 10 mg/kg of tianeptine in groups
that were psychosocially stressed blocked the fear-conditioned increase
in immobility, as evidenced by a lack of statistical significance relative
to each of the group's respective drug-treated no psychosocial stress
group.
were conducted between groups that were predicted to differ a priori.
For each stimulus intensity, except for 110 dB which was marginally
significant (p = 0.056), the vehicle-treated psychosocial stress group
exhibited significantly greater startle responses than the vehicletreated no psychosocial stress group (Fig. 3). All of the chronically
administered pharmacological agents blocked the psychosocial
stress-induced increase in startle response, except for 5 mg/kg of amitriptyline and 0.01 mg/kg of clonidine in response to 100 dB stimuli.
3.4. Novel object recognition
3.2. Elevated plus maze
3.4.1. Habituation
3.2.1. Percent time in open arms
A two-way ANOVA revealed no significant main effects or interactions (ps > 0.05). Therefore, planned comparisons were conducted
between groups that were predicted to differ a priori. The vehicletreated psychosocial stress group spent significantly less time in the
open arms of the EPM than the vehicle-treated no psychosocial stress
group, t(15)= 2.44, p b 0.05 (Fig. 2). Chronic treatment with 10 mg/kg
of amitriptyline, 0.01 or 0.05 mg/kg of clonidine or 10 mg/kg of
tianeptine in groups that were psychosocially stressed blocked the
stress-induced decrease in open arm exploration. These groups
exhibited significantly greater percent time spent in the open arms
than the vehicle-treated psychosocial stress group (ps b 0.05).
3.2.2. Ambulations
There was a significant main effect of drug, F(5,100) = 4.48,
p b 0.001. Groups treated with 10 mg/kg of amitriptyline or 10 mg/kg
of tianeptine exhibited significantly more ambulations than the
vehicle-treated group. Planned comparisons were conducted between
groups that were predicted to differ a priori. There was no significant
difference between the number of ambulations made by the vehicletreated psychosocial stress group and the vehicle-treated no psychosocial stress group on the EPM, t(16)= 0.16, p > 0.05. Chronic treatment
with 10 mg/kg of amitriptyline, t(15)= 2.67, or 10 mg/kg of tianeptine,
t(16) = 2.38, in groups that were not psychosocially stressed led to a
significantly greater number of ambulations on the EPM, relative to
the vehicle-treated no psychosocial stress group (ps b 0.05).
3.3. Startle response
The Psychosocial Stress × Drug interaction was significant for the
startle responses to 90 and 100 dB stimuli (90 dB: F(5,102) = 2.74;
100 dB: F(5,98) = 2.32; ps b 0.05). For the 110 dB stimuli, no significant effects were observed in the ANOVA, so planned comparisons
3.4.1.1. Overall locomotor activity. There were significant main effects
of psychosocial stress, F(1,104) = 8.25, and drug, F(5,104) = 9.27
(ps b 0.01). In general, rats that had been psychosocially stressed traveled significantly less distance in the open field than rats that had not
been psychosocially stressed. In addition, rats that were treated with
0.05 mg/kg of clonidine, independent of psychosocial stress, traveled
significantly less distance than all other groups.
3.4.1.2. Time spent in each area of open field. For this analysis, the open
field was divided into four square quadrants via the ANY-Maze computer program, and the amount of time that rats spent in each of the
quadrants was analyzed. No significant main effects or interactions
were observed (ps > 0.05).
3.4.2. Training
Within-group comparisons (i.e., dependent t tests) of the amount
of time that rats spent with each object replica (to rule out object
preference effects) indicated that most groups spent a comparable
amount of time with each of the objects that were placed in the
open field during object recognition training, suggesting that no object preference effects were present (ps > 0.05). Only one group, the
0.01 mg/kg clonidine-treated no psychosocial stress group, spent
more time with one object than the other (t(9) = 2.78, p b 0.05). A
between-groups comparison of the total amount of time spent with
both objects during training revealed that all main effects and interactions were not significant (ps > 0.05). These findings indicated
that all groups spent an equivalent amount of time with both objects
during training.
3.4.3. Testing
A “ratio time” score was calculated for each group by taking the
time that rats spent with the novel object and dividing it by the
No Psychosocial Stress
Psychosocial Stress
60
50
Locomotor Activity
β
β
40
30
20
*
*
10
0
No Psychosocial Stress
Psychosocial Stress
350
*
300
Total Ambulations
% Time Spent in the Open Arms
Open Arm Time
250
*
200
150
100
50
VEH
AMI-5
AMI-10 C-0.01
C-0.05 TIA-10
0
VEH
AMI-5 AMI-10 C-0.01 C-0.05 TIA-10
Fig. 2. Amount of time spent in the open arms (left) and overall locomotor activity (right) exhibited by rats on the elevated plus maze. The vehicle-treated psychosocial stress group
spent significantly less time in the open arms, without exhibiting a significant reduction of overall locomotion, compared to the control group. Clonidine, tianeptine and the higher
dose of amitriptyline were all effective in preventing the anxiogenic effects of psychosocial stress on the elevated plus maze. The data are presented as the mean ± SEM. * p b 0.05
relative to the vehicle-treated no psychosocial stress group; β = p b 0.05 relative to the vehicle-treated psychosocial stress group.
P.R. Zoladz et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 44 (2013) 1–16
100 dB Auditory Stimuli
90 dB Auditory Stimuli
0.4
1.2
No Psychosocial Stress
Psychosocial Stress
*
Startle Response (Newtons)
Startle Response (Newtons)
0.5
0.3
0.2
β
β
β
0.1
0.0
VEH
AMI-5
AMI-10 C-0.01
7
1.0
*
τ
0.8
β
0.6
β
β
0.4
0.2
0.0
C-0.05 TIA-10
No Psychosocial Stress
Psychosocial Stress
VEH
AMI-5
AMI-10 C-0.01
C-0.05 TIA-10
110 dB Auditory Stimuli
Startle Response (Newtons)
3.0
2.5
No Psychosocial Stress
Psychosocial Stress
ω
2.0
β
1.5
β
β
1.0
0.5
0.0
VEH
AMI-5
AMI-10 C-0.01 C-0.05 TIA-10
Fig. 3. Startle responses exhibited by rats following presentation of 90 dB (top left), 100 dB (top right) and 110 dB (bottom) auditory stimuli. The vehicle-treated psychosocial
stress group generally exhibited greater startle responses than the control group to all of the auditory stimuli. Tianeptine and the higher doses of amitriptyline and clonidine
were most effective in preventing this effect. The data are presented as the mean ± SEM. * p b 0.05 relative to the vehicle-treated no psychosocial stress group; β = p b 0.05 relative
to the vehicle-treated psychosocial stress group; τ = pb 0.05 relative to the respective drug-treated no psychosocial stress group; ω = p = 0.056 relative to the vehicle-treated no
psychosocial stress group.
time that rats spent with the familiar object. The analysis comparing
the ratio times of all groups revealed no significant main effects or interactions (ps > 0.05). Overall, this behavioral assessment revealed no
evidence of a preference by the rats for the novel object, nor was
there an effect of psychosocial stress or drug treatment.
amitriptyline exhibited significantly lower corticosterone levels than
the controls treated with 10 mg/kg of amitriptyline an hour following
20 min of immobilization. In contrast, 0.01 mg/kg of clonidine prevented
the reduction in corticosterone levels an hour following immobilization
in the psychosocial stress group only.
3.5. Corticosterone levels
3.6. Cardiovascular activity
There was a significant main effect of time point, F(2,194) = 487.29,
p b 0.001. Corticosterone levels, overall, increased following 20 min of
acute immobilization stress, and these levels declined, but remained
significantly elevated relative to baseline, 1 h later (Fig. 4). There was
also a significant main effect of drug, F(5,97) = 24.00, p b 0.001. Rats
treated with 5 mg/kg of amitriptyline displayed significantly lower corticosterone levels, overall, than all other groups, except for those treated
with tianeptine or 10 mg/kg of amitriptyline. In addition, rats treated
with 10 mg/kg of amitriptyline exhibited significantly lower corticosterone levels, overall, than all other groups, except for those treated with
5 mg/kg of amitriptyline. Lastly, rats that were treated with tianeptine
had significantly lower corticosterone levels, overall, than rats treated
with vehicle or 0.01 mg/kg of clonidine. The Time Point× Drug interaction was significant, F(10,194) = 8.91, p b 0.001. Both doses of amitriptyline, particularly the 10 mg/kg dose, significantly blunted the
acute immobilization-induced increase in serum corticosterone levels,
independent of exposure to chronic psychosocial stress. Tianeptine
had a similar effect, although not as pronounced as that of amitriptyline.
The Psychosocial Stress × Drug, F(5,97) = 2.70, and Time Point ×
Psychosocial Stress×Drug, F(10,194)=2.21, interactions were also significant (psb 0.05). Psychosocially stressed rats treated with 10 mg/kg of
3.6.1. Heart rate
The vehicle-treated psychosocial stress group exhibited significantly greater HR than the vehicle-treated no psychosocial stress
group (Fig. 5). The Psychosocial Stress × Drug interaction was significant, F(5,81) = 3.99, p b 0.01. Chronic treatment with 5 or 10 mg/kg
of amitriptyline, 0.01 or 0.05 mg/kg of clonidine or 10 mg/kg of
tianeptine in groups that were psychosocially stressed prevented
the chronic stress-induced increase in HR, as evidenced by the presence of significantly lower HR than the vehicle-treated psychosocial
stress group or a lack of statistical significance relative to each of
the group's respective drug-treated no psychosocial stress group.
3.6.2. Systolic blood pressure
There was a significant main effect of drug, F(5,86) = 11.80, and
the Psychosocial Stress × Drug interaction was significant, F(5,86) =
3.60 (ps b 0.01). The vehicle-treated psychosocial stress group had
significantly higher systolic BP than the vehicle-treated no psychosocial stress group (Fig. 5). Chronic treatment with 5 or 10 mg/kg of
amitriptyline in groups that were not psychosocially stressed led to significantly greater systolic BP than the vehicle-treated no psychosocial
stress group. Chronic treatment with 5 or 10 mg/kg of amitriptyline,
8
P.R. Zoladz et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 44 (2013) 1–16
Vehicle
Amitriptyline (5 mg/kg)
25
20
15
10
5
No Psychosocial Stress
Psychosocial Stress
0
20 min
Immobilization
35
30
25
20
15
10
5
No Psychosocial Stress
Psychosocial Stress
0
80 min
0 min
Home Cage
20 min
Immobilization
Corticosterone (µg/dL)
30
0 min
*
10
5
No Psychosocial Stress
Psychosocial Stress
0
0 min
20 min
Immobilization
80 min
Home Cage
20
15
10
5
*
0
0 min
20 min
Immobilization
Home Cage
80 min
Home Cage
Tianeptine (10 mg/kg)
35
30
25
20
15
10
5
No Psychosocial Stress
Psychosocial Stress
0
0 min
20 min
Immobilization
Corticosterone (µg/dL)
25
Corticosterone (µg/dL)
30
15
No Psychosocial Stress
Psychosocial Stress
25
80 min
35
20
30
Clonidine (0.05 mg/kg)
Clonidine (0.01 mg/kg)
35
Corticosterone (µg/dL)
Amitriptyline (10 mg/kg)
35
Corticosterone (µg/dL)
Corticosterone (µg/dL)
35
80 min
Home Cage
30
25
20
15
10
5
No Psychosocial Stress
Psychosocial Stress
0
0 min
20 min
Immobilization
80 min
Home Cage
Fig. 4. Serum corticosterone levels for the drug-treated psychosocial and no psychosocial stress groups. Psychosocial stress generally had no effect on corticosterone levels; however,
amitriptyline and tianeptine significantly blunted corticosterone levels. The data are presented as the mean±SEM at 3 time points: baseline (pre-stress; 0 min), stress (20 min) and
post-stress (80 min). * pb 0.05 relative to the respective no psychosocial stress group.
0.01 or 0.05 mg/kg of clonidine or 10 mg/kg of tianeptine in groups that
were psychosocially stressed prevented the chronic stress-induced
increase in systolic BP, as evidenced by the presence of significantly
lower systolic BP than the vehicle-treated psychosocial stress group or
a lack of statistical significance relative to each of the group's respective
drug-treated no psychosocial stress group.
3.6.3. Diastolic blood pressure
There were significant main effects of psychosocial stress, F(1,86) =
9.67, and drug, F(5,86)= 21.78, and the Psychosocial Stress× Drug
interaction was significant, F(5,86) = 6.11 (ps b 0.01). The vehicletreated psychosocial stress group had significantly higher diastolic BP
than the vehicle-treated no psychosocial stress group (Fig. 5). Additionally, chronic treatment with 5 or 10 mg/kg of amitriptyline in groups
that were not psychosocially stressed led to significantly greater diastolic BP than the vehicle-treated no psychosocial stress group. Chronic
treatment with 10 mg/kg of amitriptyline, 0.01 or 0.05 mg/kg of clonidine or 10 mg/kg of tianeptine in psychosocially stressed rats led to significantly lower diastolic BP than the vehicle-treated psychosocial stress
group. However, psychosocially stressed rats that were treated with
10 mg/kg of amitriptyline still displayed significantly greater diastolic
BP than the vehicle-treated no psychosocial stress group, and psychosocially stressed rats that were treated with 0.01 mg/kg of clonidine still
exhibited significantly greater diastolic BP than its respective drugtreated no psychosocial stress group.
3.7. Growth rate
Growth rate, expressed as an increase in grams per day (g/day),
was calculated for all rats by taking their total body weight gained
from the day of the first stress session through the first day of behavioral testing and dividing this by the total number of days elapsed
(i.e., 31 days). There was a significant main effect of drug,
F(5,100) = 11.78, and the Psychosocial Stress x Drug interaction was
significant, F(5,100) = 13.42 (ps b 0.001). The vehicle-treated psychosocial stress group had a significantly lower growth rate than the
vehicle-treated no psychosocial stress group (Fig. 6). Chronic treatment with 5 or 10 mg/kg of amitriptyline or 0.05 mg/kg of clonidine
in groups that were not psychosocially stressed led to significantly
lower growth rates than the vehicle-treated no psychosocial stress
group. Chronic treatment with 5 or 10 mg/kg of amitriptyline,
0.01 mg/kg of clonidine or 10 mg/kg of tianeptine prevented the
chronic stress-induced reduction of growth rate, as evidenced by
the presence of significantly greater growth rates than the vehicletreated psychosocial stress group or a lack of statistical significance
relative to each of the group's respective drug-treated no psychosocial stress group (Fig. 6). However, the psychosocial stress group
treated with 5 mg/kg of amitriptyline exhibited a significantly lower
growth rate than the vehicle-treated no psychosocial stress group.
3.8. Adrenal gland weight
The adrenal glands were weighed and expressed as milligrams per
100 g of body weight (mg/100 g b.w.). There was a significant main effect of drug, F(5,97)= 10.53, and the Psychosocial Stress× Drug interaction was significant, F(5,97) = 3.26 (ps b 0.01). The vehicle-treated
psychosocial stress group had significantly greater adrenal gland weight
than the vehicle-treated no psychosocial stress group (Fig. 6). Chronic
treatment with 10 mg/kg of amitriptyline or 0.05 mg/kg of clonidine
in groups that were not psychosocially stressed led to significantly
P.R. Zoladz et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 44 (2013) 1–16
Heart Rate (bpm)
450
425
Systolic Blood Pressure
No Psychosocial Stress
Psychosocial Stress
*
τ
400
β
β
375
350
0
VEH
AMI-5 AMI-10 C-0.01 C-0.05
TIA-10
Systolic Blood Pressure (mm Hg)
Heart Rate
9
150
No Psychosocial Stress
Psychosocial Stress
*
*
*
135
β
β
β
120
*
β
105
0
VEH
AMI-5 AMI-10 C-0.01
C-0.05 TIA-10
Diastolic Blood Pressure (mm Hg)
Diastolic Blood Pressure
110
100
No Psychosocial Stress
Psychosocial Stress
β
* *
* **
β
τ
90
β
80
β
70
0
VEH
AMI-5
AMI-10 C-0.01 C-0.05 TIA-10
Fig. 5. Cardiovascular activity exhibited by rats on the final day of testing. The vehicle-treated psychosocial stress group exhibited significantly greater heart rate (top left), systolic
blood pressure (top right) and diastolic blood pressure (bottom) than the vehicle-treated no psychosocial stress group. Although each of the drugs, in some way, were effective at
preventing this stress-induced increase in cardiovascular activity, clonidine and tianeptine were the most consistently effective pharmacological agents to prevent such effects. The
data are presented as the mean ± SEM. * p b 0.05 relative to the vehicle-treated no psychosocial stress group; β = p b 0.05 relative to the vehicle-treated psychosocial stress group;
τ = p b 0.05 relative to the respective drug-treated no psychosocial stress group.
greater adrenal gland weight than the vehicle-treated no psychosocial
stress group. Chronic treatment with 5 mg/kg of amitriptyline, 0.01 or
0.05 mg/kg of clonidine or 10 mg/kg of tianeptine in groups that were
psychosocially stressed prevented the chronic stress-induced hypertrophy of the adrenal glands, as evidenced by a lack of a statistically significant increase in adrenal gland weight relative to each of the group's
respective drug-treated no psychosocial stress group. Interestingly,
the psychosocial stress group treated with 10 mg/kg of amitriptyline
exhibited significantly reduced adrenal gland weight than its respective
drug-treated control group.
3.9. Thymus weight
The thymus was weighed and expressed as milligrams per 100 g
of body weight (mg/100 g b.w.). There was a significant main effect
of psychosocial stress, F(1,91) = 17.12, and the Psychosocial Stress ×
Drug interaction was significant, F(5,91) = 6.49 (ps b 0.001). The difference in thymus weight between the vehicle-treated psychosocial
stress group and the vehicle-treated no psychosocial stress group
was marginally significant (p = 0.051). Chronic treatment with
10 mg/kg of amitriptyline in groups that were not psychosocially
stressed led to significantly reduced thymus weight than the vehicletreated no psychosocial stress group. Chronic treatment with 5 mg/kg
of amitriptyline or 0.01 or 0.05 mg/kg of clonidine in groups that
were psychosocially stressed led to significantly reduced thymus
weight than the vehicle-treated no psychosocial stress group. Chronic
treatment with 10 mg/kg of amitriptyline or 10 mg/kg of tianeptine
prevented the slight decrease in thymus weight induced by chronic
psychosocial stress, as evidenced by significantly greater thymus
weight than the vehicle-treated psychosocial stress group and/or a
lack of a statistically significant decrease in thymus weight relative to
each of the group's respective drug-treated no psychosocial stress
group (Fig. 6).
4. Discussion
We have extended our established animal model of PTSD by evaluating the influence of conventional, as well as potentially novel, pharmacological treatments on psychosocial stress-induced sequelae in
psychosocially rats. We have emphasized the clinical relevance in our
design by initiating treatment 24 h after initial stress exposure and continuing the treatment daily throughout the duration of the experiment.
Treatment beginning 24 h after exposing rats to an intense stressor is
potentially relevant to treatment applications initiated in people
who seek treatment within 24 h of a traumatic experience. The relative
effectiveness of the treatments described here may highlight the importance of beginning a treatment regimen soon after a person experiences
intense trauma. Our approach may be useful not only for understanding
the physiological and behavioral mechanisms underlying PTSD, but also
for discerning how certain treatments act on the brain to ameliorate
PTSD-like symptom development.
Consistent with our previous work (Zoladz et al., 2008a, 2012),
vehicle-treated psychosocially stressed rats exhibited reduced growth
rate, greater adrenal gland weight, heightened anxiety, an exaggerated
startle response, greater BP reactivity to an acute stressor and strong,
fear conditioned “traumatic” memory for the context and cue that
were associated with the two cat exposures. While amitriptyline and
clonidine were effective in ameliorating a subset of the PTSD-like
10
P.R. Zoladz et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 44 (2013) 1–16
Adrenal Gland Weight
No Psychosocial Stress
Psychosocial Stress
6
Growth Rate (g/day)
Adrenal Gland Weight (mg / 100 g b.w.)
Growth Rate
β
5
* βτ
*
* **
4
3
#
2
1
0
VEH
AMI-5
AMI-10 C-0.01
C-0.05 TIA-10
No Psychosocial Stress
Psychosocial Stress
*
15
14
13
τ
*
12
*
*
*
11
10
9
0
VEH
AMI-5 AMI-10 C-0.01 C-0.05
TIA-10
Thymus Weight (mg / 100 g b.w.)
Thymus Gland Weight
135
No Psychosocial Stress
Psychosocial Stress
120
τ
105
τ
* *
90
β
τ
*
τ
*
75
0
VEH
AMI-5
AMI-10 C-0.01 C-0.05 TIA-10
Fig. 6. Growth rate and organ weights for all groups. The vehicle-treated psychosocial stress group exhibited a significantly lower growth rate, significantly larger adrenal gland
weight and a marginally significant reduction of thymus gland weight. Tianeptine was the most consistent pharmacological agent in blocking these stress-induced effects. The
data are presented as the mean ± SEM. * p b 0.05 relative to the vehicle-treated no psychosocial stress group; β = p b 0.05 relative to the vehicle-treated psychosocial stress
group; τ = p b 0.05 relative to the respective drug-treated no psychosocial stress group; # = p b 0.05 relative to all other groups.
symptoms, tianeptine was the only pharmacological agent that
prevented the effects of psychosocial stress on all physiological and behavioral endpoints without having adverse effects of its own in control
animals. Thus, our findings highlight tianeptine, more than the other
agents, as a potential component of pharmacotherapy to include in
early post-trauma treatment. In Table 1 we have provided a comparison
of the effectiveness of each pharmacological agent in ameliorating the
psychosocial stress effects on the behavioral and physiological measurements under study.
Table 1
Efficacy of amitriptyline, clonidine and tianeptine in ameliorating PTSD-like sequelae in
rats.
Endpoint
AMI-5
AMI-10
C-0.01
C-0.05
TIA
Context test
Cue test
EPM — open arms
Startle — 90 dB
Startle — 100 dB
Startle — 110 dB
Heart rate
Systolic BP
Diastolic BP
Growth rate
Adrenal gland weight
Thymus gland weight
+
N
−
+
−
+
+
C
C
C
+
−
+
+
+
+
+
+
+
C
C
C
C
C
N
−
+
+
N
+
+
+
N
+
+
−
−
−
+
+
+
+
+
+
+
C
C
−
+
+
+
+
+
+
+
+
+
+
+
+
+ = prevented chronic psychosocial stress effects on endpoint; − = did not prevent
chronic psychosocial stress effects on endpoint; N = insufficient basis for determining
if chronic psychosocial stress effects on endpoint were prevented; C = compromised
assessment due to adverse side effects in controls.
4.1. Amitriptyline
Both doses of the TCA amitriptyline blocked the expression of traumatic memory in psychosocially stressed rats in response to the context
and cue that were paired with the two cat exposures (see the 5 mg/kg
dose response for a caveat; Table 1). These fear responses served as a
measure of memory for the acute stress experiences and rat analogs
of a traumatic memory in humans (Zoladz et al., 2012). As traumatic
memories are a source of psychological distress in people with PTSD,
these findings suggest that amitriptyline could serve to effectively reduce the strength of traumatic memories and consequentially diminish
the intrusion and re-experiencing symptoms endured by PTSD patients.
One caveat to this interpretation, however, is that extensive work has
reported amitriptyline-induced memory impairments in both humans
(Kerr et al., 1996; Liljequist et al., 1978; Mattila et al., 1978; Spring et
al., 1992; van Laar et al., 2002) and rodents (Everss et al., 2005;
Gonzalez-Pardo et al., 2008; Kumar and Kulkarni, 1996), findings that
may be related to the drug's anti-cholinergic side effects (Pavone et
al., 1997). Therefore, the attenuation of contextual and cue fear conditioning in psychosocially stressed rats treated with amitriptyline could
be due to its amnestic side effects, rather than a specific amelioration
of the chronic stress-induced behavioral sequelae. On the other hand,
studies reporting amitriptyline-induced memory impairments have administered the drug prior to learning. In the present experiment, amitriptyline treatment did not begin until 24 h after the first pairing of
the context and cue with the cat exposure. Therefore, a more likely explanation of the present findings is that amitriptyline blunted the augmentation of contextual and cue fear conditioning in psychosocially
stressed rats that occurred in response to the second cat exposure on
P.R. Zoladz et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 44 (2013) 1–16
Day 11 of the paradigm. Another possible explanation of these findings
is that amitriptyline increased general locomotor activity, thus reducing
overall immobility. Amitriptyline-treated control animals did display a
significantly greater amount of motor activity on the EPM than
vehicle-treated animals, but the same effect was not observed during
the open field habituation period on the following day.
Both doses of amitriptyline were at least partially effective in
preventing the chronic stress-induced increase in startle response,
but only the 10 mg/kg dose of amitriptyline blocked the effects of
chronic psychosocial stress on anxiety, as measured by rat behavior
on the EPM. These findings are consistent with other work in the rodent literature reporting that amitriptyline exerts anxiolytic effects in
control animals (Bodnoff et al., 1988; Zajaczkowski and Gorka, 1993)
and blocks stress-induced increases in anxiety-like behavior and
startle response (Orsetti et al., 2007; Poltyrev and Weinstock, 2004;
West and Weiss, 2005). Research in humans has also shown that
amitriptyline significantly blunts the startle response (Phillips et al.,
2000). Thus, amitriptyline appears to have potent anxiolytic effects
that may effectively ameliorate the hyperarousal symptoms related
to PTSD.
Amitriptyline also led to significantly lower serum corticosterone
levels in rats and was particularly effective in blunting the
immobilization-induced increase in these levels on the final day of
testing. This finding is consistent with several studies in the rodent
literature reporting that chronic amitriptyline administration results
in significantly reduced basal and stress-induced levels of adrenocorticotropic hormone (ACTH) and corticosterone in rats (Barden, 1999;
Reul et al., 1993). Amitriptyline appears to accomplish these effects
by enhancing the negative feedback inhibition of the hypothalamus–
pituitary–adrenal (HPA) axis. Investigators have shown that chronic
amitriptyline administration leads to an up-regulation of glucocorticoid
receptor expression and enhanced glucocorticoid receptor binding
in several brain regions (Barden, 1999; Pariante and Miller, 2001;
Przegalinski and Budziszewska, 1993; Reul et al., 1993). Interestingly, in
the present study, there was an additive effect of amitriptyline and psychosocial stress on the recovery of serum corticosterone levels following
immobilization. Psychosocially stressed rats that were treated with amitriptyline, particularly the 10 mg/kg dose, exhibited significantly lower
corticosterone levels at the 80 minute time point than amitriptylinetreated controls. In theory, chronic amitriptyline and psychosocial stress
(Zoladz et al., 2012) synergistically facilitated the production of enhanced
negative feedback of the HPA axis, which led to a more rapid recovery of
stress-induced serum corticosterone levels in these rats.
Despite the positive effects of amitriptyline on the chronic stressinduced physiological and behavioral sequelae in rats, there were adverse side effects of the drug that should be considered. For instance,
chronic amitriptyline treatment resulted in significantly greater acute
stress-induced increases in systolic and diastolic BP than vehicle.
Most work in both humans and rodents has reported that chronic
amitriptyline treatment results in increased HR, postural hypotension
and increased cardiotoxicity (Balcioglu et al., 1991; Fiedler et al.,
1986; Hong et al., 1974; Joubert et al., 1985; Kopera, 1978; Low and
Opfer-Gehrking, 1992; Yokota et al., 1987). The results presented
here reveal that chronic amitriptyline treatment has unfavorable
effects on cardiovascular activity in rats exposed to psychosocial
stress, as well.
Amitriptyline also led to a significant reduction in growth rate,
particularly at the higher dose of the drug. Although counterintuitive,
10 mg/kg of amitriptyline significantly reduced growth rates in the
control rats, an effect that was reversed by exposure to chronic psychosocial stress. Similar findings were observed with regard to adrenal gland and thymus weights. The higher dose of amitriptyline
led to a significantly larger adrenal gland and a significantly smaller
thymus than vehicle-treated control (i.e., non-stressed) animals,
and exposure to chronic psychosocial stress significantly blunted
each of these effects. These findings suggest that, in the present
11
experiment, there was an interaction between amitriptyline treatment and chronic psychosocial stress, in which the physiological consequences of the drug were more prominent in those rats that were
not stressed.
Another finding of interest is that upon dissection of the
amitriptyline-treated animals, we observed a large number of adhesions on the internal organs, such as the liver, intestines and spleen.
In addition, the mortality rate for amitriptyline-treated rats (3/40,
7.5%) was greater than the mortality rates for rats treated with clonidine (0/40, 0%) or tianeptine (0/20, 0%). Thus, despite its prevention
of effects of psychosocial stress on anxiety-like behavior, startle response and fear memory, the adverse physiological side effects of
amitriptyline could be a major limitation to its use in the treatment
of people with PTSD.
4.2. Clonidine
Neither dose of clonidine prevented the expression of traumatic
memory in psychosocially stressed rats in response to the context
and cue that were paired with the two cat exposures. Although
psychosocially stressed rats chronically treated with 0.01 mg/kg of
clonidine did not display significantly greater immobility during the
context test than vehicle-treated control rats, the within-drug contrast
(i.e., clonidine-treated psychosocial stress group vs. clonidine-treated
controls) was marginally significant. However, these findings should
be interpreted cautiously. Since clonidine is an α2-adrenergic receptor
agonist and significantly reduces central noradrenergic activity, it can
have sedative side effects at higher doses (Millan et al., 2000). In the
present study, the higher dose of clonidine did result in a significant reduction of locomotor activity in the open field during NOR habituation.
On the other hand, clonidine had no significant effects on motor activity
on the EPM, and psychosocially stressed rats treated with 0.05 mg/kg of
clonidine still produced significantly more fecal boli during the context
test than the no psychosocial stress group treated with 0.05 mg/kg of
clonidine (data not shown). One study also reported that clonidine's
sedative effects are not observed until doses greater than 0.1 mg/kg
are employed (Millan et al., 2000). Therefore, the present data support
the notion that clonidine is ineffective in blunting the expression of a
traumatic memory in rats.
Both doses of clonidine, and in particular the 0.05 mg/kg dose,
blocked the effects of psychosocial stress on anxiety and startle, as
well as cardiovascular responses to acute immobilization. However,
the higher dose of clonidine led to a significantly reduced growth rate
in controls, which was exacerbated by chronic psychosocial stress. It
also resulted in significantly increased adrenal gland weight in control
animals. Lastly, neither dose of clonidine prevented the chronic psychosocial stress-induced decrease in thymus weight. Thus, despite its
amelioration of a subset of the chronic stress-induced behavioral and
cardiovascular sequelae, clonidine was ineffective at preventing the
remaining physiological changes induced by the psychosocial stress
regimen.
Since people with PTSD have significantly elevated baseline NE
levels and demonstrate adverse reactions (e.g., panic attacks, flashbacks) to agents that increase these levels (e.g., yohimbine), pharmacological agents that reduce noradrenergic activity could be effective
treatments for people with PTSD (Boehnlein and Kinzie, 2007; Strawn
and Geracioti, 2008). Some studies have reported that propranolol,
a β-adrenergic receptor antagonist, may be an effective treatment
for PTSD if administered after the traumatic event or after the
re-experiencing of a traumatic event (Brunet et al., 2011; Hoge et al.,
2012; Pitman et al., 2002; Taylor and Raskind, 2002; Vaiva et al., 2003).
Other work has found that prazosin, an α1-adrenergic receptor antagonist, reduces hyperarousal symptoms, intrusive thoughts, recurrent
distressing dreams and sleep disturbances in PTSD (Brkanac et al.,
2003; Peskind et al., 2003; Raskind et al., 2002, 2003; Taylor and
Raskind, 2002; Taylor et al., 2006). The present findings suggest that
12
P.R. Zoladz et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 44 (2013) 1–16
clonidine, as well as propranolol and prazosin, can serve as adjunct contributions to pharmacotherapy, specifically in targeting the anxiety,
hyperarousal (e.g., exaggerated startle response) and cardiovascular
components of PTSD.
4.3. Tianeptine
Tianeptine was the only pharmacological agent that prevented the
effects of chronic psychosocial stress on all physiological and behavioral measures and did not produce adverse side effects in control
animals. Tianeptine completely blocked the expression of fearrelated behaviors in psychosocially stressed rats in response to the
context and cue that were paired with the two cat exposures. It also
prevented the effects of psychosocial stress on anxiety, startle, cardiovascular reactivity to an acute stressor, growth rate, adrenal gland
and thymus weights.
The present findings are consistent with previous work which
reported that chronic, but not acute, administration of tianeptine significantly reduced the expression of conditioned fear in rats (Burghardt et
al., 2004). These effects appear to be more related to a reduction of fear
expression by tianeptine, rather than a memory-impairing effect, per se,
as numerous studies have shown that tianeptine treatment enhances,
rather than impairs, hippocampus-dependent learning and memory
(Jaffard et al., 1991; Meneses, 2002; Munoz et al., 2005). Indeed, we
have reported that tianeptine enhanced spatial memory and synaptic
plasticity by blocking the adverse effects of acute stress on both measures (Campbell et al., 2008; Diamond et al., 2004; Vouimba et al.,
2006; Zoladz and Diamond, 2010; Zoladz et al., 2008b). Thus, tianeptine
appears to demonstrate long-term anxiolytic effects that are similar to
SSRIs, without the initial anxiogenic effects typically induced by SSRIs
(Burghardt et al., 2004, 2007; Ravinder et al., 2011; Robert et al.,
2011; Zienowicz et al., 2006).
We also observed that tianeptine, unlike clonidine, significantly
attenuated the immobilization-induced increase in serum corticosterone levels. This finding is consistent with previous work indicating
that chronically administered tianeptine reduced the stress-induced
activation of the HPA axis (Delbende et al., 1994). In contrast, we
have reported that acute treatment with tianeptine blocked predator
stress effects on spatial memory without affecting corticosterone
levels (Campbell et al., 2008). Furthermore, we have demonstrated
that tianeptine blocked the memory-impairing effects of predator
stress in adrenalectomized rats (Zoladz et al., 2008b). This observation confirmed that stress effects on memory are not dependent on
a stress-evoked increase in corticosterone levels, and more importantly, that the memory-protective effect of tianeptine is independent
of corticosterone levels. The current work adds to our understanding of
tianeptine-HPA interactions by demonstrating that chronic tianeptine
treatment attenuated HPA axis activity in rats exposed to a novel acute
stressor.
In a related finding, tianeptine, relative to vehicle, resulted in significantly lower systolic BP in control animals following 20 min of immobilization stress. Few studies have examined the effects of tianeptine on
cardiovascular activity, but those that have investigated the phenomenon have reported either no effect on HR or BP (Juvent et al., 1990;
Lasnier et al., 1991) or reduced diastolic BP (Lechin et al., 2006). The effects of tianeptine on cardiovascular activity in the present study are not
likely due to acute effects of the drug. In recent work we have found that
tianeptine does not prevent an acute stress-induced increase in BP
(unpublished findings). In this pilot study, rats were treated with
10 mg/kg of tianeptine or vehicle 30 min prior to a 15-min exposure
to predator stress. Rats exposed to the cat for 15 min exhibited significant elevations of systolic and diastolic BP which were unaffected by
tianeptine treatment. Hence, tianeptine did not exert a direct influence
on the acute stress-induced increase in BP. Therefore, the tianeptinemediated prevention of the effects of chronic psychosocial stress on cardiovascular reactivity to an acute stressor is more likely attributable to
its attenuation of anxiety in response to stress, rather than a direct effect
on blood pressure regulation. It is also possible that the tianeptine effects on stabilizing blood pressure (as well as the HPA axis) under stress
may be mediated indirectly via a strengthening of coping strategies involving the tianeptine-mediated enhancement of higher cortical functioning (Dupin et al., 2006; Qi et al., 2009; Sacchetti et al., 1993).
Chronic treatment with tianeptine also prevented the effects of
chronic psychosocial stress on all of the other physiological endpoints, including growth rate, adrenal gland weight and thymus
weight. Previous work in rodents has reported that tianeptine had
no effect on the chronic stress-induced adrenal gland hypertrophy
or reduction in growth rate (Magarinos et al., 1999; Watanabe et al.,
1992b). However, these studies utilized a different stressor (restraint
stress, 6 h per day for 21 days) than that which was employed here,
which could account for the discrepancies. The finding that tianeptine
prevented chronic stress-induced atrophy of the thymus is consistent
with work demonstrating that tianeptine insulates the immune system from adverse effects of stress. For instance, several studies have
shown that tianeptine prevents the adverse effects of cytokines on
brain biochemistry and peripheral measures of inflammation in
the rat (Castanon et al., 2001; Plaisant et al., 2003a, 2003b). Thus,
an important avenue of study in clinical trials would involve the administration of tianeptine to block auto-immune related pathology,
which is commonly observed in traumatized people (Boscarino,
2004, 2010; Heim et al., 2000).
Recent work has suggested that tianeptine's therapeutic effects
may be associated with enhanced neuroplasticity and stabilization
of glutamatergic neurotransmission (Brink et al., 2006; Kasper and
McEwen, 2008; Kole et al., 2002; McEwen et al., 2010; Zoladz et al.,
2008b). For example, the deleterious effects of stress on brain structure and function are, at least in part, a result of glutamatergic hyperactivity (Bagley and Moghaddam, 1997; Bartanusz et al., 1995; Joels
et al., 2003; Krugers et al., 1993; Lowy et al., 1993, 1995; McEwen
et al., 2002; Moghaddam, 1993; Reznikov et al., 2007; Yang et al.,
2005), and tianeptine appears to normalize this alteration (Kole
et al., 2002; Reznikov et al., 2007), resulting in normal physiology
and behavior during stress (Campbell et al., 2008; Diamond et al.,
2004; Kasper and McEwen, 2008; McEwen et al., 2010; Shakesby
et al., 2002; Vouimba et al., 2006; Zoladz et al., 2008b). It is therefore
likely that the PTSD-like sequelae observed in our rats were associated with alterations of glutamate function which were normalized
by daily tianeptine treatment. In related work, investigators have
suggested that abnormalities in glutamatergic function could underlie
the pathology of PTSD (Chambers et al., 1999; Nair and Singh, 2008;
Reul and Nutt, 2008). Together, these findings highlight the potential
importance of stabilizing glutamate neurotransmission in future research on PTSD pharmacotherapy.
4.4. Caveats
In contrast to our previous work (Zoladz et al., 2008a, 2012),
vehicle-treated psychosocially stressed rats did not display lower
levels of baseline corticosterone, a significant impairment of object
recognition memory or significantly reduced thymus weights, relative to vehicle-treated control animals. One possible explanation for
the lack of statistical significance is that the chronic injections in the
present study acted as a persistent mild stressor in control rats,
which added variability to their data on these measures. Indeed,
other work in rodents has reported that chronic mild stress in the
form of repeated injections can significantly alter rat physiology and
behavior (Weaver et al., 2005; Wellman, 2001). In addition, we
have previously reported significantly reduced baseline corticosterone levels in rats that were exposed to our psychosocial stress paradigm (Zoladz et al., 2012), a finding that is consistent with much of
the PTSD literature on HPA axis alterations (Yehuda, 2005). However,
we observed this effect only when psychosocially stressed rats had
P.R. Zoladz et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 44 (2013) 1–16
not been exposed to repeated injections prior to blood sampling.
Thus, in the present study, it is likely that the chronic injections and
numerous behavioral manipulations that were performed on rats hindered our ability to detect some of the previously-observed effects on
stress on rat physiology and behavior.
Another finding that diverges from our previous work is that the
vehicle-treated psychosocially stressed rats demonstrated significantly greater HR than vehicle-treated control rats following exposure to an acute stressor on the final day of testing. Previously, we
reported that the present psychosocial stress paradigm resulted in
significantly lower HR, compared to controls, following acute stress
on the final day of testing. Nevertheless, the HR exhibited by psychosocially stressed rats in the present study (409.85 ± 7.30 bpm) was
very similar to the HR exhibited by psychosocially stressed rats in
our previous work (413.25 ± 9.93 bpm). The basis of the inconsistent
effects between the findings of the present study and those of our
previous work is the HR exhibited by the control animals in each
case. Vehicle-treated control rats displayed lower HR in the present
study (385.61 ± 8.11 bpm) than that which was displayed by controls
in the prior study (462.88 ± 11.43 bpm). In theory, vehicle-treated
controls could have exhibited lower HR in the present study because
the chronic mild stress of repeated injections desensitized them
from responding as strongly to the acute stressor as the more naïve
animals that were utilized in our prior work.
The design of the present experiment did not afford us the opportunity to make within-group comparisons (i.e., pre-stress vs. poststress) on measures of physiology and behavior in rats that were exposed to chronic psychosocial stress and administered the various
pharmacological agents. We chose not to expose rats in the present
study to any pre-stress physiological or behavioral testing to eliminate any influence pre-stress manipulations could have had on the
battery of tests administered at the conclusion of the experiment.
Moreover, we did not want to expose rats to the same apparatus multiple times, as prior exposures could influence behavior on subsequent tests. For example, in the current study the no Psychosocial
Stress vehicle- and drug-treated groups were exposed twice to the
fear conditioning chamber without reinforcement (cat exposure).
This repeated unreinforced exposure resulted in the rats in these
groups developing greater immobility with each exposure, which indicates that their initial exploratory response habituated with repeated
exposures to the same environment. Although this observation is of
value to the study of habituation behavior, it illustrates the problem
with repeatedly exposing rats to the same environment where subsequent critical behavioral testing, specifically fear memory-induced immobility, will occur. Overall, our findings should be interpreted within
the context of a design involving the strictest control over factors that
might have influenced how the various drug treatments interacted
with psychosocial stress.
5. Summary and conclusions
A subset of people with PTSD shows an improvement in their
symptoms following treatment with SSRIs. However, SSRIs tend to
blunt the depressive components of PTSD, while having insufficient
effect on the memory- and anxiety-related symptoms of the disorder.
In addition, some forms of PTSD, such as combat-related PTSD, are
highly resistant to SSRI treatment. Moreover, these agents can have
anxiogenic effects early in the treatment phase and exert their antidepressant effects only after a substantial delay. Thus, there is an urgent
need to develop alternative pharmacotherapeutic interventions for
the treatment of PTSD.
We have found that the TCA amitriptyline was effective in reducing
the magnitude of the fear-conditioned memory for the context and cue
that were associated with the acute cat exposures, and it ameliorated
the stress-induced increase in anxiety and startle. However, amitriptyline had adverse side effects, as it significantly increased cardiovascular
13
reactivity in control animals and led to adverse physiological reactions,
including reduced growth rate, increase adrenal gland weight and internal adhesions. Thus, despite some positive effects, the adverse side effects of amitriptyline could be a significant limitation to its use in the
treatment of people with PTSD.
Clonidine blocked the effects of chronic psychosocial stress on
anxiety and startle, and in contrast to amitriptyline, prevented the
stress-induced changes in cardiovascular reactivity to the acute
stressor, as well. However, clonidine did not blunt the expression of
conditioned fear memory in psychosocially stressed rats in response
to re-exposure to the context and cue that were paired with the
acute cat exposures. Clonidine had adverse side effects as well, including a significant reduction in overall growth rate and an idiopathic increase in adrenal gland weight in the control (non-stress) group.
Thus, the current work indicates that clonidine can serve as an effective anxiolytic agent and reduces the hyperarousal (e.g., exaggerated
startle response) and cardiovascular components of PTSD, but it
would have little effect on the strength (and intrusiveness) of a traumatic memory.
Tianeptine was the only pharmacological agent to prevent the effects of chronic psychosocial stress on our entire battery of physiological
and behavioral endpoints. Specifically, tianeptine blocked the expression of fear-conditioned traumatic memory in psychosocially stressed
rats when they were re-exposed to the context and cue that were
paired with the two cat exposures. Tianeptine also prevented the effects
of psychosocial stress on anxiety, startle, cardiovascular reactivity to an
acute stressor, growth rate, and adrenal gland and thymus weights.
These salutary effects of tianeptine occurred in the absence of any observed adverse side effects.
In summary, the present experiment examined the influence of
amitriptyline, clonidine and tianeptine on the expression of PTSD-like
sequelae in rats. Our findings provide guidance in the development of
treatments for traumatized people by demonstrating the differential
effectiveness of each of these three agents in blocking the behavioral
and physiological consequences of psychosocial stress in rats.
Acknowledgments
This research was supported by Career Scientist and Merit Review
Awards from the Veterans Affairs Department to David Diamond. The
opinions expressed in this publication are those of the authors and
not of the Department of Veterans Affairs or the US government.
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