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The Exhibition of Behavioral Despair following Repeated Social Defeat Stress: Implications for Major Depression

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The Exhibition of Behavioral Despair following Repeated Social Defeat Stress: Implications for Major Depression
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Naccarato, Andrea
Devine, Darragh ( Mentor )
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Gainesville, Fla.
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University of Florida
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The Exhibition of Behavioral Despair following Repeated Social Defeat
Stress: Implications for Major Depression

Andrea Naccarato


ABSTRACT


Emotional stress is associated with the etiology and expression of major depression. In order to evaluate

the emotional impact of the social defeat model of stress, rats were tested in the Porsolt forced swim test. Rats

were exposed to either acute or repeated social defeat stress, while a control group was unstressed. Following

the final social defeat session (or equivalent time in the unstressed rats), all the rats were tested in a forced

swim, and behavioral despair (floating) was assayed. After one exposure to the emotional stressor, the rats

showed transient behavioral despair during the forced swim. After repeated exposure to the same

emotional stressor, the rats showed persistent behavioral despair during the forced swim. This outcome parallels

the behavioral despair that is seen in major depression. Therefore, this animal model is suitable for study of

the neurobiology of emotional stress-induced depression.



INTRODUCTION


Major depression is a common disorder, with 6.6% of Americans suffering from symptoms each year (Kessler et

al., 2003). According to the most recent Diagnostic and Statistical Manual of Mental Disorders (DSM),

characteristics of an adult individual suffering from major depressive disorder include, but are not limited

to, depressed mood, anhedonia, insomnia or hypersomnia, fatigue, psychomotor stimulation or

retardation, decreased concentration, and thoughts of suicide. In major depression, these symptoms are

not explained by another condition and are not substance-induced (American Psychiatric Association, 2000, pg

356). Major depressive disorder can exacerbate the manifestation of other medical diseases (Brown et. al,

2004), the combination of which can lead to increased occupational absences and increased health care costs.

Major depression is also the most prevalent cause of suicide in the United States (for review, see Mann et. al,

2005). Since major depressive disorder typically recurs over the lifetime of patients, a basic pathophysiology is

likely (for review, see McEwen, 2005).



Emotional stress is positively associated with the etiology and expression of many psychiatric disorders,

including major depression (for review, see Herman and Cullinan, 1997). These stressors elicit

characteristic physiological responses (e.g. increased respiration or heart rate) that may help an organism to

cope with alterations in its surrounding environment. However, not all stressors are of the emotional






variety. Stressors may be classified as systemic or processive. Systemic stressors (e.g. water deprivation)

may immediately jeopardize survival of the organism. The presentation of such events activates a response from

the hypothalamus without initial intervention by cortical and limbic brain structures (i.e., brain systems that

are involved in processing emotional valence and planning appropriate responses), and these stressors are

generally not associated with the etiology and expression of human psychopathology. Conversely,

processive stressors do not immediately jeopardize survival of the organism. These stressors comprise

emotional stimuli that may be considered stressful only when processed by cortical and limbic structures (e.

g. instability in social hierarchy). Since this class of stressor is associated with major psychopathology, it

suggests that these pathologies result from functional alterations in cortical and limbic circuits.



The hypothalamic-pituitary-adrenal (HPA) axis regulates humans' and other animals' responses to stress.

This cascade begins with catecholamine neurotransmitters acting on the hypothalamus in the brain to

release corticotropin-releasing hormone (CRH). CRH travels to the pituitary gland, which is situated near the

brain, to stimulate release of adrenocorticotropic hormone (ACTH). ACTH enters the general blood circulation

to arrive at the adrenal glands found above the kidneys. ACTH stimulates the adrenal glands to

release glucocorticoids (GCs), such as cortisol or corticosterone (for review, see Sapolsky et al., 2000). Cortisol

is primarily measured in humans and corticosterone is primarily measured in rats. GCs are capable of

negative feedback regulation of the HPA axis by acting on a variety of brain structures, including the hippocampus

or hypothalamus, thereby turning off the stress response when appropriate (for review, see Brown et al.,

1999). However, if the animal does not sufficiently cope with the presented stressor, this negative

feedback mechanism is inefficient (for review, see De Kloet, 2003).



A dysregulation of the hormones of the HPA axis is associated with major depressive disorder (for review, see

Parker et al., 2003). Specifically, major depression has been linked to adrenocortical hypertrophy (Parker et

al., 2003) and an excess of circulating cortisol (Brown et al., 1999; Parker et al., 2003). Furthermore, some

patients with major depression exhibit non-responsiveness to the dexamethasone suppression test (Brown et

al., 2004), which suggests an inefficiency of the negative feedback mechanism.

An animal model of stress is the social defeat model in rats (Miczek, 1979). This model has been shown to

produce hyperactivation of the HPA axis. In the social defeat model, a young naive male rat is introduced into

the home cage of an experienced, socially dominant male rat. The experienced resident male defeats the

naive intruder. Rats repeatedly exposed to social defeat stress exhibit elevated circulating

corticosterone concentrations 24 hours after the final social defeat session as compared with the

corticosterone concentrations in the unstressed controls (Lopes and Devine, 2004). The present study utilizes

the social defeat model followed by the Porsolt swim test to observe behavioral despair.



The Porsolt swim test is a commonly used animal model to test antidepressant actions of drugs. In this model, a

rat is placed into a tank of water and forced to swim. Rats treated with medications that have antidepressant

effects in humans exhibit less floating behavior compared with control rats (Porsolt et al., 1978). Conversely,

rats given corticosterone injections exhibit more floating behavior in a dose-orderly fashion, compared with






control rats (Johnson et al., 2006). The expression of floating behavior is interpreted as a behavioral measure

of despair.



The association between repeated emotional stress and depression has not been sufficiently characterized using

a social defeat - forced swim protocol. In this experiment, the Porsolt forced swim test will be used to

evaluate whether the socially defeated rats exhibit more helpless, floating behavior than undefeated, control rats do.



MATERIALS AND METHODS


Animals


A total of 34 Long-Evans male rats and 10 Long-Evans female rats were purchased from Harlan Co. of

Indianapolis, IN. Initially, the experimental rats were housed as sex-matched pairs in polycarbonate cages (43

x 21.5 x 25.5cm) for a one-week period of acclimation. The housing room was maintained on a 12hr/12hr light/

dark cycle, with lights on at 7:00a.m. Standard rat chow (LabDiet 5001) and tap water were available ad libitum

for the duration of the study.



Sixteen male rats (225-250g) were used as intruders that were pair-housed for the duration of the experiment.

Eight male rats were unstressed controls that were pair-housed for the duration of the experiment. Ten of the

male rats (300-325g) were used as dominant residents for the social defeat procedure. Ten female rats were used

as cage-mates for the resident rats.



The 10 male resident rats were vasectomized, singly-housed for 10 days after surgery, then pair-housed with

female rats for one week. Then, the resident male rats were screened for dominance behaviors. The male-

female pairings were then maintained throughout the duration of the experiment. The resident male and female

rats remained in a housing room separate from the room in which the intruder rats were housed. At the time of

the experiments, the residents weighed 600-800g and the intruders weighed 275-325g. All of the procedures

were pre-approved by the Institutional Animal Care and Use Committee at the University of Florida and

were conducted in accordance with the Guide for the Care and Use of Laboratory Animals.



Surgical Procedure


The 10 male resident rats were anesthetized with an intraperitoneal injection of ketamine:xylazine (50mg/

kg:5mg/kg). The lower abdomen (rostral to the scrotum) was shaved and washed three times with Betadine.

A small incision was made, each vas deferens was located, and a small portion of the duct was removed using

a micro-cautery knife. The incision was sutured and stapled closed. A 1ml subcutaneous injection of 0.9%

warm saline and keterolac was administered as an analgesic. The sedated rat was placed in an empty cage over

a heating pad until it recovered from anesthesia. The 10 vasectomized rats were singly housed during the

recovery period. The staples were removed after 10 days of recovery.







Experimental Procedure


The schedule of the experimental procedure is detailed in Table 1. The 16 intruder rats were randomly assigned

to two groups (n=8 rats per group). The repeated social stress group experienced social defeat once a day for

five consecutive days. The acute social stress group experienced social defeat stress once on the fifth day

only. Eight male rats of the same size and age as the intruder rats constituted a control group, which experienced

no social defeat stress. The control rats remained in their home cages during the five days of the experiment.







Table 1.
Experimental schedule for control, acutely stressed, and repeatedly stressed groups of rats
for the social defeat phase and Porsolt swim test phase

Social Defeat Phase Porsolt Swim Test
Phase

Group Day 1 Day 2 Day 3 Day 4 Day 5 Day 6

Control PS T

Acute SD PS T

Repeat SD SD SD SD SD PS T

Key: SD = Social Defeat; PS T = Porsolt Swim Test Phase


Before each social defeat session (in both the screening and experimental phases), the female rat was removed

from the male resident's cage and a smaller male intruder rat was placed into the resident's cage. The resident

rat and intruder rat were allowed to interact until the intruder rat displayed submissive, supine posture three times

or until five minutes elapsed (whichever occurred first). In the experimental phase, the intruder rat was

then removed from the resident's cage and placed in a small wire mesh cage (10 x 10 x 15 cm). The intruder

rat (inside the wire mesh cage) was placed back into the resident's cage until a total of 10 minutes elapsed from

the start of the encounter. The wire mesh cage served as physical protection for the intruder from the resident,

yet the intruder still had visual and olfactory contact with the resident. Both of the pair-housed intruder rats

from any particular cage were exposed to social defeat at the same time, with one intruder placed into one

resident's cage and the other intruder into another resident's cage. The repeatedly stressed rats were exposed to

the resident males in a pre-determined order so that each intruder was exposed to each resident, and there were

no repeat pairings. All social defeat sessions occurred between 8:00am and 10:00am.



Twenty-four hours after the final social defeat session was completed, the two groups of intruder rats and the

one control group of rats were exposed to the Porsolt swim test, which is a gauge of behavioral despair. The

swim test apparatus consisted of a cylindrical, plastic bucket positioned beneath a video camera, which

was suspended from the ceiling. Tap water was poured into the bucket until the water reached a depth of 25






cm, which allows the rat to balance on the end of its tail if it chooses to float. The water was prepared at

room temperature (24-270C). A bucket of fresh water was provided for each rat. The swim sessions began when

the rat was placed into the center of the water by an experimenter who was blind to the rat's previous

stress experience. The rat's exposure to the swim test lasted 15 minutes. Upon removal from the swim test, each

rat was towel dried and returned to its home cage. The order of intruder and control rats to be exposed to the

swim test was randomized, but pair-housed rats were exposed sequentially. All forced swim sessions

occurred between 8:00am and 12:00pm.



Behavioral Assays


Both social defeat and swim test sessions were videotaped and the rats' behaviors were scored without reference

to the treatment group to which the rat belonged. Regarding the Porsolt swim test, floating and swimming

behaviors were quantified. Floating was defined as a rat making no motions other than minimal paw movements

that were necessary to keep the head above water. The rat could remain in one location or slowly drift around

the bucket's edge. Swimming was defined as a rat vigorously moving with direction, such as swimming from

one side of the bucket to another or climbing at the sides of the bucket.



Total times (seconds) floating and swimming were determined using the Observer computer program.

Behavioral despair was inferred in groups that exhibited significantly more floating behavior. Two observers

scored the swim test videos to determine interobserver reliability.



Statistical Analyses


Potential between-group differences in total floating times from the Porsolt swim test were analyzed with a one-

way ANOVA. These results were then analyzed with Newman-Keuls multiple comparison tests comparing each

social defeat stress group with the control group. The floating time data were then divided into three 5-minute

bins, and were re-analyzed using a 3x3 (group x bin) repeated-measures ANOVA. These data were analyzed

with individual t-tests to determine if there were significant differences between groups in each bin.



RESULTS


A significant between groups difference in floating times was found (F (2, 21) = 5.363, p < 0.0131). The rats

that experienced the repeated social defeat schedule spent significantly more time floating during the 15-

minute Porsolt swim test, compared with the total floating time for the no-stress control group (Fig. 1).






900-
800-

oC 600-
LL 500-
400-
300-
200-
100
0
No Stress Acute Stress Repeated Stress


Figure 1. Floating during the Porsolt swim test. Total time floating (sec) for the no-stress control
group, acute stress group, and repeated stress group. Values expressed are group means � SEM (n =
8 rats per group). *Significantly different from control group at p < 0.05.


When the 15-minute swim session was divided into three 5-minute bins, a significant between groups effect (F(2,
42) = 2.424, p < 0.0150) and a significant time effect (F (2, 42) = 72.74, p < 0.0001) were found. There was not
a significant group x time interaction effect (F (4, 42) = 1.270, p < 0.2970). The duration of floating for the
acute stress group was compared with that of the control group for the 0-5 min, 5-10 min, and 10-15 min
bins, respectively (t = 0.1795, p < 0.8601; t = 2.171, p < 0.0476; t = 0.1770, p < 0.8621), with a
significant difference found for the 5-10 min bin only (Fig. 2). The duration of floating for the repeated stress
group was compared with that of the control group for the 0-5 min, 5-10 min, and 10-15 min bins, respectively (t
= 2.325, p < 0.0356; t = 3.598, p < 0.0029; t = 2.228, p < 0.0428), with significant differences found for all
bins (Fig. 2). The duration of floating for the acute stress group was compared with that of the repeated stress
group for the 0-5 min, 5-10 min, and 10-15 min bins, respectively (t = 1.677, p < 0.1157; t = 1.027, p < 0.3218;
t = 1.401, p < 0.1831), with no significant differences found (Fig. 2).






+

300- -



S200-



i -0- No Stress
S 100- -F- Acute Stress

-A- Repeated Stress


0-5 min 5-10 min 10-15 min


Figure 2. Floating during Porsolt swim test in 5-minute bins. Total floating time divided into three
5-minute bins for the no-stress control group, acute stress group, and repeated stress group of
rats. Values expressed are group means � SEM (n = 8 rats per group). *Repeated stress group
is significantly different from control group at p < 0.05. +Acute stress group significantly different
from control group at p < 0.05.



The floating times recorded by both observers differed by less than 40 seconds in 92% of the 15-minute
swim sessions scored. Furthermore, the two observers floating scores never differed by more than 43 seconds.



DISCUSSION


A deficit in emotional coping for the acutely stressed rats was revealed as increased floating behaviors when
the forced swim test was analyzed in 5-minute bins. A similar deficit in emotional coping for the repeatedly
stressed rats was revealed as increased floating behaviors when the forced swim test was analyzed for overall
effect and in 5-minute bins. These results suggest that rats exposed to a single, severe emotional stressor
(social defeat) develop a deficit in emotional coping. After repeated exposures to the same emotional stressor,
this deficit in emotional coping appears to persist and increase. Accordingly, this study suggests that the number
of experiences an animal has with an emotional stressor may affect its ability to cope in other stressful,
yet dissimilar, situations.


The social defeat - forced swim protocol appears to have face validity, predictive validity, and etiological validity
to be an appropriate model of major depression. Regarding face validity, the floating observed in the forced swim
is similar to behavioral depression or anhedonia symptoms of major depression. Regarding predictive validity,
many other studies have demonstrated the capability of antidepressants to decrease floating in the forced swim
test (for review, see Cryan et al., 2002). Regarding etiological validity, emotional stressors are commonly
accepted as contributors to depressive disorders (for review, see Anisman and Matheson, 2005) and the social





defeat model does induce a physiological stress response (elevated corticosterone) in rats (Sgoifo et al.,

1996). Neurochemical changes associated with the social defeat - forced swim protocol need investigation

to determine this model's construct validity.



The results obtained in this study are similar to those obtained by Rygula et al. (2005), in that the

repeatedly stressed rats demonstrated greater floating in the forced swim test. However, the procedure followed

by Rygula et al. consisted of a five-week schedule of social defeat stress, with hour-long defeat sessions.

The present study demonstrated that significant behavioral despair is evoked with only five days of social

defeat stress, with 10-minute-long defeat sessions. However, the more severe schedule induced greater

behavioral despair in the stressed rats compared to controls; therefore, a schedule of intermediate intensity may

be preferable for future use of the social defeat-forced swim model.



Although other researchers have used a 5-minute swim test session (Porsolt et al., 1978; Gavioli, et al.,

2003; Rygula et al., 2005; Hinojosa et al., 2006), the 15-minute swim test of the present study divided into three

5-minute bins revealed subtle differences between repeatedly stressed, acutely stressed, and no-stress control

rats. The acutely stressed rats had significantly increased floating in the middle 5-10 minute bin while the

floating was significantly elevated in all bins for the repeatedly stressed rats. If similar subtleties in

behavioral variations are to be studied, perhaps a 10- or 15-minute swim test session is preferable.



Future Directions


As this animal model successfully demonstrates a parallel of some symptoms of major depression, future

studies using an identical procedure could be used to investigate the neurobiology and endocrinology of

emotional stress and behavioral despair. There is a possibility that cortical and limbic plasticity may underlie

such stress-induced psychopathology. Furthermore, this combination of animal models could be used to screen

drugs for anti-stress or anti-depressant effects.



A similar study should be repeated with the addition of surveillance of intruder rats' locomotor behavior in an

open arena and a test of intruder rats' preference for sucrose solution compared with water. Rygula et al.

(2005) determined that intruder rats had significantly decreased locomotor activity in an open arena and

significantly decreased preference for sucrose solution during weeks three through five of their experiment,

compared with control rats. The present study's abbreviated schedule should be utilized and locomotor behavior

and sucrose preferences measured for intruder rats to determine if this shorter schedule can lead to such signs

of anhedonia.



Future research may include development of a similar female rat model of emotional stress that may be used

with the Porsolt swim test. Development of an appropriate female model of emotional stress is critical

because women are more likely than men to suffer from major depression (Gorman, 2006). The social

defeat procedure may not be suitable for a female rat model of social stress because male resident rats may





not commence a social defeat interaction with female intruders (personal observation), female rats may not mount

a stress reaction to social defeat (Haller et al., 1999), and social hierarchies are not as apparent in female

rats (Tamashiro et al., 2005). Some evidence suggests that social instability (crowding vs. isolation) may serve

as an emotional stressor for female rats (Haller et al., 1999).



CONCLUSIONS


This social defeat - forced swim protocol demonstrates that repeated emotional stress is likely to induce

behavioral despair in rats. In parallel, humans that repeatedly encounter emotional stressors may develop

major depressive disorder. The significant behavioral changes noted during the forced swim test for the stressed

rats potentially resemble symptoms of major depression (e.g. behavioral depression). Future use of this

animal model should contribute to research endeavors to alleviate major depression in the human population.






ACKNOWLEDGEMENTS


I would like to especially thank my research mentor Dr. Darragh Devine for his superb guidance and Kristen Stone

for her excellent work during the social defeat tests and assistance during the Porsolt swim tests. I would also like

to thank the other members of the Devine lab for their support during my time of undergraduate research.






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