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Published online before print February 8, 2007, 10.1097/PSY.0b013e31802f054b
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Psychosomatic Medicine 69:149-157 (2007)
© 2007 American Psychosomatic Society

ORIGINAL ARTICLES

Depression-Like Behavior and Stressor-Induced Neuroendocrine Activation in Female Prairie Voles Exposed to Chronic Social Isolation

Angela J. Grippo, PhD, Bruce S. Cushing, PhD and C. Sue Carter, PhD

From the Department of Psychiatry and Brain-Body Center, University of Illinois at Chicago, Chicago, IL.

Address correspondence and reprint requests to Angela J. Grippo, PhD, Department of Psychiatry, University of Illinois at Chicago, 1601 W. Taylor St. (MC 912), Chicago, IL 60612. E-mail: agrippo{at}psych.uic.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 NOTES
 REFERENCES
 
Objective: To assess whether the responses of prairie voles to social stressors play a mechanistic role in the behavioral and physiological changes associated with affective disorders such as depression, as suggested in previous studies. Prairie voles (Microtus ochrogaster) are socially monogamous rodents that share features of social behavior with humans; therefore, they may serve as useful models for examining social behavioral regulations and physiological responses related to depression. In this study, we hypothesized that social isolation in female prairie voles would induce depression-relevant behaviors and alter their neuroendocrine responses to an acute social stressor.

Methods: Twenty adult female prairie voles were exposed to either 60 days of social isolation or paired (control) housing. They were tested and observed for a depression-like behavior (anhedonia). The levels of corticotropin-releasing factor- and oxytocin-immunoreactive cells in the paraventricular nucleus of the hypothalamus and circulating levels of hormones and peptide were measured in response to an acute social stressor (resident-intruder test).

Results: Chronic social isolation produced anhedonia, measured by reduced sucrose intake and sucrose preference relative to the control animals. Compared with the paired animals, the isolated prairie voles displayed increased plasma hormone and peptide levels (oxytocin, arginine vasopressin, and corticosterone) after a 5-minute resident-intruder test, mirrored by an increased number of oxytocin- and corticotropin-releasing factor-immunoreactive cells in the hypothalamic paraventricular nucleus.

Conclusions: These findings suggest that isolation in a socially monogamous rodent model induces both behavioral and neuroendocrine changes that are relevant to depression. These results may provide insight into the mechanisms that underlie the development and/or maintenance of depressive disorders in humans.

Key Words: affective disorders • corticotropin-releasing factor • hypothalamic-pituitary-adrenal axis • oxytocin • paraventricular nucleus • stress

Abbreviations: ACTH = Adrenocorticotropic hormone; ANOVA = analysis of variance; AVP = arginine vasopressin; CRF = corticotropin releasing factor; DAB = diaminobenzadine; HPA = hypothalamic-pituitary-adrenal; KPBS = potassium phosphate buffered saline; PVN = paraventricular nucleus; SEM = standard error of the mean.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 NOTES
 REFERENCES
 
Evidence from animal research, including humans, documents an important overlap of affective disorders and physiological dysfunction (1–6). Disorders of negative effect, such as depression, are associated with several neuroendocrine and autonomic alterations including dysfunction of the hypothalamic-pituitary-adrenal (HPA) axis and activation of the sympathetic nervous system. For instance, alterations in corticotropin-releasing factor (CRF) are found in the cerebrospinal fluid (7) and hypothalamus (2) of depressed patients, and central CRF administration induces several depression-like effects in rodents and primates, including decreased food intake and sexual activity, disturbed sleep, altered motor behavior, and impaired learning (8). Also, cortisol may be hypersecreted in many depressed patients (9). Chronic activation of the HPA axis leads to activation of the sympathetic nervous system. To this end, our previous laboratory research demonstrated increased sympathetic tone to the heart in a rodent model of depression (5,10). These physiological changes associated with affective disorders are not unlike those accompanying exposure to stressors.

Exposure to and increased reactivity to environmental stressors are widely recognized to be associated with affective disorders (11–14). Furthermore, there is a growing body of literature that discusses the importance of the social environment in mediating behavioral and neuroendocrine dysfunction associated with affective disorders, as well as the role of positive social interactions in buffering against detrimental or prolonged behavioral and physiological responses to stressors (15–19). For instance, a recent study involving middle-aged individuals found that perceived loneliness (including social isolation) is directly related to symptoms of depression and cardiovascular responses to a mental stressor (15). Also, a combination of oxytocin treatment and social support reduces cortisol responses and subjective anxiety reactions after a social stressor in men (17). Early life trauma, such as abuse, is associated with increased adrenocorticotropic hormone (ACTH) and cortisol responses in adult female patients with depression (20). Similarly, rats subjected to maternal separation as pups exhibit activation of the HPA axis (21) and exaggerated stressor-induced corticosterone responses in adulthood (22) versus the control groups. These previous findings suggested that behavioral and physiological responsiveness to acute stressors might be an important mechanism underlying the signs and symptoms of depression.

To understand the mechanisms of the behavioral and physiological characteristics related to affective disorders and the underlying neurobiological processes, it is useful to conduct integrative research involving animal models. The prairie vole (Microtus ochrogaster), a unique rodent species, provides a valuable model for studying the mechanisms of psychiatric conditions and the role of social experiences in regulating behavior and physiology (23). This species exhibits traits of social monogamy that are similar to humans and other primates, including an active engagement in and reliance on their social environment, the formation of adult pair bonds, biparental care, and family group living styles (19,24).

The present study used the prairie vole model to examine potential mechanisms that underlie behavioral and physiological responses related to depression. Our laboratory previously demonstrated depression-like behaviors and neuroendocrine disturbances in a rodent model of depression that involved exposure to environmental and social stressors (5,6,10). Furthermore, previous evidence suggested that behavioral and physiological reactivity to stressors might play a role in mediating signs and symptoms of depression (15,17,22). Therefore, in the current study, we investigated specifically behavioral and neuroendocrine responses to chronic social isolation in adult female prairie voles, with a focus on depression-like behaviors and acute stressor responsiveness. We hypothesized that prairie voles exposed to chronic social isolation would display anhedonia, a common behavioral sign of depression characterized by the reduced responsiveness to a pleasurable stimulus. We predicted that isolated prairie voles would display exaggerated neuroendocrine activation after an acute social stressor (resident-intruder test), including increased HPA axis and oxytocin levels, and that these peripheral changes would be mirrored by increased stressor-associated hormone and peptide levels in the hypothalamic paraventricular nucleus (PVN).


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 NOTES
 REFERENCES
 
Animals
Twenty adult, reproductively naïve female prairie voles (35–45 g) were used for the experimental procedures. Animals were descendants of a wild stock originally caught near Champaign, Illinois. Animals were maintained on a 14/10 hours light/dark cycle (lights on at 6 AM), with a temperature of 25 ± 1°C, and relative humidity of 21 ± 4 g/m3. All animals were allowed food (Purina rabbit chow) and water ad libitum, unless otherwise specified. Offspring were housed with breeding pairs in large polycarbonate cages (25 x 45 x 60 cm) with cotton nesting material until 21 days of age, at which time they were removed and housed in same-sex sibling pairs in smaller cages (12 x 18 x 28 cm). All procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee, University of Illinois at Chicago.

Female prairie voles were used as our model system for several reasons. First, depression is more common in women than in men (25), yet female rodents are an understudied group in both behavioral and physiological investigations relating to depression (26,27). Second, female prairie voles have been studied extensively for their social behavior in our laboratory and they may be especially sensitive to the effects of social stressors (28–30). Third, female prairie voles do not show a spontaneous puberty or estrous cycle; in this species, the ovaries remain inactive until the female has physical contact with a male (31), allowing for the use of reproductively intact animals without the need for controlling the estrous cycle.

Social Isolation
Animals were divided randomly into paired (control; n =10) or isolated (n = 10) conditions. Animals were subjected to social isolation after living with a female sibling in a standard-sized cage since weaning; the isolation period began when the animals were between 60 and 120 days of age (modal age = 90 days) and lasted for 60 days. Isolation involved removing the experimental animal from the home cage and placing it in an isolated cage identical in size to the home cage. The paired (control) animals were also moved into new cages at the same time as the isolated animals, and then they were continually housed with the siblings for the length of the respective isolation period. Handling and cage changing throughout the isolation period were matched between both groups.

Fluid Intake
After 50 days of social isolation, an acute fluid intake test was conducted during the light period (approximately 4 hours after the light onset) to operationally define anhedonia, using a modification of procedures described elsewhere (5). Anhedonia was operationally defined as reduced absolute sucrose intake and sucrose preference, relative to control values. To allow for adaptation to the taste of the sucrose, all animals were allowed ad libitum access to 1% sucrose, along with food and water, for 1 week before beginning any experimental procedures. Food and water were removed from the cage for 20 hours before the sucrose preference test. One hour before beginning the test, all animals were moved into clean, individual cages to ensure accurate fluid intake measurements of the control animals. Both groups (paired and isolated animals) were moved into clean cages, thus avoiding potentially differential responses to a novel environment. Tap water and 1% sucrose were placed in premeasured bottles in the cages, and fluid intake was monitored for 1 hour. All animals were returned to the home cages immediately after the test.

Resident-Intruder Test
After 60 days of isolation, all animals participated in a resident-intruder test during the light period (approximately 3–5 hours after light onset), consisting of placing the paired or isolated animal (intruder) into the cage of an unrelated, unfamiliar female (resident) for 5 minutes. Residents and intruders had no prior contact before the test and did not share parentage. The resident-intruder paradigm was used here because it had previously been demonstrated to be a stressor in female rodents (32,33). To ensure the acute nature of the stressor in prairie voles, the time period here was shorter than the previous 10-minute periods reported for rats and mice (34,35). An experimentally blind observer scored the intruder's aggressive behavior during the 5-minute paradigm. Aggressive behavior was defined as aggressive grooming or posture, swatting, biting, thrusting, pulling, and/or attack behavior directed toward the other animal (34). An overall score of aggressive behavior was determined for each animal by summing the number of episodes of each behavior. The results are reported as mean number of aggressive episodes and the percentage of animals displaying aggressive behaviors during the 5-minute test.

Immediately after completion of the test, all animals in both groups were placed into individual cages to facilitate poststressor observations and avoid possible effects of reuniting the paired animals with the siblings. At 5 minutes after the test, an experimentally blind observer monitored the animal's behavior for a total of 1 minute (immediately before sacrificing). Signs of behavioral agitation were recorded and defined as running around the perimeter of the cage, jumping, repeated self-grooming, and/or performing repeated, stereotypy-like behaviors. An overall score of behavioral agitation was determined for each animal by summing the number of episodes of each behavior. The results are reported as mean number of agitated behaviors and the percentage of animals displaying agitated behaviors during the 1-minute observation period after the resident-intruder test.

Plasma Collection
Ten minutes after the completion of the resident-intruder test (4 minutes after the 1-minute poststressor observation period), the animals were anesthetized with a mixture of ketamine (67 mg/kg, sc; NLS Animal Health, Owings Mills, MD) and xylazine (13.33 mg/kg, sc; NLS Animal Health, Owings Mills, MD). To avoid potential anesthesia-induced hormone alterations, blood was sampled within 2 minutes of the anesthetic injection, from the periorbital sinus via a heparanized capillary tube, and was collected during a period not exceeding 1 minute. The blood sample was placed immediately on ice, and then centrifuged at 4°C, 3500 rpm, for 15 minutes to obtain plasma. Plasma aliquots were stored at –80°C until assayed for circulating hormones and peptides.

Circulating Hormone and Peptide Analysis
Plasma levels of oxytocin and arginine vasopressin (AVP) were determined using commercially available enzyme-linked immunosorbent assay kits (Assay Designs, Ann Arbor, MI), which have been validated previously by our laboratory for use in prairie voles (36). Inter- and intra-assay coefficients of variation for oxytocin are 19.2% and 2.9%, respectively, and for AVP are 5.7% and 2.5%, respectively. The minimum detection limits for the assays are 4.68 pg/ml for oxytocin and 3.39 pg/ml for AVP. The antibodies have negligible cross-reactivity (<0.001%) with similar mammalian peptides.

Plasma levels of ACTH were determined by radioimmunoassay, according to procedures described elsewhere (37). The inter- and intra-assay coefficients of variation are 14.6% and 4.2%, respectively. The sensitivity of this assay is 0.25 pg/tube.

Plasma levels of corticosterone were determined using a commercially available radioimmunoassay kit (MP Biomedicals, Irvine, CA). The plasma sample was diluted in assay buffer as necessary (1:2000) to give results reliably within the linear portion of the standard curve. The inter- and intra-assay coefficients of variation for corticosterone are <5%, and cross-reactivity with other steroids is <1%. The minimum detectable dose for this assay is 7.7 ng/ml.

Tissue Collection
Immediately after blood collection, the anesthetized animals were sacrificed via cervical dislocation. Brains were carefully removed from the skulls and were processed with a spin immersion technique (38). Brains were immersed in a fixative solution consisting of 4% paraformaldehyde containing 5% acrolein (pH 8.6) for a total of 4 hours. Brains were first spun for 10 minutes in the fixative solution, and then removed and blocked, exposing the lateral ventricles. After blocking, brains were replaced in the fixative solution and spun gently for 1 hour 50 minutes, at which time the fixative solution was replaced with fresh solution, and brains were gently spun for an additional 2 hours. Brains were postfixed for 24 hours in 4% paraformaldehyde, and sunk in 25% sucrose. Tissue was stored in 25% sucrose at 4°C until it was sectioned at 40 µm on a freezing sliding microtome. Sliced serial sections were stored in wells in cryoprotectant antifreeze solution at –20°C until assayed for CRF and oxytocin, using standard avidin:biotinylated enzyme complex immunocytochemistry.

Immunocytochemistry
Free-floating sections were rinsed six times during a 1-hour period with potassium phosphate buffered saline (KPBS) to remove the cryoprotectant. Sections were then incubated in 1% sodium borohydride for 20 minutes at room temperature. After multiple washes in KPBS, sections were incubated in 0.014% phenylhydrazine for 15 minutes at room temperature. Tissue was rinsed six times during 1 hour in KPBS. Sections were then incubated in primary antibody for either CRF or oxytocin (anti-CRF, 1:50,000, provided by Dr. Ann-Judith Silverman; anti-oxytocin, 1:200,000, provided by Dr. Mariana Morris; both antibodies were generated in rabbit) diluted in KPBS + 0.4% Triton X-100 for 1 hour at room temperature, and then incubated for 42 hours at 4°C. After this incubation, sections were rinsed 10 times with KPBS during a 1-hour period. Sections were then incubated in antirabbit IgG (BA-1000; Vector Laboratories, Burlingame, CA; 1:600) for 1 hour at room temperature. Sections were rinsed five times with KPBS during a 50-minute period and then incubated in A/B solution (Vectastain Elite PK-6100; Vector Laboratories, Burlingame, CA; 45 µl A, 45 µl B per 10 ml KPBS + 0.4% Triton X-100) for 1 hour in room temperature. Sections were rinsed three times in KPBS and then three times in either 0.175 M sodium acetate (for CRF) or Tris buffered saline (for oxytocin). CRF was visualized by incubation in nickel-diaminobenzadine (DAB) solution, dissolved in 0.175 M sodium acetate, for 15 minutes at room temperature, and then sections were rinsed three times with 0.175 M sodium acetate and three times with KPBS. Oxytocin was visualized by incubation in DAB dissolved in Tris buffered saline, for 15 minutes at room temperature, and then sections were rinsed three times with Tris buffered saline and three times with KPBS.

Stained sections were mounted on gelatin-coated slides, air-dried, dehydrated in a series of ethanol dilutions, cleared with Histoclear (National Diagnostics, Atlanta, GA), and then coverslipped using Histomount mounting medium (National Diagnostics, Atlanta, GA).

Image Analysis
Brain sections were matched across subjects, captured using a Nikon Eclipse E 800 microscope, Sensi-cam camera, and IP Lab Software (Scanalytics, Inc., Fairfax, VA), and scored by a trained, experimentally blind observer. The numbers of oxytocin- and CRF-immunoreactive cell bodies were manually counted bilaterally in the PVN and averaged. Only cell bodies were counted; stained fibers were excluded from the analysis.

Data Analysis
All data are presented as mean ± (or +) standard error of the mean (SEM). All data were analyzed using a single factor analysis of variance (ANOVA) and a priori Student's t tests. The parameters recorded during and after the resident-intruder test also were analyzed with tests to determine a significant difference between two proportions (z test). A Bonferroni correction was used for any multiple comparisons, and p < .05 was considered to be statistically significant.

Sample sizes of the analyses varied slightly because several animals were excluded from specified analyses based on a priori criteria. One animal from the isolated group was excluded from the resident-intruder test and all subsequent analyses due to an apparent illness before the test (this animal was painlessly euthanized according to Animal Care and Use Committee guidelines). One animal from the isolated group was excluded from the plasma and tissue analyses due to handling problems before the anesthesia was administered. If a blood sample was not collected within 3 minutes after the anesthesia was injected, plasma hormones and peptides were not analyzed to avoid potential handling- and/or anesthetic-induced stressor responses (two paired animals were excluded for this reason). Finally, if there was an insufficient number of intact brain slices from the PVN after the immunohistochemical assays, these data were excluded (one paired and two isolated animals were excluded for this reason).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 NOTES
 REFERENCES
 
Fluid Intake
Figure 1 A displays fluid intake in the paired and isolated groups after the isolation period. Single factor ANOVAs were performed on water and sucrose intake separately. The ANOVA performed on water intake was not significant (p > .05), indicating that there was no difference in water intake between the paired and isolated groups; no follow-up tests were performed. The ANOVA performed on sucrose intake yielded a significant main effect (F(1,18) = 27.75, p < .05). The isolated group drank significantly less sucrose than did the paired group after isolation (t(18) = 5.27, p <. 05).


Figure 16
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  		Figure 1. Mean (±SEM) absolute water and sucrose intake (A) and mean sucrose preference (B) in paired and isolated prairie voles after 50 days of social isolation. *p < .05 versus respective paired value.

 

Social isolation also led to reduced sucrose preference, relative to social pairing. Figure 1B shows the sucrose preference, relative to total fluid intake, in paired and isolated groups after the isolation period. The ANOVA yielded a significant main effect (F(1,18) = 12.04, p < .05). The preference for sucrose was significantly lower in the isolated group versus the paired group (t(18) = 3.45, p < .05).

Resident-Intruder Test
During the resident-intruder test (5-minute test period), there was no significant difference in the number of aggressive behaviors exhibited between the paired and isolated intruders (Figure 2A; p > .05). Furthermore, the percentage of animals in each group displaying aggressive behaviors during the test was not significantly different (60% and 67% in the paired and isolated groups, respectively; p > .05).


Figure 26
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  		Figure 2. (±SEM) number of aggressive episodes during the resident-intruder test (A) and number of agitated behaviors after the resident-intruder test (B) in paired and isolated groups. Note the scale differences in A and B. *p < .05 versus paired value.

 

After the resident-intruder test (1-minute poststressor observation period), the isolated group displayed significantly greater numbers of agitated behaviors versus the paired group (Figure 2B; t(17) = 6.12; p < .05). Also, the isolated animals were significantly more likely to display agitated behaviors compared with the paired animals (22% and 89% in paired and isolated groups, respectively; z = 3.00, p < .05).

Circulating Hormone and Peptide Levels
Figure 3 shows the circulating levels of oxytocin (panel A), AVP (panel B), ACTH (panel C), and corticosterone (panel D) in the paired and isolated animals 10 minutes after the end of the resident-intruder paradigm. Compared with the paired animals, the socially isolated animals displayed significantly increased oxytocin (t(14) = 2.01, p < .05), AVP (t(8) = 2.18, p < .05; these data were compared with a t test assuming unequal variances), and corticosterone (t(14) = 2.00, p < .05). There were no significant differences in ACTH levels after the resident-intruder test (p > .05; these data were compared with a t test assuming unequal variances).


Figure 36
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  		Figure 3. Mean (±SEM) circulating levels of oxytocin (A), arginine vasopressin (B), ACTH (C), and corticosterone (D) in paired and isolated prairie voles at 10 minutes after a 5-minute resident-intruder test. Note the scale differences among the four panels. *p < .05 versus paired value.

 

Tissue Hormone and Peptide Levels
Figure 4 shows the oxytocin- (panels A and B) and CRF-immunoreactivity (panels C and D) distribution in the PVN of the hypothalamus in a representative paired and isolated prairie vole, and Figure 5 displays the mean number of oxytocin- (panel A) and CRF-immunoreactive (panel B) cells in the PVN. The isolated group displayed significantly greater numbers of oxytocin-immunoreactive cells (t(13) = 1.78, p < .05) and CRF-immunoreactive cells (t(13) = 2.55, p < .05) in the PVN versus the paired group.


Figure 46
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  		Figure 4. Brain sections (40 µm) showing oxytocin- (A and B) and CRF-immunoreactivity (C and D) in representative paired (A and C) and isolated (B and D) prairie voles in the hypothalamic PVN. Pictures are shown at 100x magnification. Scale bars = 100 µm. Stained fibers were excluded from analysis.

 

Figure 56
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  		Figure 5. Mean (±SEM) number of oxytocin- (A) and CRF-immunoreactive (B) cells in the hypothalamic PVN in paired and isolated prairie voles. Note the scale differences in A and B. *p < .05 versus paired value. Stained fibers were excluded from analysis.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
NOTES
 REFERENCES
 
The present study investigated the role of the social environment in mediating behavioral and physiological processes relating to depression. The results suggest that social isolation induces anhedonia and increased levels of stress-related hormones and peptides in the PVN. Furthermore, social isolation is associated with increased behavioral and neuroendocrine reactivity to an acute social stressor (resident-intruder paradigm). These results extend previous findings from our laboratory showing that chronic exposure to environmental and social stressors in rats induces depression-relevant behaviors and neuroendocrine dysfunction (5,6,10) and provide further insight into mechanisms that may underlie the development of behavioral and physiological signs of affective disorders.

Socially isolated prairie voles, in contrast to paired prairie voles, displayed reduced sucrose intake and sucrose preference indicative of anhedonia. Anhedonia is a characteristic sign of depression and may be present in approximately 95% of depressed patients (39). The sucrose deficit in isolated prairie voles represents a specific hedonic deficit rather than a generalized attenuation of fluid ingestion because water intake was unaffected by the isolation procedure. Also, the reduced preference for sucrose in the isolated group was attributed entirely to a reduced sucrose consumption, rather than an increased water consumption. These data are consistent with previous studies, from our laboratory and others, that examined hedonic behaviors in animal models of depression (5,10,27,40,41). To our knowledge, this is the first demonstration of a depression-like behavior after chronic social isolation in prairie voles using a validated, observable behavioral index. A shorter isolation period may be sufficient to induce anhedonia in prairie voles. Previous research suggested that 28 days of chronic mild stress induced anhedonia in rats (5), and 3 days of isolation altered forced swim test behavior in male prairie voles separated from a pair-bonded female (41).

Exposure to an acute social stressor increased circulating oxytocin, AVP, and corticosterone in socially isolated prairie voles, indicative of stressor-induced neuroendocrine activation. However, ACTH was not altered, which may be attributable to the high variability of responses in the isolated group (Figure 3C). Consistent with these results is the observation that isolated prairie voles versus paired animals displayed a greater number of agitated behaviors after the acute stressor. Paired and isolated animals displayed the same number of aggressive behaviors during the resident-intruder test, indicating that the generalized activity level and aggression did not lead to increased agitation and neuroendocrine responses after this stressor. It is possible that the threatening situation (i.e., being the intruder) led to the behavioral and physiological responses to this stressor in the isolated animals. The findings described here are in agreement with previous results from our laboratory that showed increased corticosterone levels in the chronic mild stress model of depression (6), as well as previous reports of peripheral oxytocin release in response to stressful stimuli in male and female rodents (42–44). Additionally, these changes mirror those observed in patients with affective disorders and in individuals who have experienced social stressors (7,17,20,45). However, AVP responses to stressors have been inconsistent in rodents (42,43,46) and may merit additional experimentation.

The increased circulating oxytocin levels observed in this study may be indicative of a compensatory response whereby this peptide is secreted to counteract the excess corticosterone and AVP. It is possible that corticosterone and AVP levels, although initially increased, would be reduced at a later time point after the resident-intruder stressor, suggesting that oxytocin levels are increased in a compensatory manner that leads to a subsequent reduction in the HPA axis response. Oxytocin can suppress the activity of the HPA axis in the natural and experimental settings in humans (47,48). Recent studies showed that intracerebroventricular oxytocin treatment attenuated the central and peripheral responses to restraint stress in rats (49). Neumann and colleagues (50,51) reviewed the role of oxytocin in mediating the physiological stress response, suggesting that its involvement is both brain region- and stressor-specific.

Social isolation in prairie voles is associated with increased circulating stressor-reactive hormones and peptides in the presence of an acute stressor, mirrored by increased oxytocin- and CRF-immunoreactivity in the PVN. These findings are in agreement with findings of Bosch and associates (32), who demonstrated increased oxytocin release in the PVN in female rats (lactating residents and virgin intruders) during a 10-minute resident-intruder test. Increases in oxytocin and CRF levels in the hypothalamus may represent long-term responses to social isolation and may be linked to the release of peripheral oxytocin and/or activation of the HPA axis in response to an acute stressor. Acute stressors in both male and female rats have been shown to induce the release of oxytocin in the amygdala (52) and hypothalamus (32,43,53,54), measured via microdialysis and in situ hybridization. Therefore, it will be important to investigate whether an acute social stressor can lead to differential changes in oxytocin, CRF, and AVP release in the central nervous system in isolated versus paired prairie voles. Furthermore, in previous studies, social isolation has been shown to increase circulating corticosterone and tissue CRF levels (but not circulating or tissue oxytocin levels) of juvenile prairie voles (23; M. Ruscio, unpublished observations) and reduce neurogenesis in the hypothalamus of adult prairie voles (55). Considering these findings, it may be possible for chronic social isolation alone to induce basal changes in some circulating or central factors in prairie voles. However, the increased levels of CRF and oxytocin cells observed in the PVN may shed some light on this issue, as the time course of the acute stressor employed here (5-minute stressor with tissue collection 10 minutes after the stressor) may not be sufficient to produce changes in the number of oxytocin- and CRF-producing cells in the central nervous system. Future research should investigate basal changes in stressor-reactive hormones and peptides after isolation.

Specific limitations of this research may have had an impact on the present findings. Some results could have been influenced by differential age-related responses. However, although not all animals were the same age in the current study (modal age = 90 days), the age range shown here has been used in several studies examining the behavior, stress responses, and physiological function in prairie voles and produced reliable differences (30,56,57); therefore, we feel that using this age range was acceptable. A second limitation in studies of social interactions is that the experimental design may add uncontrolled variables associated with temporary isolation experienced by the paired animals. However, if this was the case, differences between paired and isolated groups may have been less pronounced or absent. The present results suggest several robust differences in both behavior and physiology between paired and isolated animals. A third limitation involves the difficulty in studying female rodents as a model for understanding processes that mediate behavior and physiology in women. The lack of spontaneous puberty and estrous cycle in female prairie voles (31) allows for conducting experiments without controlling for female-specific hormonal influences on the dependent variables in question, but it also limits the translation of results to human conditions during which the menstrual cycle cannot be controlled (i.e., naturalistic settings in women). Future research might investigate the effects of social stressors in reproductively primed prairie voles (or other rodents that show a spontaneous estrous cycle, such as rats or mice). To this end, a previous study from our laboratory suggested that exposure to chronic environmental and social stressors induced anhedonia and a disruption of the estrous cycle in adult female rats (27).

The current study demonstrates that neuroendocrine dysfunction and behavioral alterations can result from exposure to a combination of chronic and acute social stressors. These experiments, which include measurements of behavioral and neuroendocrine responses in the same animal, are relevant to understanding human mood disorders. Prairie voles may be especially valuable for understanding the mechanisms underlying depression because this species demonstrates social behaviors, such as social bonds, and physiological parameters, including high levels of parasympathetic nervous system activity, that mirror those of humans (19,23,24,30). The prairie vole also may be a useful model for preclinical investigation of novel treatments for affective disorders, such as pharmacological treatments that focus on CRF, AVP, oxytocin, or glucocorticoid systems. An increased understanding of the behavioral and neurobiological processes that underlie affective disorders can lead to the development of comprehensive treatments that target the mechanisms, rather than the symptoms, of these important mental disorders.

We would like to thank Drs. Mariana Morris and Ann-Judith Silverman for generously donating the antibodies used in these experiments. We are grateful to Francisca Garcia for help with the ACTH assay, and Davida Gerena, Narmda Kumar, Lisa Sanzenbacher, and Raj Ughreja for technical assistance.


   NOTES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 NOTES
REFERENCES
 
Received for publication April 27, 2006; revision received July 28, 2006.

This research was funded by the National Institute of Mental Health Grant MH 73233 (A.J.G.) and Grant MH 01992 (B.S.C.), and the National Institute of Child Health and Human Development Grant HD 48390 (C.S.C.).

DOI:10.1097/PSY.0b013e31802f054b


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 NOTES
 REFERENCES
 

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