Morphological changes in the basolateral amygdala and behavioral disruptions associated with social isolation

Abstract

Social isolation and the disruption of established social bonds contribute to several physical and psychological health issues. Animal models are a useful tool for investigating consequences of social stress, including social isolation. The current study examined morphological changes in the basolateral amygdala (BLA) and affect-related behavioral and endocrine changes, due to prolonged social isolation using the translational prairie vole model (Microtus ochrogaster). Adult male prairie voles were either socially paired (control) or isolated from a same-sex sibling for 4 weeks. Following this 4-week period, a subset of animals (n = 6 per condition) underwent a series of behavioral tasks to assess affective, social, and stress-coping behaviors. Plasma was collected following the last behavioral task for stressor-induced endocrine assays. Brains were collected from a separate subset of animals (n = 10 per condition) following the 4-week social housing period for dendritic structure analyses in the BLA. Social isolation was associated with depressive- and anxiety-like behaviors, as well as elevated oxytocin reactivity following a social stressor. Social isolation was also associated with altered amount of dendritic material in the BLA, with an increase in spine density. These results provide further evidence that social isolation may lead to the development of affective disorders. Dysfunction in the oxytocin system and BLA remodeling may mediate these behavioral changes. Further research will promote an understanding of the connections between oxytocin function and structural changes in the BLA in the context of social stress. This research can facilitate novel treatments for alleviating or preventing behavioral and physiological consequences of social stressors in humans.

1. INTRODUCTION

In a modern industrialized society where the advancement of technology can connect people from opposite sides of the globe with only a few clicks, a surprising number of people report objective social isolation, feelings of loneliness, or disconnection from others [1,2]. Further, perceptions of loneliness and objective social isolation have become front and center during the global COVID-19 pandemic [3–5]. Feelings of loneliness are associated with affective symptoms such as depression and anxiety [6,7], as well as health issues including heart disease, metabolic syndrome, and immune disorders [8–10]. Similar to loneliness, objective social isolation contributes to psychological and physiological disturbances and reduced mortality [11]. For example, relocation due to work or education oftentimes requires physical seperation between loved ones (e.g., military deployment) or between children and parents (e.g., first year university students living on their own); these physical seperations from familiar social partners and environments are associated with increased mental and physical health issues [12,13].

In searching for an animal model to investigate the neurobiological and behavioral effects of chronic social stressors, the prairie vole (Microtus ochrogaster) offers a unique perspective. The prairie vole is a rodent species that exhibits a similar social structure to humans but unlike other laboratory rodents, including forming long-term pair-bonds with partners, displaying biparental care of offspring, and living in large family units in the wild [14,15]. Exposure to social stressors in this species – including both long-term social isolation from family members and the disruption of established pair-bonds between males and females – elicits several behavioral and physiological disruptions similar to humans. For example, prairie voles that are socially isolated from a same-sex sibling for at least 4 weeks or that are removed from a pair-bonded partner of the opposite sex for at least 5 days display maladaptive behaviors such as poor stress coping strategies, anhedonic, and anxiogenic behaviors [16–22]. Neuroendocrine changes accompany these behaviors, including hyperactive and reactive hypothalamic-pituitary-adrenal (HPA) processes and altered oxytocin (OT) function [16,17,20,23]. These findings demonstrate that the prairie vole is an ideal translational animal model to investigate behavioral and neuroendocrine changes as a function of social isolation and the disruption of established social bonds.

Previous research from laboratory rodents, including prairie voles, indicates that prolonged social isolation is associated with morphological and functional changes in the HPA axis [16–18,20]. The HPA axis works intimately with other systems and brain regions to maintain biological homeostasis [24]. Therefore, an activation of these HPA-related regions can alter HPA function [25,26], and dysfunction in these regions can have downstream effects on the HPA axis [27]. For example, the neuropeptides OT and arginine vasopressin (AVP) interact with HPA-related processes. OT and AVP are both synthesized primarily by the magnocellular neurons of the hypothalamic paraventricular nucleus (PVN), which is an important region for HPA signaling [28]. Further, central and peripheral release of OT and AVP mediate stress responses by interacting directly and indirectly with the HPA axis [29]. Prairie voles that were allowed to recover from a restraint stressor with a pair-bonded partner displayed elevated OT levels in the PVN, with attenuated plasma corticosterone levels, compared to animals that recovered alone [30]. Moreover, this HPA-buffering effect by the pair-bonded partner was rendered ineffective following administration of OT receptor antagonist, suggesting that partner-mediated release of central OT had a downregulating effect on the HPA axis [30]. Lastly, central release of OT and AVP mediate prosocial and reproductive behaviors in socially monogamous rodent species [28,31], and separation from a pair-bonded partner or a same-sex sibling can result in altered OT, AVP, and HPA function [16,32–34]. For example, male prairie voles that were socially isolated from a pair-bonded partner for 4 weeks displayed elevated levels of OT-, AVP-, and corticotropin-releasing hormone-immunoreactivity in the PVN [23]. These previous data indicate that social isolation and social buffering can influence many neuroendocrine systems. The interactions among these systems are complex, and long-term health effects are not fully understood.

In addition to these peptide systems, the amygdala—which mediates emotional processing, memory consolidation, and fear conditioning [35]—plays an important role within the HPA-related network. The amygdala can disinhibit the PVN indirectly from the controls of the bed nucleus of stria terminalis and from the nucleus of the solitary tract in the brain stem through GABA-ergic projections [26,27]. Additionally, the amygdala is rich in OT receptors [36,37], and binding of OT in this region may mediate fear conditioning and fear-related behavior [38,39]. Furthermore, the amygdala also integrates cognitive, emotional, and autonomic information from the prefrontal cortex, hippocampus, and brainstem [40]. Therefore, dysfunction of the amygdala can result in alterations of emotional processing and neuroendocrine signaling, which may produce long-term health effects [41,42].

Chronic stress influences the structure and function of the amygdala. In particular, the basolateral region of the amygdala (BLA) has received attention in recent years due to its association with social stressors and environmental cues [43,44]. For example, increased dendritic arborization and spine density in the BLA are observed in mice exposed to repeated social defeat stress [45,46]. Interestingly, repeated corticosterone administration does not alter BLA spine density or morphology in male rats [47,48], but a single dose of corticosterone appears to induce dendritic hypertrophy in the BLA, suggesting a fast neuronal reorganization and adaptation in this region [47]. There are limited data on BLA arborization and morphology in socially isolated rodents, with existing findings showing conflicting results. For example, post-weaning isolation in male rats was associated with increased dendritic arborization in the BLA, with shorter dendritic lengths [49]. However, adolescent social isolation did not augment dendritic branching or morphology in the BLA of mice [50]. Furthermore, prolonged social isolation leads to abnormal neuronal firing in the amygdala of male rats [51], and is associated with decreased cell proliferation, survival, differentiation, and increased FosB/ΔFosB-immunoreactivity in the BLA of female prairie voles [18,52]. These changes in the BLA may contribute to hyperactivated HPA function as well as depressive- and anxiety-related behaviors often observed in lonely individuals and socially isolated animals [25,43].

Given the importance of the social environment in mediating several aspects of behavior and neurobiological functioning, it is important to gain a better understanding of stress circuitry, HPA axis reactivity, and behavior in the context of social stress. The current study was designed to examine the effects of prolonged social isolation on the development of depressive- and anxiety-like behaviors, as well as plasma corticosterone, OT, and AVP responses to an acute social stressor in male prairie voles. The present study also evaluated amygdaloid morphology in the prairie vole model. We predicted that social isolation (relative to control conditions) would be associated with a.) depressive- and anxiety-like phenotypes; b.) altered corticosterone, OT, and AVP reactivity following a brief social stressor; and c.) greater branching and altered neuronal connectivity in the BLA.

2. MATERIALS & METHODS

2.1. General Methods

2.1.1. Animals

Thirty-two adult male prairie voles, with a mean (± standard error of the mean; SEM) age of 120 ± 7 days and a mean (± SEM) body weight of 52 ± 1 g, were used for the present experiments. Animals were descendants of a wild stock originating from Champaign, Illinois. Animals were housed on a 14/10 hour light/dark cycle with lights on at 6:30AM. The temperature of the housing environment was maintained at 25 ± 2°C and a relative humidity of 40 ± 5%. Animals were allowed food (Purina rabbit chow) and water ad libitum, unless otherwise noted in the experimental procedures. Offspring were housed at birth with breeding pairs in polycarbonate cages (25 × 45 × 60 cm) with cotton nesting material. Animals were separated from the family group at 21 days of age into sibling pairs housed in smaller cages (12 × 18 × 28 cm), until the commencement of experiments during adulthood. One animal from each sibling pair was studied in the experiments described here. All procedures were conducted in accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals and approved by the Northern Illinois University Institutional Animal Care and Use Committee.

2.1.2. Experimental Conditions

Animals were randomly assigned to either a socially isolated or socially paired (control) condition across 2 experiments. Animals in the isolated condition were separated into individual cages in separate rooms, without visual, auditory, or olfactory cues from the respective sibling. Animals in the socially paired condition remained paired with the sibling for the duration of the experiment. Social isolation and pairing conditions were maintained for 4 weeks in each experiment. Handling, cage changes, and measuring of body weight were standardized across the experimental conditions.

2.2. Experiment 1 Methods

2.2.1. General Design & Timeline

Experiment 1 consisted of 12 adult, male prairie voles. The general experimental timeline is described here, with specific procedures described in the following sections. Prior to the beginning of the experiment, all animals were allowed ad libitum access to a 1% sucrose solution, to allow for adaptation to the taste of the sucrose. Following this period, all animals were exposed to a 1-hour baseline fluid intake test. Animals were then assigned to either socially paired (n = 6) or isolated conditions (n = 6) for a total of 4 weeks. Following this 4-week housing period, all animals were exposed to a second 1-hour fluid intake test. Four hours following the fluid intake test, all animals were exposed to a 5-minute elevated plus maze (EPM). Twenty-four hours following the EPM, all animals were exposed to a 5-minute resident-intruder stressor, followed by the collection of plasma for enzyme-linked immunosorbent assay (ELISA) analysis of stressor-induced hormones and peptides.

2.2.2. Fluid Intake

The fluid intake test was used to operationally define anhedonia [53]. A 1-hour baseline fluid intake test was conducted prior to the pairing/isolation period, and an additional 1-hour fluid intake test was conducted following 4 weeks of the housing period. The procedures followed those outlined previously [16]. Twenty hours prior to testing, food and water were removed from all cages. Two hours prior to testing, all animals were relocated to clean, individual cages, to ensure accurate intake values of paired animals. Both paired and isolated animals were moved into clean, individual cages, and left undisturbed for a 2-hour period, to prevent differential consumption responses as a function of a novel environment [17]. Tap water and 1% sucrose were pre-measured and placed on the cages, with the positions of the bottles counterbalanced across conditions. Animals were allowed to freely consume the fluids for 1 hour, after which time the animals were returned to their home cages (paired or isolated conditions). A reduction in sucrose intake and preference, relative to baseline and control intake values, was used as an operational index of anhedonia, the reduced responsiveness to a previously-demonstrated pleasurable stimulus [53,54].

2.2.3. Elevated Plus Maze

The EPM was used to operationally define anxiogenic and exploratory behaviors [55]. A 5-minute EPM was conducted 4 hours following the post-housing fluid intake test. The maze consisted of two opposing open arms made of clear Plexiglass (49.5 × 10 cm), two opposing closed arms made of black Plexiglass (49.5 × 10 × 30.5 cm) with an open roof, and a center section of clear Plexiglass (10 × 10 cm). All sections were 57 cm high. The test was conducted in a brightly lit room. The maze was cleaned prior to each test. The animal was placed in the center of the maze and allowed to freely explore for 5 minutes. Immediately after testing, the animal was returned to its home cage (paired or isolated conditions). All behaviors were recorded using a digital video camera for offline analysis. The behaviors included: a.) the duration spent in each section of the maze – open, closed, and center; and b.) the number of crosses into the center section. Entering a section was defined as all four paws of the animal being in the respective section; and the animal must necessarily cross the center section prior to entering an open or closed arm. The number of crosses into the center section was used to identify general activity levels, while the time spent in the open arms of the maze was used to operationally define anxiogenic behaviors [17,55].

2.2.4. Resident-Intruder Test

Twenty-four hours following the EPM, all animals were exposed to a 5-minute resident-intruder test [16], which has been described previously as a stressor in rodents [56,57]. The test consisted of an experimental paired or isolated animal (the intruder) and an animal of the same sex, of similar size and body weight, unrelated and unfamiliar to the experimental animal (the resident). The resident’s cage mate was removed 1 minute prior to testing and remained housed in a separate cage until the test was completed. The resident remained in its home cage for the duration of the test; the intruder was placed into the resident’s cage for a period of 5 minutes.

Behaviors during the resident-intruder test were recorded using a digital video camera for offline analysis. The behaviors observed between the resident and intruder included: a.) one or both animals exhibiting aggressive grooming or posture toward the other animal; b.) one or both animals biting; c.) one or both animals thrusting; d.) one or both animals swatting at each other; e.) one or both animals pulling on each other; and/or f.) one or both animals exhibiting attack behavior [17,58,59]. These behaviors were summed to provide one overall index of aggressive behaviors during the resident-intruder test [16], and are reported as mean aggressive behaviors in each group, as well as the proportion of animals in each group that displayed at least 1 aggressive behavior during the 5-minute test.

Following testing in the resident’s cage, all experimental animals were placed into clean, individual cages for post-stressor observations. The animal was housed alone for 10 minutes, with post-stressor behavior analyzed for the first 1 minute of this period.

The behaviors during the post-stressor period were recorded using a digital video camera for offline analysis. The behaviors observed during the post-stressor period included: a.) running around the perimeter of the cage; b.) jumping; c.) repeated self-grooming; and/or d.) performing repeated, stereotypic behaviors, such as moving in a circle, digging in the shavings, scratching at the side of the cage, or chewing on the metal bars of the cage top [16]. These behaviors were summed to provide one overall index of activated behaviors during the one-minute post-stressor period [16], and are reported as the proportion of animals in each group that displayed at least 1 instance of activated behaviors during the 1-minute observation period.

2.2.5. Behavioral Analyses

All behaviors during the EPM and during and following the resident-intruder paradigm were recorded using a digital video camera and analyzed offline using The Observer XT version 8.0 (Noldus, Wageningen, the Netherlands). Behaviors were analyzed by 2 observers who were blind to the experimental conditions and trained to a level of at least 90% inter-rater reliability. All behaviors were averaged between the 2 raters.

2.2.6. Collection of Plasma

Nine minutes following the 1-minute post-stressor observation period (10 minutes following the end of the resident-intruder test), all experimental paired and isolated animals were anesthetized using a mixture of ketamine (67mg/kg, sc; NLS Animal Health, Ownings Mills, MD) and xylazine (13.33 mg/kg, sc; NLS Animal Health). Blood was sampled within 2 minutes of the anesthetic injection. Blood samples—collected over a period not exceeding 1 minute—were obtained from the periorbital sinus via a heparinized capillary tube. The time period of sample collection (10 minutes following a 5-minute behavioral test), and specific collection procedures following a behavioral test, have been validated previously by our laboratory to represent peak increases in circulating hormone and peptide levels following the previous short-term test, without confounds from short-term alterations as a function of anesthetic administration or shifts across time [17,60]. The samples were placed immediately on ice and centrifuged at 4°C, 3500 rpm, for 15 minutes to obtain plasma from the blood. The plasma aliquots were stored at −80 °C until used for ELISA analysis.

2.2.7. Circulating Hormone & Peptide Analyses

Analyses of circulating OT, AVP, and corticosterone were conducted with procedures previously validated in prairie vole experiments [16,61]. Commercially available ELISA kits were used to evaluate plasma levels of OT and AVP (Assay Designs, Ann Arbor, MI) and corticosterone (Enzo Life Sciences, Farmingdale, NY). Inter- and intra-assay coefficients of variation for these factors are the following (respectively): 19.2% and 2.9% for OT; 5.7% and 2.5% for AVP; and 5% and 5% for corticosterone. The minimum detection limit for each of these factors is the following: 34.68 pg/ml for OT; 3.39 pg/ml for AVP; and 0.027 ng/ml for corticosterone. The cross-reactivity of each of these factors with similar mammalian peptides and hormones is the following: < .001% for both OT and AVP; and < 1.7% for corticosterone.

2.3. Experiment 2 Methods

2.3.1. General Design

Experiment 2 consisted of 20 adult, male prairie voles. The general experimental timeline is described here, with specific procedures described in the following sections. Animals were assigned to either socially paired (n = 10) or isolated conditions (n = 10) for 4 weeks. Twenty-four hours following the end of housing period, brain tissue was collected and preserved using a Golgi-Cox staining solution, for the analysis of dendritic morphology and spine density.

2.3.2. Collection of Brain Tissue

Twenty-four hours following the end of the housing period, all experimental paired and isolated animals were anesthetized using ketamine (67mg/kg, sc; NLS Animal Health) and xylazine (13.33 mg/kg, sc; NLS Animal Health), and transcardially perfused using phosphate buffered saline and 4% paraformaldehyde.

2.3.3. Golgi-Cox Staining

The Golgi-Cox staining procedure followed the methodologies set forth by a previous experiment [62] (Figure 1). Brains were carefully removed from the skull, placed in 20 ml of Golgi-Cox solution [63] and stored in the dark. The brains were stored in this solution for 14 days, after which they were placed in 30% sucrose and again stored in a dark environment. The brains remained in the sucrose solution for a minimum of 2 days before being sectioned. Brains were sectioned into 200 μm thick sections using a vibratome. Sections were placed on 2% gelatinized microscope slides. The slides were processed utilizing a previously outlined procedure [62].

Figure 1.

Example images of 200 μm brain sections of the basolateral amygdala showing the Golgi staining procedure of interneurons, displayed at 4x (Panel A) and 40x (Panel B) magnification.

2.3.4. Golgi Analysis

Individual interneurons from the BLA were manually selected at random, ensuring that the neurons did not overlap, and drawn by hand using an Olympus CX31 microscope (Olympus Corporation, Center Valley, PA). Five interneurons per hemisphere were drawn per brain. The images were analyzed using ImageJ (NIH, Bethesda, MD). Concentric circles spaced 10 μm apart were overlaid on the scanned image of the neuron drawing (Figure 2). The amount of dendritic branching within each concentric circle was recorded, as well as the number of dendrites that crossed each concentric circle. This procedure was conducted to provide an estimation of dendritic length. First order branches were defined as branches originating from the cell body.

Figure 2.

Example drawing of a neuron in the basolateral amygdala with concentric circles (spaced 10 μm apart) overlaid on the scanned image of the neuron drawing.

2.3.5. Spine Analysis

An analysis of spine density was conducted using an Olympus BX51 microscope (Olympus Corporation) and Ocular software (Teledyne Photometrics, Tuscon, AZ) for picture capturing. The oil immersion technique was used for analysis following procedures previously outlined [63]. The experiment calculated both proximal and terminal spine densities from interneurons in the BLA. Proximal spines were obtained from non-terminating first and second order branches. Terminal spines were counted from the ends of higher-order branches (4^th ordered branches or higher), 30–40 μm from the center of the soma, that did not bifurcate further. Spines were counted from 5 BLA interneurons per hemisphere per brain.

2.4. Data Analyses

Behavioral analyses from the EPM, during and following the resident-intruder test, and body weight from Experiment 1 were conducted using mixed-design analyses of variance (ANOVA) with group as the independent factor and time as the repeated measures factor, and/or Student’s t-tests. The proportion of animals displaying aggressive behaviors during the resident-intruder test or behavioral activation following the test was calculated from summing the behaviors for each animal and compared using z-tests for a difference between two proportions. Hormone and peptide analyses from Experiment 1 were conducted using Student’s t-tests. Dendritic analyses from Experiment 2 were conducted using mixed-design ANOVAs with group as the independent factor and either distance from soma or branch order as repeated factors, and/or Student’s t-tests. A probability level of p < 0.05 was considered to be statistically significant, and all data are presented as means ± SEM.

3. RESULTS

3.1. Experiment 1

3.1.1. Body Weight

Body weight was analyzed using a mixed-design ANOVA. Body weight did not differ between paired and isolated groups at either the baseline period (53 ± 2 g in paired group vs. 52 ± 2 g in isolated group) or following 4 weeks of the housing period (51 ± 2 g in paired group vs. 51 ± 3 g in isolated group). There were no significant main effects of condition or time, and no condition by time interaction (p > 0.05 for all analyses).

3.1.2. Fluid Intake

Sucrose and water intake were analyzed using mixed design ANOVAs (Table 1). Social isolation resulted in a significant decrease in sucrose intake (F(1, 10) = 14.755, p < 0.003). There was a significant main effect of time (F(1, 10) = 12.031, p < 0.006), as well as an interaction of group and time of measurement (F(1, 10) = 13.992, p < 0.004). The isolated group displayed a marked decrease in the percentage of total fluid intake from sucrose at the 4-week measurement compared to the baseline measurement (Figure 3). No differences were observed in water consumption with respect to time, group, or an interaction thereof (p > 0.05 for all comparisons).

Table 1.

Fluid intake during a 1-hour consumption test in paired and isolated prairie voles at baseline and following 4 weeks of social housing manipulations.

	Water Intake (ml)		1% Sucrose Intake (ml)
	Baseline	Week 4	Baseline	Week 4
Paired	2.1 ± 2.2	2.8 ± 2.1	6.8 ± 2.4	7.0 ± 1.3
Isolated	2.4 ± 2.1	2.0 ± 1.3	6.5 ± 3.1	2.1 ± 1.8*#

Note: Data are shown as means ± SEM

^*p < 0.05 vs. socially paired control condition at the same time point;

^#p < 0.05 vs. respective baseline sucrose intake value.

Figure 3.

Percentage of total fluid intake from 1% sucrose in paired and isolated prairie voles at baseline and following 4 weeks of social housing manipulations. Both groups displayed the same preference for sucrose at baseline. Socially isolated animals displayed a decreased preference for sucrose following 4 weeks of social isolation, while socially paired animals maintained their sucrose preference. Data are shown as means ± SEM (* p < 0.05, vs. socially paired control condition at the same time point; ^# p < 0.05 vs. respective baseline value).

3.1.3. Elevated Plus Maze

Absolute duration spent, and percentage of total time spent, in each section of the 5-minute EPM were compared using Student’s t-tests for each category (Table 2). Social isolation resulted in a reduction in the absolute duration spent exploring the open arms (t(10) = 2.665, p < 0.02), and percentage of total time spent exploring these arms (t(10) = 3.070, p < 0.01). There was no significant group difference in absolute duration spent exploring the closed arms (p > 0.05), but social isolation was associated with an increase in the percentage of total time spent exploring these arms (t(10) = 3.412, p < 0.02). Social isolation did not significantly influence absolute duration spent exploring the center section of the maze, the percentage of total time spent exploring this section, or the number of entries into this section (p > 0.05 for all comparisons).

Table 2.

Exploration of open, closed, and center sections of a 5-minute elevated plus maze in paired and isolated prairie voles following 4 weeks of social housing manipulations.

	Open		Closed		Center
	Absolute (s)	Percent (%)	Absolute (s)	Percent (%)	Absolute (s)	Percent (%)
Paired	53.8 ± 16.5	17.9 ± 5.5	166.0 ± 26.7	55.3 ± 8.9	80.2 ± 24.0	26.7 ± 8.0
Isolated	9.3 ± 2.6*	3.1 ± 0.9*	222.5 ± 9.8	74.2 ± 3.3*	68.2 ± 9.5	22.7 ± 3.2

Note: Data are shown as means ± SEM for absolute time spent in each section (s) and percentage of total time spent in each section (%)

^*p < 0.05 vs. socially paired control condition.

3.1.4. Resident-Intruder Test

Total aggressive behaviors during the resident-intruder test were compared using a Student’s t-test. During the 5-minute resident-intruder test, no significant differences in aggressive behaviors were observed between the isolated and paired prairie voles. The mean number of aggressive episodes in each group was similar (7.8 ± 0.6 and 7.9 ± 1.2 instances in isolated and paired groups, respectively, p > 0.05), as was the percentage of animals in each group that displayed at least 1 instance of aggression during the 5-minute period [100% of animals in both isolated (6/6 animals) and paired groups (6/6 animals), p > 0.05].

Following the resident-intruder test during the 1-minute observation period, a significant difference in the percentage of animals displaying visible behavioral activation was observed. At least 1 instance of of visible behavioral activation was displayed in 83% of the isolated group (5/6 animals), but in 17% of the paired group (1/6 animals) (z = 2.31, p < 0.05).

3.1.5. Circulating Hormone & Peptides

Ten minutes following the resident-intruder test, the socially isolated prairie voles displayed significantly greater circulating levels of OT compared to paired prairie voles (t(10) = 8.425, p < 0.001; Figure 4A). AVP and corticosterone levels did not significantly differ between the groups (p > 0.05 for both comparisons; Figures 4B and 4C).

Figure 4.

Plasma oxytocin (Panel A), arginine vasopressin (Panel B), and corticosterone (Panel C) of socially isolated and paired prairie voles, 10 minutes following a 5-minute resident intruding test. Socially isolated prairie voles displayed significantly higher plasma oxytocin reactivity compared to socially paired animals; social housing conditions did not significantly influence plasma arginine vasopressin or corticosterone reactivity. Data are shown as means + SEM (* p < 0.05 vs. socially paired control condition).

3.2. Experiment 2

3.2.1. Dendritic Branching

Figure 5 shows a representative image of BLA interneurons from a paired and isolated prairie vole. An independent samples t-test was conducted on the total amount of dendritic branching (Figure 6A). The isolated group demonstrated a significantly lower amount of total branching than the paired group (t(18) = 3.137, p < 0.006).

Figure 5.

Representative images of stained interneurons in the basolateral amygdala (40x magnification) following 4 weeks of socially paired control conditions (Panel A) or social isolation (Panel B).

Figure 6.

Total number of dendritic branches in the basolateral amygdala (Panel A), number of branches as a function of branch order (Panel B), and number of branches as a function of distance from the soma (Panel C) following 4 weeks of social housing manipulations. Socially isolated animals displayed significantly fewer dendritic branches in the basolateral amygdala compared to socially paired animals, including fewer branches as a function of branch order and distance from the soma. Data are shown as means ± SEM (* p < 0.05 vs. socially paired control condition (main effect)).

A mixed-design ANOVA was conducted to examine the effects of isolation on specific branch orders (Figure 6B). While there were significant effects of isolation on the overall number of branches (F(1,90) = 9.288, p < 0.007), there was no significant interaction between group and branch order (p > 0.05). Fewer branches occurred depending on the branch order analyzed (F(5,90) = 810.915, p < 0.001).

A mixed-design ANOVA was conducted to examine the effects of isolation on the amount of branching at 10 μm intervals from the center of the soma (Figure 6C). There was a significant main effect of group such that there was a decrease in the number of branches radiating from the soma in isolated animals (F(1,54) = 9.288, p < 0.007), and a main effect of branch order such that there were fewer branches with increasing distance from the soma (F(1,54) = 977.191, p < 0.001). However, there was no significant interaction between distance from soma and group on the number of branches (p > 0.05).

3.2.2. Dendritic Length

The results of an independent samples t-test regarding dendritic length yielded significant effects of group. The isolated group displayed significantly lower overall dendritic length relative to the paired group (t(18) = 2.633, p < 0.02, Figure 7A). A mixed-design ANOVA was also conducted examining the effects of isolation on distance from the center of the soma. A significant main effect of group was observed such that isolation was associated with decreased dendritic length (F(1,72) = 6.933, p < 0.02). A significant main effect of distance from the soma was observed, such that branching decreased with increased distance from the soma (F(1,72) = 758.772, p < 0.001). There was a significant interaction between group and distance from the soma (F(4,72) = 2.775, p < 0.03). Post-hoc pairwise comparisons indicated greater differences between the groups the closer to the soma the length was analyzed, with significant decreases occurring in isolated animals at 10 μm (p < .05) and 20 μm (p < 0.05) from the soma (Figure 7B).

Figure 7.

Overall dendritic length in the basolateral amygdala (Panel A) and dendritic length as a function of distance from the soma (Panel B), following 4 weeks of social housing manipulations. Socially isolated animals displayed shorter dendritic length in the basolateral amygdala compared to socially paired animals, as well as significantly shorter dendritic length at 10 and 20 μm away from the soma. Data are shown as means ± SEM (* p < 0.05 vs. socially paired control condition).

3.2.3. Spine Density

Student’s t-tests were performed on proximal and terminal spine density. The isolated group demonstrated a significant increase in proximal spine density compared to the paired group (t(17) = 2.441, p < 0.03, Figure 8A). However, social isolation did not influence terminal spine density (p > 0.05, Figure 8B). Terminal branches are typically found at further distances from the cell soma, 30–40 μm from the center of the soma.

Figure 8.

Density of proximal (Panel A) and terminal (Panel B) spines (number of spines per 10 μm) in the basolateral amygdala, following 4 weeks of social housing manipulations. Socially isolated animals displayed significantly higher proximal, but not terminal, spine density compared to socially paired animals. Data are shown as means + SEM (* p < 0.05 vs. socially paired control condition).

4. DISCUSSION

The current study investigated the effects of prolonged social isolation in male prairie voles on maladaptive emotion-related behaviors, neuroendocrine markers of stress reactivity, and structural BLA changes. Four weeks of social isolation was associated with increased anxiolytic and anhedonic behaviors, as well as elevated plasma OT reactivity following the resident-intruder test. Social isolation also was associated with decreased amount of dendritic material in the BLA, both in branching and length, and an increase in spine density. The current findings provide insight into behavioral and neurobiological consequences of social isolation.

Social isolation was associated with several behavioral changes that are relevant to emotional and affective disruptions. As predicted, social isolation for 4 weeks in male prairie voles was associated with significantly less exploratory behavior in the EPM relative to paired animals, indicated by a reduction in open arm exploration time without a change in general activity. Isolated animals also lost their preference for sucrose by the end of the experiment, while their pair-housed counterparts maintained their preference for sucrose. This reduction in sucrose preference represents a specific hedonic deficit rather than a generalized consummatory change [54], evidenced by a specific decrease in sucrose intake without a change in water intake across 4 weeks of social isolation. Isolation-induced affective behaviors have previously been reported in rodents, including in male and female isolated prairie voles [17,23,59,64,65]. For example, male prairie voles isolated from a male sibling for 4 weeks displayed similar reductions in sucrose to those in the present study [17]. Although our laboratory has not previously studied anxiety-related behaviors using the EPM in adult male prairie voles following 4 weeks of isolation from a male sibling, the current EPM data are in line with reduced open arm exploration time displayed by adult female prairie voles isolated for 4 weeks from a female sibling [59,66,67], and in adult male prairie voles isolated for 4 weeks from a female social partner [23]. However, the present EPM data are inconsistent with a previous study demonstrating that neither isolation from a male sibling nor from a female social partner for 5 days altered open arm exploration time in the EPM in adult male prairie voles [21]. It is possible that longer periods of social isolation may be necessary to induce anxiety-related behaviors in the EPM in prairie voles. The current findings, coupled with those of previous social isolation paradigms in prairie voles, support correlations between loneliness and affective symptoms [6]; and may inform our understanding of the comorbidity of anxiety and depression described in clinical populations [68].

In addition to the EPM, prairie voles in the present study were exposed to the resident-intruder test for the analysis of behaviors and short-term physiological reactivity. Social isolation did not influence aggressive behaviors between the resident and intruder during the test. This lack of a group difference in aggressive behaviors supports previous results from male and female prairie vole intruders in similar resident-intruder paradigms [16,17], yet the behaviors are different from defensive behaviors exhibited by prairie vole intruders during a social defeat paradigm [69]. Separate from aggressive behaviors during the resident-intruder test, a greater proportion of the isolated condition (vs. paired) displayed behavioral activation following this test, indicated by running around in the cage, jumping, repeated self-grooming, and exhibiting repetitive and stereotypic-like behaviors during the 1-minute post-test observation period. Although males were specifically chosen as the subjects for the present design, the behavioral activation exhibited by isolated males here is similar to that reported previously in isolated female prairie voles [16]. This increased behavioral activation following the resident-intruder task may indicate that prior social isolation promotes maladaptive short-term stress responses. This hypothesis is supported by previous observations of impaired autonomic regulation of the heart associated with short-term stressors in socially isolated prairie voles [59,70]. For instance, isolated female prairie voles (vs. paired) displayed greater heart rate reactivity during a 5-minute resident-intruder test, and an approximate 5-fold increase in recovery time for heart rate to return to pre-stressor levels following the test [59]. Further investigation of potential sex differences in response to long- and short-term social tasks in prairie voles is warranted.

In addition to behavioral disturbances associated with affect and stress, prolonged social isolation was associated with elevated plasma OT reactivity—but not AVP or corticosterone reactivity—following the resident-intruder test. Circulating levels of OT and corticosterone observed in the present study replicate those described previously in male paired and isolated prairie voles after the resident-intruder test [17]. Further, the stressor-induced corticosterone levels here are higher than previously-described baseline corticosterone levels in male paired and isolated prairie voles, providing support for the hypothesis that the resident-intruder test serves as a stressor in prairie voles [17].

Although OT was increased following the resident-intruder test in isolated prairie voles, the present study design cannot address the question of whether OT was elevated in a chronic manner as a function of social isolation, or whether it was activated acutely as a function of the resident-intruder paradigm (or both). It is possible that social isolation interacts with disrupted behavior following the resident-intruder test and consequently increases circulating OT, as neither hypothalamic PVN nor circulating peripheral OT levels were increased following social isolation alone in male prairie voles in a previous study, yet both were elevated following social isolation plus a resident-intruder stressor [17]. Given that OT in the present study was elevated in the isolated group 10 minutes following the resident-intruder test, but corticosterone and AVP were not significantly elevated at the same time point, it is also possible that OT was released in a compensatory manner to down-regulate a short- or long-term HPA axis response. This hypothesis is supported by previous evidence from multiple species suggesting that OT has stress-buffering effects [30,71–73]. These stress-buffering consequences may be partly mediated by central OT activity, as a recent meta-analysis illustrated that plasma OT measures reflect central OT activity following a stress response [74]. However, it has also been suggested that chronic stress may lead to disrupted OT-HPA communication, such that the HPA axis may be desensitized or unresponsive to the downregulatory effects of OT [75,76].

Based on previous changes in the structure and function of central circuitry following stress in rats and mice [43,44], the present study also predicted that prolonged social isolation in prairie voles would increase dendritic arborization and spine density in the BLA. While this prediction was partially supported with an increased spine density in the BLA observed in socially isolated animals, a significant decrease in amount of dendritic material (both branching and length) was associated with social isolation. Dendritic pruning has been reported in rats and mice exposed to acute stress [77], yet the present data suggest a decrease in dendritic material as a function of chronic stress. The difference between the current findings and previous studies may be due to differences in species (socially monogamous vs. non-monogamous rodents) [78–80], the nature or duration of stressors (social isolation vs. restraint or social defeat) [79–81], sex [82,83], or a combination of these factors. Investigating the interactions of species, sex, stressor type and duration, and BLA function will enhance our understanding of the amygdala in the context of social stress.

The present pattern of dendritic plasticity observed in socially isolated animals is intriguing. Dendritic pruning suggests a decrease in communication to and from the BLA, while increased spine density suggests an increase in synaptic strength and stability in this region [84]. Several mechanisms may influence BLA function. BLA remodeling may be mediated by the influence of OT in various regions of the amygdala. For example, intracerebroventricular administration of OT decreased dendritic length and complexity in the BLA of male rats [85]. Further, behavioral changes (post-traumatic-like symptoms) interacted with dendritic alterations in the BLA of rats exposed to a single prolonged stressor, and central OT administration prevented behavioral dysfunction and dendritic pruning in stress-exposed animals [85]. Additionally, male and female OT knockout mice displayed higher dendritic spine density in the posterodorsal medial amygdala compared to wild type mice [86,87]. The elevated plasma OT levels and reduced dendritic material in the BLA support these previous findings from other rodent models. It is possible that the pattern of BLA remodeling observed in isolated animals was a result of chronic social stress, increased OT interactions with the BLA, or a combination of both. As OT is hypothesized to be anxiolytic [76,88], increased OT and associated reduced dendritic material in the BLA may be compensatory responses to the stress of social isolation.

Second, it is possible that OT interacts with other neural structures that communicate with the amygdala. For example, OT and OT receptors play important roles in hippocampal functions, including neurogenesis, cytoskeletal rearrangement, and synaptic formation [89]. Changes in receptor binding may lead to changes in synaptic transmissions, either via excitation or inhibition, and lead to synaptic strengthening via long-term potentiation or synaptic weakening via long-term depression [89,90]. For example, hippocampal neurons of OT receptor knockout mice (vs. wildtypes) were more pharmacologically excitable, with decreased numbers of GABAergic synapses [91]. Taken together, it is possible that isolation-related OT function may influence both hippocampal and amygdala communication; this may impact synaptic transmission and lead to changes in dendritic material in the BLA. Future studies will benefit from investigating the mechanisms that underlie altered dendritic material in the BLA in socially isolated animals, for instance by observing the influence of artificially manipulating OT on behavior and morphology.

5. CONCLUSIONS

In conclusion, the current study investigated the associations among chronic social isolation, affective behaviors, endocrine reactivity, and structural changes in the BLA of male prairie voles. Prolonged social isolation induced anxiety-related and anhedonic behaviors. These data indicate that prolonged social isolation increases affective behaviors, potentially influencing the development of depression and/or anxiety disorders. Social isolation also was associated with increased behavioral activation and elevated plasma OT reactivity following the resident-intruder test. These results demonstrate potential interactions among OT, HPA reactivity, and emotion-related behavior. For example, OT may be released in an attempt to down-regulate a short-term HPA axis response; or alternatively social isolation may disrupt OT signaling such that the release of OT during stress may not have appropriate compensatory behavioral stress-buffering effects in socially isolated individuals. Finally, social isolation was associated with reduced dendritic material, coupled with increased dendritic spine density, in the BLA. Considered together, the present findings suggest that alterations in OT function as well as BLA remodeling may contribute to affective behaviors observed in socially isolated prairie voles. The current findings also highlight the complexity of neural circuitry and plasticity of loneliness, anxiety, and depression. Prairie voles are a useful translational model for further investigating these inter-relationships, as well as related questions focused on fear memory and amygdala-related disorders [92]. A better understanding of these neurobiological and behavioral interactions can lead to improved intervention strategies to mitigate psychological and physiological health consequences of social stressors in humans.

HIGHLIGHTS.

• Social isolation induced anxiety-like and anhedonic behaviors in male prairie voles

• Isolation was associated with elevated plasma oxytocin following a social stressor

• Isolation altered dendritic material and branching in the basolateral amygdala

• This research informs prevention and treatment strategies for isolated humans