Decision letter: Prolonged partner separation erodes nucleus accumbens transcriptional signatures of pair bonding in male prairie voles

Abstract

Article Figures and data Abstract Editor's evaluation eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The loss of a spouse is often cited as the most traumatic event in a person’s life. However, for most people, the severity of grief and its maladaptive effects subside over time via an understudied adaptive process. Like humans, socially monogamous prairie voles (Microtus ochrogaster) form opposite-sex pair bonds, and upon partner separation, show stress phenotypes that diminish over time. We test the hypothesis that extended partner separation diminishes pair bond-associated behaviors and causes pair bond transcriptional signatures to erode. Opposite-sex or same-sex paired males were cohoused for 2 weeks and then either remained paired or were separated for 48 hours or 4 weeks before collecting fresh nucleus accumbens tissue for RNAseq. In a separate cohort, we assessed partner-directed affiliation at these time points. We found that these behaviors persist despite prolonged separation in both same-sex and opposite-sex paired voles. Opposite-sex pair bonding led to changes in accumbal transcription that were stably maintained while animals remained paired but eroded following prolonged partner separation. Eroded genes are associated with gliogenesis and myelination, suggesting a previously undescribed role for glia in pair bonding and loss. Further, we pioneered neuron-specific translating ribosomal affinity purification in voles. Neuronally enriched transcriptional changes revealed dopaminergic-, mitochondrial-, and steroid hormone signaling-associated gene clusters sensitive to acute pair bond disruption and loss adaptation. Our results suggest that partner separation erodes transcriptomic signatures of pair bonding despite core behavioral features of the bond remaining intact, revealing potential molecular processes priming a vole to be able to form a new bond. Editor's evaluation This study focuses on an important but understudied topic, the biological basis of pair bonding and associated stress when the bond is lost. The authors present convincing evidence that transcriptional changes associated with the formation of a pair bond in voles degrade during prolonged separation but do not precisely mirror behavioral responses. Gene expression changes were associated with glia, steroid hormone signaling, and myelination, among others, providing critical guideposts for future investigations. This work will be of interest to behavioral neuroscientists and/or those interested in social behavior. https://doi.org/10.7554/eLife.80517.sa0 Decision letter Reviews on Sciety eLife's review process eLife digest Losing a spouse or life partner is a deeply traumatic event that can have long-term repercussions. Given enough time, however, most surviving partners are able to process their grief. The neural processes that enable people to adapt to their loss remain unknown. To explore this question, scientists often turn to animals that form long-term mating based pair bonds and can be raised in the laboratory. Monogamous prairie voles enter lifelong partnerships where the two individuals live together, prefer to cuddle with each other, and take care of their pups as a team. After having lost their mate, they show signs of distress that eventually subside with time. Sadino et al. examined the biological impact of partner loss in these animals by focusing on the nucleus accumbens, a brain region important for social connections. This involved tracking gene expression – which genes were switched on and off in this area – as the voles established their pair bonds, and then at different time points after one of the partners had been removed. The experiments revealed that establishing a relationship leads to a stable shift in nucleus accumbens gene expression, which may help maintain bonds over time. In particular, genes related to glia (the non-neuronal cells which assist neurons in their tasks) see their expression levels increase, indicating a previously undescribed role for this cell type in regulating pair bonding. Having their partner removed led to an erosion of the gene expression pattern that had emerged during pair bonding; this may help the remaining vole adapt to its loss and go on to form a new bond. In addition, Sadino et al. explored the gene expression of only neurons in the nucleus accumbens and uncovered biological processes distinct from those that occur in glia after partner separation. Together, these results shed light on the genetic and neuronal mechanisms which underlie adaptation to loss; this knowledge could one day inform how to better support individuals during this time. Introduction The death of a romantic partner is cited as one of the most traumatic experiences in a person’s life and results in grief and corresponding indicators of mental and physiological distress (Keyes et al., 2014; Holmes and Rahe, 1967). However, for the majority of people acute grief subsides within approximately six months as the bereaved integrates and adapts to the loss (Shear et al., 2011). Most people will also eventually form a new pair bond, which provides a behavioral indicator of loss adaptation (Shear and Shair, 2005). The processes that enable such adaptation remain poorly understood but likely occur via the same neural systems that are uniquely engaged by pair bonding (Blocker and Ophir, 2016). Socially monogamous prairie voles are a laboratory-amenable species that recapitulates many aspects of human social bonds, making them ideal for interrogating the neurobiology of bonding and loss. Pair bonding in this species results in a shift in sociobehavioral state where specific behaviors are only evident after the transition from living in a same-sex pair (i.e. family group) to an opposite-sex bonded pair. Pair bonded voles will prefer to affiliate with their partner, display selective aggression towards non-partner individuals, and exhibit robust and organized biparental care (Williams, 1992; Getz et al., 1981; Williams et al., 1992; Insel and Young, 2001; Carter et al., 1995; Winslow et al., 1993). Considering that other stable behavioral shifts, such as reproductive status and dominance hierarchies, are underwritten by changes in transcription, we aimed to determine if a mature pair bond—and the process of adapting to loss—are underwritten by stable changes in gene expression in the nucleus accumbens (Cardoso et al., 2015; Zayed and Robinson, 2012; Tripp et al., 2018). While an extended network of brain regions mediates social processing and decision making, the nucleus accumbens (NAc)—a region important for reward, motivation, and action selection—is a critical hub that is engaged when forming a bond and is implicated in loss processing (Aragona and Wang, 2004; Lim and Young, 2004; Walum and Young, 2018). In humans, holding hands with a romantic partner enhanced blood oxygenation levels (BOLD) in the NAc and successful adaptation to spousal loss is associated with a reduced partner-associated BOLD signal in this region (O’Connor et al., 2008; Kreuder et al., 2017). Thus, the NAc contributes to pair bonding and loss and broad scale changes in this brain region potentially mediate shifts in transcription which may be required to adapt to partner loss. Similarly, in prairie voles, pair bond-associated behavioral changes are underwritten by several known neuromolecular changes within the NAc which maintain and reinforce pair bonds over time (Insel and Young, 2001; Aragona and Wang, 2004; Young and Wang, 2004). Further, when separated from their partner, prairie voles exhibit behavioral and physiological distress that mirrors what is seen upon bond disruption in humans and other species, such as titi monkeys (Carter et al., 1995). These include increased circulating glucocortioids levels, activation of the HPA axis, increased anxiety, decreased pain thresholds, and autonomic dysfunction (McNeal et al., 2014; Grippo et al., 2007; Bosch et al., 2016; Pohl et al., 2019; Osako et al., 2018). However, we have previously shown that if a pair bonded prairie vole loses their partner, given enough time, they will form a new bond that supersedes the original bond (Harbert et al., 2020; Tamarin et al., 1990). As in humans, the ability to form a new bond indicates that prairie voles can adapt to partner loss. Here, we map the trajectory of the pair bond transcriptional profile by comparing opposite-sex paired males to same-sex sibling male pairs. In the wild, sexually naive male voles can cohabitate with other males, especially siblings, although these relationships do not persist after males form an opposite-sex pair bond and establish a nest/territory with their partner (Getz et al., 1981; Carter et al., 1995). By comparing opposite-sex pair bonded voles to their same-sex paired counterparts before and after partner separation, we have an ethologically relevant means to isolate the unique biology of bonding and loss independent of those that support general affiliative interactions (e.g. peer relationships) or the stressful effects of social isolation more broadly. Thus, we compared behavior and NAc transcriptional profiles of opposite-sex pair bonded and same-sex cohoused naïve male voles to explicitly define the pair bond both behaviorally and transcriptionally. Then we examined how affiliative pair bond characteristics were altered by partner separation to test the hypothesis that pair bond transcriptional signatures and bond-associated behaviors would erode as a function of time since partner separation. We reasoned that these changes represent key components of loss adaptation that, together, may prime the vole to be able to form a new bond. We found that pairing induced a reliable affiliative preference for a peer or a pair bonded partner, and that this preference is remarkably stable, persisting even after four weeks of separation. We further show that pair bond-associated changes in accumbal gene expression were consistent at two and six weeks post-pairing. However, once opposite-sex pairs are separated, the pair bond transcriptional signature erodes as a function of separation time. To further home in on the transcriptional changes associated with partner separation specifically in NAc neurons, we pioneered translating ribosomal affinity purification in voles (vTRAP). Using vTRAP, we identified clusters of genes associated with dopaminergic signaling, mitochondrial organization, and steroid hormone signaling whose expression patterns are sensitive to acute pair bond disruption and loss adaptation. In sum, our parallel behavioral and transcriptional data suggests that erosion of pair bond transcriptional signatures in the NAc precedes changes in affiliative partner preference, providing insight into time-dependent neuromolecular changes that may contribute to loss adaptation. Results We determined how bonding and extended separation affects social behavior and NAc transcription in opposite-sex and same-sex paired males. We employed timepoints that are experimentally validated and ethologically relevant (Figure 1A). We paired all study animals for 2 weeks, a duration that reliably produces mature pair bonds (Scribner et al., 2020; Brusman et al., 2021). For the separation timepoints, the ability to form a new bond after loss serves as a behavioral metric of loss adaptation. Prior work has shown that male voles are able to form a new pair bond 4 weeks post-separation, but not 2 weeks or earlier (Harbert et al., 2020). Figure 1 with 3 supplements see all Download asset Open asset Males in either opposite-sex or same-sex pairs retain their partner preferences for at least four weeks following partner separation. Additional stats including post-hocs provided in Supplementary file 1. (A) Schematic of a partner preference test (PPT) and timelines for behavioral experiments. Opposite-sex (n=8/cohort) and same-sex (n=8/cohort) pairs were paired for 2 weeks prior to a baseline PPT. Pairs were then either in a remain paired or separated cohort. Remain paired animals stayed with their partners throughout the experiment and underwent PPT 48 hours and 4 weeks after baseline. Separated animals were separated from their partners for either 48 hours (short-term) or 4 weeks (long-term) prior to the endpoint PPT. (B) Partner preference scores (% partner huddle/total huddle) from baseline, short-term, and long-term PPTs of opposite-sex (dark green) and same-sex (light green) paired males. Opposite- and same-sex paired males showed a baseline partner preference that remained evident for both groups after short-term separation and long-term separation (one-way t-tests relative to 50%, *** p<0.001, ** p<0.01). Black dotted line at 50% indicates no preference for partner or novel. (C) Partner (left) and novel (right) huddle duration (seconds) for remain paired animals over time during each PPT (two-way RM-ANOVA). (D) Partner huddle for short-term (left) and long-term (right) separated animals between baseline and the endpoint PPT—48 hours or 4 weeks post-baseline, respectively (two-way RM-ANOVA). (E) Novel huddle for short-term (left) and long-term (right) separated animals between baseline and the endpoint PPT—48 hours or 4 weeks post-baseline respectively (two-way RM-ANOVA). Consistent with our laboratory observations, wild male voles who lose a partner will remain at the nest for ~17 days (Tamarin et al., 1990). Thus, we assessed behavior and transcription in paired voles two days and four weeks after separation—timepoints before and after the animal can form a new bond. Extended separation disrupts the strengthening of bonds but does not baseline erode partner preference We measured partner-directed affiliation via a partner preference test (PPT) across time in animals that remained paired and before and after short- or long-term partner separation (Figure 1A). The PPT measures the amount of time spent huddling either with the partner or a novel vole tethered at opposite ends of an arena (Figure 1A, right side). To ensure that the experimental males could not hear, smell, or see their separated partners during separation, all partners were moved to a separate vivarium room. All sample sizes and comprehensive statistical results, including effect size estimates, are reported in Supplementary file 1. We initially hypothesized that opposite-sex pairs would have a partner preference at baseline and after short-term separation, when the pair bond is still intact, but not after long-term separation (Harbert et al., 2020; Sun et al., 2014). Similarly, we anticipated that same-sex sibling pairs would have a partner preference at baseline due to familiarity but would exhibit a faster and more robust loss of partner preference when separated (Lee et al., 2019). In contrast to our initial hypothesis, we found that opposite-sex and same-sex paired males demonstrated a partner preference even after extended separation (Figure 1B; Lee et al., 2019; Goodwin et al., 2019). In pairs that remained with their partners throughout the 6-week timeline, both opposite-sex and same-sex pairs form a selective preference for their partner after 2 weeks of pairing (Figure 1B, one sample t-test relative to 50%: opposite-sex p=0.00564, same–sex p=0.00922). The baseline partner preference for both opposite- and same-sex pairs is retained—and even moderately strengthened—after 4 additional weeks of cohabitation (one sample t-test relative to 50% for 48 hr PPT: opposite-sex p=0.0107, same-sex p=0.01478; for 4 weeks PPT: opposite-sex p=5.19 × 10-7, same-sex p=6.49 × 10–7, two-way RM-ANOVA in Supplementary file 1). In separated animals, both opposite-sex and same-sex pairs form a selective preference for their partner after 2 weeks of cohabitation (one sample t-test relative to 50% short-term separated animals: opposite-sex p=3.30 × 10–12, same-sex p=7.72 × 10–5; long-term separated animals: opposite-sex p=1.50 × 10–3, same-sex p=3.04 × 10–5) and retain their partner preference after short-term and long-term separation (one sample t-test relative to 50%: short-term separated animals: opposite-sex p=4.10 × 10–7, same-sex p=3.66 × 10–8; long-term separated animals: opposite-sex p=0.001846, same-sex p=3.14 × 10–8). Additionally, there was no significant difference in partner preference score due to either separation duration or partner type (Figure 1B; two-way RM-ANOVA with repeated measures for timepoint: short-term separation: Main effect of partner (opposite- vs same-sex): F(1,28) = 0.38, p=0.54; Main effect of time: F(1,28) = 0.22, p=0.64; Partner X time: F(1, 28)=1.87, p=0.18; long-term separation: Main effect of partner (opposite- vs same-sex): F(1,26) = 1.70, p=0.20; Main effect of time: F(1,26) = 0.16, p=0.70; Partner X time: F(1, 26)=0.057, p=0.81). Together, these results indicate that prairie voles are capable of forming an affiliative preference for an opposite- or same-sex partner and that preference remains intact despite prolonged separation. Next, we examined how partner- and novel-directed affiliative behavior, respectively, changed over time. For males that remain paired there were no significant differences in novel huddle over time for either pairing type. Of note, partner huddle increases with extended co-housing in both pairing types indicting a strengthening of partner-directed affiliation (2-way RM-ANOVA: main effect of timepoint (4 wks:base): F(2, 42)=4.92, p=0.012, η=0.170) (Figure 1C). This increase was not observed in opposite-sex or same-sex paired males that had been separated from their partner (Figure 1D and E; two-way RM-ANOVA in Supplementary file 1). This suggests that long-term separation effectively impedes the maturation and strengthening of the pair bond without necessarily dissolving the baseline preference. We also quantified how long it took to begin huddling in the partner preference test but failed to observe any consistent differences for partner or novel huddle latencies over time regardless of experimental condition or pairing type (Figure 1—figure supplement 1A–F, log-rank test in Supplementary file 1). Additionally, we quantified how much each animal’s partner preference and partner huddle changed between timepoints (endpoint minus baseline; Figure 1—figure supplement 2A, B, stats in Supplementary file 1). These change scores revealed that partner preference and partner huddle were relatively stable between the baseline and short-term timepoint regardless pairing status or pairing type. However, at the long-term timepoint, we observed another indicator that separation impedes ongoing pair bond strengthening: opposite-sex males that remain paired exhibited increased partner huddle duration at 4 weeks relative to baseline, which was not observed in opposite-sex separated males (Figure 1—figure supplement 2B). In contrast, in same sex paired males, ongoing pairing was not required for increased partner huddle at the long-term timepoint (Figure 1—figure supplement 2B). Finally, we assessed behavioral consistency across PPTs by determining the correlation of partner preference test metrics (huddle times, partner preference score, and chamber times) between baseline to short-term and baseline to long-term timepoints in all cohorts (Figure 1—figure supplement 3). Pairing duration, separation, and partner type did not yield reliable differences in correlated patterns of behavior. Together, these results indicate that prairie voles form an affiliative preference for an opposite- or same-sex partner that remains intact despite prolonged separation. However, separation impedes the normal, strengthening of partner-directed affiliation that occurs over time. The pair bond transcriptional signature is stable in intact bonds Changes in behavioral states, such as a shift in dominance or reproductive status, are supported by stable changes in transcription, although this has not been examined in the context of pair bonds (Cardoso et al., 2015; Zayed and Robinson, 2012; Tripp et al., 2018). While prior work has demonstrated that mating and cohabitation in prairie voles result in transcriptional changes within the NAc, the consistency of these changes as long as the bond remains intact has yet to be assessed (Duclot et al., 2020; Tripp et al., 2021; Resendez et al., 2016; Resendez et al., 2012; Aragona et al., 2006). Thus, we compared NAc transcription in opposite- versus same-sex-paired voles following either 2 or 6 weeks of pairing/cohabitation (Figure 2A). By comparing opposite-sex to same-sex-paired animals, we identified transcripts specific to pair bonds compared with those associated with affiliative behavior more generally. For consistency, we limited transcriptional assessment to voles that had a baseline partner preference >50% (Figure 2B; Supplementary file 1; OS n=15; SS n=11. Excluded for PPT <50% OS n=3; SS n=8). Figure 2 with 2 supplements see all Download asset Open asset Pair bonding leads to persistent and consistent changes in NAc transcription. (A) Opposite- and same-sex pairs were paired for 2 weeks prior to a baseline partner preference test. Pairs then remain paired for either 48 hours (short-term; ~2 weeks total pairing) or 4 weeks (long-term; ~6 weeks total pairing) prior to collecting fresh nucleus accumbens tissue (dissection sites in red boxes) for RNA sequencing. (B) Baseline partner preference scores of males included in RNA sequencing for the opposite- and same-sex groups (one-tailed t-test relative to 50%: opposite-sex T14=11.76, p=1.21 X 10–8; same-sex T10=7.78, p=1.50 X 10–5). Black dotted line indicates a 50% partner preference score and the grey dotted line indicates 66%. There were no differences in partner preference score between opposite- and same-sex paired animals used for RNAseq (two-tailed t-test: T21.016 = 1.374, p=0.184). (C) Transcriptional analysis workflow. (D) Gene list from both timepoints ordered from the smallest to largest log2FoldChange after short-term pairing with color indicating up- or down-regulation in opposite- vs same-sex pairs. Expression patterns are strongly correlated across timepoints (Rho = 0.38, p=2.2 X 10–16). (E) Schematic of RRHO analysis. The heatmap is arranged into quadrants of genes upregulated in both lists (up-up: quadrant UU), downregulated in both lists (down-down: quadrant DD), or genes that have opposite regulation (up in list 1-down in list 2: quadrant UD; down in list 1-up in list 2: quadrant DU). Genes that are found in both lists at a similar ranked position result in higher p-values and are represented by a yellow color. (F) RRHO comparing short-term and long-term pair bonding (from 2D) indicates a stable pair bond gene signature over time as evidenced by concordant up- or downregulated genes at the two timepoints. (G) The short-term and long-term time points were pooled for opposite- and same-sex pairs to define the combined pair bond gene signature. (H) We compared GO analysis Mus musculus ontology terms between the combined pair bond DEGs and the RRHO quadrants (from 2 F) with strong correspondence between the two analyses. We used DESeq2 to identify transcripts up- or down-regulated in opposite- relative to same-sex paired males after short-term and long-term pairing (Figure 3—figure supplement 2A, B; Love et al., 2014). When ordering transcripts based on their log2FoldChange at the short-term timepoint, the global transcriptional differences for opposite- versus same-sex pairs were strikingly similar following either 2 or 6 weeks of pairing, suggesting stable pair bond associated transcription across these timepoints (Figure 2D; Spearman’s Rho = 0.38, p=2.2 × 10–16). We further determined that the observed correlation across timepoints is stronger than what would be expected by chance. We shuffled each animal’s cohort identity at the long-term timepoint and calculated Rho values of the log2FoldChange of differential gene expression between the observed short-term pair bond and the shuffled long-term pair bond over 1000 iterations (Figure 2—figure supplement 1A–D). The cohort identity is a combination of three variables: pairing type (opposite-sex or same-sex), separation condition (remain paired or separated), and timepoint (short- or long-term). For example, long-term separated opposite-sex animals is one cohort identity of 8 total. This approach effectively randomized timepoint, partner type, and pairing status without disrupting underlying structure that exists within our transcriptional dataset due to correlations in expression across genes. Our observed Rho value was greater than 100% of iterations, indicating that the similarity in gene expression across short and long-term pairing timepoints is greater than would be expected by chance (Figure 2—figure supplement 1D). Next, we asked whether our observed association across timepoints was driven by moderately but consistently expressed genes. Eliminating transcripts whose expression difference fell within the middle two quartiles in the short-term group resulted in a greater Rho value (R=0.44, p<2.2 × 10–16), indicating that the correlation across short- and long-term timepoints is more strongly driven by transcripts with larger expression differences between opposite- and same-sex pairs. To further interrogate the biological function of altered transcripts, we set nominal thresholds of log2FoldChange >0.30 or<–0.30, which are sufficient to identify replicable differences detectable via alternate methods (Walker et al., 2018), along with a p-value threshold of p<0.05 to identify up- and down-regulated gene lists (DEGs). This nominal p-value threshold was used with the aim of identifying gene families regulating molecular pathways responsive to bonding instead of specific gene candidates. Prior studies in animal brain tissue using this p-value have yielded biologically meaningful insights (Walker et al., 2018; Mukamel, 2022; Kronman et al., 2021; Walker et al., 2022; Labonté et al., 2017; Krishnan et al., 2007; Peña et al., 2017; Seney et al., 2018; DEGs based on these thresholds are listed in Supplementary file 2). There was significant overlap in the upregulated (104 shared, Fisher’s Exact Test: χ2=30.24, p=5.36 X 10–31) and downregulated (46 shared, Fisher’s Exact Test: χ2=13.32, p=7.05 X 10–14) transcripts across timepoints (Figure 3—figure supplement 2E; Wang et al., 2015). To further examine similarity in transcriptional patterns between short-term and long-term pair bonds we employed Rank-Rank Hypergeometric Overlap (RRHO; Figure 2E; Plaisier et al., 2010; Cahill et al., 2018). This threshold free approach allows us to determine if similar global transcription patterns are observed between two comparisons. The resulting RRHO heatmap is arranged into quadrants based on the direction of gene expression and each point represents the significance derived from the number of overlapping genes via the hypergeometric distribution (Figure 2E). We determined how similar global transcription is between short-term and long-term pair bonds by comparing differential gene expression in the opposite- vs same-sex 2-week paired to opposite-sex vs same-sex 6-week paired groups. We observed extensive concordance of transcriptional patterns between 2- and 6-week paired voles, further supporting that the transcriptional profile of pair bonds is stable (Figure 2F). Confirming that our observed RRHO signal was unlikely to be attributable to chance, we randomly shuffled cohort identity (Figure 3—figure supplement 4E) and, separately, shuffled the gene ranking of the combined pair bond genes (Figure 3—figure supplement 4G), both of which ablated the RRHO signal. Since the pair bond transcriptional profiles are concordant at both time points, the 2-week and 6-week animals from each pairing type were pooled to create a single, well-powered opposite-sex- vs same-sex-paired comparison that defines the combined pair bond transcriptional signature (Figure 2G). To determine which biological pathways might underlie pair bonding we employed Gene Ontology (GO) and Ingenuity Pathway Analysis (IPA) (Yu et al., 2012). Gene lists for GO terms or IPA were generated either from DEG lists or from genes in concordant (UU or DD) RRHO quadrants. GO terms that passed a false discovery rate of p<0.05 were retained while IPA terms that had activation scores of at least –2 or 2 and whose enrichment for genes predicted to be regulated was significant (p-value <0.05) were kept (Supplementary file 3). Combined pair bond GO terms associated with the upregulated DEGs and the quadrant UU genes strongly implicate changes in glial cells and extracellular matrix organization (Figure 2H), which is mirrored by activation of glioblastoma signaling via IPA (Figure 2—figure supplement 2A). IPA additionally identified upregulation of Synaptogenesis, CREB signaling, and Endocannabinoid Neuronal Synapse pathways. All these pathways are consistent with pair bonding as a form of complex learning that is mediated by neuromodulatory signaling (Walum and Young, 2018; Loth and Donaldson, 2021; Modi and Young, 2011; Johnson and Young, 2017). Predicted upstream regulators of these pathways broadly support a role for learning and neuromodulation, including Creb1, Estrogen receptor alpha (Esr1), and various growth-factor and developmental genes (Figure 2—figure supplement 2A). Combined pair bond GO terms associated with the downregulated DEGs and the quadrant DD genes are implicated in nucleoside synthesis and synaptic plasticity (Figure 2H). IPA indicates a suppression of corticotropin releasing hormone signaling, potentially reflecting the strong social buffering of stressors that occurs in pair bonded voles specif