Sexual differentiation of neural mechanisms of stress sensitivity during puberty.

Anxiety disorders are a major public health concern and current treatments are inadequate for many individuals. Anxiety is more common in women than men and this difference arises during puberty. Sex differences in physiological stress responses may contribute to this variability. During puberty, gonadal hormones shape brain structure and function, but the extent to which these changes affect stress sensitivity is unknown. We examined how pubertal androgens shape behavioral and neural responses to social stress in California mice (Peromyscus californicus), a model species for studying sex differences in stress responses. In adults, social defeat reduces social approach and increases social vigilance in females but not males. We show this sex difference is absent in juveniles, and that prepubertal castration sensitizes adult males to social defeat. Adult gonadectomy does not alter behavioral responses to defeat, indicating that gonadal hormones act during puberty to program behavioral responses to stress in adulthood. Calcium imaging in the medioventral bed nucleus of the stria terminalis (BNST) showed that social threats increased neural activity and that prepubertal castration generalized these responses to less threatening social contexts. These results support recent hypotheses that the BNST responds to immediate threats. Prepubertal treatment with the nonaromatizable androgen dihydrotestosterone acts in males and females to reduce the effects of defeat on social approach and vigilance in adults. These data indicate that activation of androgen receptors during puberty is critical for programming behavioral responses to stress in adulthood.

Leveraging the unique social organization of California mice to study circuit-specific effects of oxytocin on behavior.

Oxytocin is a versatile neuropeptide that modulates many different forms of social behavior. Recent hypotheses pose that oxytocin enhances the salience of rewarding and aversive social experiences, and the field has been working to identify mechanisms that allow oxytocin to have diverse effects on behavior. Here we review studies conducted on the California mouse (Peromyscus californicus) that shed light on how oxytocin modulates social behavior following stressful experiences. In this species, both males and females exhibit high levels of aggression, which has facilitated the study of how social stress impacts both sexes. We review findings of short- and long-term effects of social stress on the reactivity of oxytocin neurons. We also consider the results of pharmacological studies which show that oxytocin receptors in the bed nucleus of the stria terminalis and nucleus accumbens have distinct but overlapping effects on social approach behaviors. These findings help explain how social stress can have different behavioral effects in males and females, and how oxytocin can have such divergent effects on behavior. Finally, we consider how new technological developments and innovative research programs take advantage of the unique social organization of California mice to address questions that can be difficult to study in conventional rodent model species. These new methods and questions have opened new avenues for studying the neurobiology of social behavior.

Sex differences in responses to aggressive encounters among California mice.

Despite how widespread female aggression is across the animal kingdom, there remains much unknown about its neuroendocrine mechanisms, especially in females that engage in aggression outside the peripartum period. Although the impact of aggressive experience on steroid hormone responses have been described, little is known about the impact of these experiences on female behavior or the subsequent neuropeptide responses to performing aggression. In this study, we compared behavioral responses in both male and female adult California mice based on if they had 0, 1, or 3 aggressive encounters using a resident intruder paradigm. We measured how arginine vasopressin and oxytocin cells in the paraventricular nucleus responded to aggression using c-fos immunohistochemistry. We saw that both sexes disengaged from intruders with repeated aggressive encounters, but that on the final day of testing females were more likely to freeze when they encountered intruders compared to no aggression controls - which was not significant in males. Finally, we saw that percent of arginine vasopressin and c-fos co-localizations in the posterior region of the paraventricular nucleus increased in males who fought compared to no aggression controls. No difference was observed in females. Overall, there is evidence that engaging in aggression induces stress responses in both sexes, and that females may be more sensitive to the effects of fighting.

Extrahypothalamic oxytocin neurons drive stress-induced social vigilance and avoidance.

Oxytocin increases the salience of both positive and negative social contexts and it is thought that these diverse actions on behavior are mediated in part through circuit-specific action. This hypothesis is based primarily on manipulations of oxytocin receptor function, leaving open the question of whether different populations of oxytocin neurons mediate different effects on behavior. Here we inhibited oxytocin synthesis in a stress-sensitive population of oxytocin neurons specifically within the medioventral bed nucleus of the stria terminalis (BNSTmv). Oxytocin knockdown prevented social stress-induced increases in social vigilance and decreases in social approach. Viral tracing of BNSTmv oxytocin neurons revealed fibers in regions controlling defensive behaviors, including lateral hypothalamus, anterior hypothalamus, and anteromedial BNST (BNSTam). Oxytocin infusion into BNSTam in stress naïve mice increased social vigilance and reduced social approach. These results show that a population of extrahypothalamic oxytocin neurons plays a key role in controlling stress-induced social anxiety behaviors.

Oxytocin receptor behavioral effects and cell types in the bed nucleus of the stria terminalis.

Oxytocin is a neuropeptide that can produce anxiolytic effects and promote social approach. However, emerging evidence shows that under some conditions, oxytocin can instead induce anxiety-related behaviors. These diverse effects of oxytocin appear to be mediated by circuit-specific actions. Recent data showed that inhibition of oxytocin receptors (OTRs) in the bed nucleus of the stria terminalis (BNST) was sufficient to increase social approach and decrease social vigilance in female California mice (Peromyscus californicus) exposed to social defeat stress. As a member of the G-protein coupled receptor family, OTRs can induce distinct downstream pathways by coupling to different G-protein isoforms. We show that infusion of carbetocin, a biased OTR-Gq agonist, in the BNST reduced social approach in both female and male California mice. In both females and males, carbetocin also increased social vigilance. To gain insight into cell types that could be mediating this effect, we analyzed previously published single-cell RNAseq data from the BNST and nucleus accumbens (NAc). In the NAc, we and others showed that OTR activation promotes social approach behaviors. In the BNST, Oxtr was expressed in over 40 cell types, that span both posterior and anterior subregions of the BNST. The majority of Oxtr-expressing neurons were GABAergic. In the anterior regions of BNST targeted in our carbetocin experiments, Cyp26b1-expressing neurons had high average Oxtr expression. In the NAc, most Oxtr+ cells were D1 dopamine receptor-expressing neurons and interneurons. These differences in Oxtr cell type distribution may help explain how activation of OTR in BNST versus NAc can have different effects on social approach and social vigilance.

Conserved transcriptomic profiles underpin monogamy across vertebrates.

Social monogamy, typically characterized by the formation of a pair bond, increased territorial defense, and often biparental care, has independently evolved multiple times in animals. Despite the independent evolutionary origins of monogamous mating systems, several homologous brain regions and neuropeptides and their receptors have been shown to play a conserved role in regulating social affiliation and parental care, but little is known about the neuromolecular mechanisms underlying monogamy on a genomic scale. Here, we compare neural transcriptomes of reproductive males in monogamous and nonmonogamous species pairs of Peromyscus mice, Microtus voles, parid songbirds, dendrobatid frogs, and Xenotilapia species of cichlid fishes. We find that, while evolutionary divergence time between species or clades did not explain gene expression similarity, characteristics of the mating system correlated with neural gene expression patterns, and neural gene expression varied concordantly across vertebrates when species transition to monogamy. Our study provides evidence of a universal transcriptomic mechanism underlying the evolution of monogamy in vertebrates.

Convergent neuroendocrine mechanisms of social buffering and stress contagion.

Social interactions play a key role in modulating the impact of stressful experiences. In some cases, social interactions can result in social buffering, the process in which the presence of one individual reduces the physiological and behavioral impact of stress in another individual. On the other hand, there is growing evidence that a key initiating factor of social buffering behaviors is the initiation of an anxiogenic state in the individual that was not directly exposed to the stress. This is referred to as stress contagion (a form of emotion contagion). Both processes involve the transmission of social information, suggesting that contagion and buffering could share similar neural mechanisms. In general, mechanistic studies of contagion and buffering are considered separately, even though behavioral studies show that a degree of contagion is usually necessary for social buffering behaviors to occur. Here we consider the extent to which the neuropeptides corticotropin releasing hormone and oxytocin are involved in contagion and stress buffering. We also assess the importance that frontal cortical areas such as the anterior cingulate cortex and infralimbic cortex play in these behavioral processes. We suggest that further work that directly compares neural mechanisms during stress contagion and stress buffering will be important for identifying what appear to be distinct but overlapping circuits mediating these processes.

Disruption of global hypothalamic microRNA (miR) profiles and associated behavioral changes in California mice (Peromyscus californicus) developmentally exposed to endocrine disrupting chemicals.

Developmental exposure to endocrine disrupting chemicals (EDCs), e.g., bisphenol A (BPA) or genistein (GEN), causes longstanding epigenome effects. MicroRNAs (miRs) regulate which mRNAs will be translated to proteins and thereby serve as the final checkpoint in epigenetic control. Scant amount is known, however, whether EDCs affect neural miRNA (miR) patterns. We aimed to test the hypothesis that developmental exposure of California mice (Peromyscus californicus) to GEN, BPA, or both chemicals influences hypothalamic miR/small RNA profiles and ascertain the extent such biomolecular alterations correlate with behavioral and metabolic changes. California mice were developmentally exposed to GEN (250 mg/kg feed weight, FW), GEN (250 mg/kg FW)+BPA (5 mg/kg FW), low dose (LD) BPA (5 mg/kg FW), or upper dose (UD) BPA (50 mg/kg FW). Adult offspring were tested in a battery of behavioral and metabolic tests; whereupon, mice were euthanized, brains were collected and frozen, small RNAs were isolated from hypothalamic punches, and subsequently sequenced. California mice exposed to one or both EDCs engaged in one or more repetitive behaviors. GEN, LD BPA, and UD BPA altered aspects of ultrasonic and audible vocalizations. Each EDC exposure led to sex-dependent differences in differentially expressed miR/small RNAs with miR7-2, miR146, and miR148a being increased in all female and male EDC exposed groups. Current findings reveal that developmental exposure to GEN and/or BPA affects hypothalamic miR/small RNA expression patterns, and such changes correlate with EDC-induced behavioral and metabolic alterations. miR146 is likely an important mediator and biomarker of EDC exposure in mammals, including humans.

Enriched laboratory housing increases sensitivity to social stress in female California mice (Peromyscus californicus).

Domesticated mice and rats have shown to be powerful model systems for biomedical research, but there are cases in which the biology of species is a poor match for the hypotheses under study. The California mouse (Peromyscus californicus) has unique traits that make it an ideal model for studying biological mechanisms underlying human-relevant behaviors such as intra-female aggression, biparental care, and monogamy. Indeed, peer-reviewed scientific publications using California mouse as a model for behavioral research have more than doubled in the past decade. Critically, behavioral outcomes in captive animals can be profoundly affected by housing conditions, but there is very limited knowledge regarding species-specific housing needs in California mice. Currently, California mouse investigators have to rely on guidelines aimed for more common laboratory species that show vastly different physiology, behavior, and/or ecological niche. This not only could be suboptimal for animals' welfare, but also result in lack of standardization that could potentially compromise experimental reproducibility and replicability across laboratories. With the aim of assessing how different housing systems can affect California mouse behavior both in the home cage as well as the open field and social interaction tests before and after social defeat stress, here we tested three different caging systems: 1. Standard mouse cage, 2. Large cage, and 3. Large cage + environmental enrichment (EE), which focused on increasing vertical complexity based on observations that California mice are semiarboreal in the wild. We found that the effects of housing were largely sex specific: compared to standard cages, in females large + EE reduced home cage stereotypic-like backflipping and rearing behaviors, while large cage increased social interactions. In males, the large+EE cage reduced rearing and digging but did not significantly affect backflipping behavior. Interestingly, while there were no significant differences in the open field and social interaction pre-stress behaviors, large and large+EE housing increased the sensitivity of these tests to detect stress induced phenotypes in females. Together, these results suggest that increasing social and environmental complexity affects home cage behaviors in male and female California mice without interfering with, but rather increasing the magnitude of, the effects of defeat stress on the open field and social interaction tests.

Anxious to see you: Neuroendocrine mechanisms of social vigilance and anxiety during adolescence.

Social vigilance is a behavioral strategy commonly used in adverse or changing social environments. In animals, a combination of avoidance and vigilance allows an individual to evade potentially dangerous confrontations while monitoring the social environment to identify favorable changes. However, prolonged use of this behavioral strategy in humans is associated with increased risk of anxiety disorders, a major burden for human health. Elucidating the mechanisms of social vigilance in animals could provide important clues for new treatment strategies for social anxiety. Importantly, during adolescence the prevalence of social anxiety increases significantly. We hypothesize that many of the actions typically characterized as anxiety behaviors begin to emerge during this time as strategies for navigating more complex social structures. Here, we consider how the social environment and the pubertal transition shape neural circuits that modulate social vigilance, focusing on the bed nucleus of the stria terminalis and prefrontal cortex. The emergence of gonadal hormone secretion during adolescence has important effects on the function and structure of these circuits, and may play a role in the emergence of a notable sex difference in anxiety rates across adolescence. However, the significance of these changes in the context of anxiety is still uncertain, as not enough studies are sufficiently powered to evaluate sex as a biological variable. We conclude that greater integration between human and animal models will aid the development of more effective strategies for treating social anxiety.

The challenge hypothesis revisited: Focus on reproductive experience and neural mechanisms.

Our review focuses on findings from mammals as part of a Special Issue "30th Anniversary of the Challenge Hypothesis". Here we put forth an integration of the mechanisms through which testosterone controls territorial behavior and consider how reproductive experience may alter these mechanisms. The emphasis is placed on the function of socially induced increases in testosterone (T) pulses, which occur in response to social interactions, as elegantly developed by Wingfield and colleagues. We focus on findings from the monogamous California mouse, as data from this species shows that reproductive status is a key factor influencing social interactions, site fidelity, and vigilance for offspring defense. Specifically, we examine differences in T pulses in sexually naïve versus sexually experienced pair bonded males. Testosterone pulses influence processes such as social decision making, the winner-challenge effect, and location preferences through rewarding effects of T. We also consider how social and predatory vigilance contribute to T pulses and how these interactions contribute to a territory centered around maximizing reproduction. Possible underlying mechanisms for these effects include the nucleus accumbens (rewarding effects of testosterone), hippocampus (spatial memories for territories), and the bed nucleus of the stria terminalis (social vigilance). The development of the challenge effect has provided an ideal framework for understanding the complex network of behavioral, environmental, physiological and neural mechanisms that ultimately relates to competition and territoriality across taxa. The opportunity to merge research on the challenge effect using both laboratory and field research to understand social behavior is unparalleled.

Sex differences in the effects of social defeat on brain and behavior in the California mouse: Insights from a monogamous rodent.

Women are nearly twice as likely as men to be diagnosed with major depressive disorder, yet the use of female animal models in studying the biological basis of depression lags behind that of males. The social defeat model uses social stress to generate depression-like symptoms in order to study the neurobiological mechanisms. In general, social defeat is difficult to apply in female rodents. However, male and female California mice (Peromyscus californicus) are territorial. This allows defeat to be studied in both sexes. Males exposed to defeat tend to exhibit proactive coping mechanisms and demonstrate aggression and reduced cognitive flexibility. Females exposed to defeat engage more in reactive coping mechanisms which is highlighted by social avoidance and low aggression. Importantly, effects of defeat on social interaction behavior in females is independent of adult gonadal steroids. These behavioral phenotypes are associated with sex-specific changes in arginine vasopressin (AVP) and oxytocin (OT), closely related peptides that regulate social behavior and stress reactivity. In brain regions associated with stress responses and social behavior, defeat induced long term decreases in AVP activity and increases in OT activity in males and females respectively. Intranasal OT administration was shown to mimic the effects of defeat-induced increases in endogenous OT activity, causing social withdrawal in undefeated females. This suggests that inhibition of OT activity could reduce the impact of stress on behavior in females. These results highlight the value of maintaining diverse rodent models in the search for sex-specific pharmacological approaches to treating mood disorders.

The impact of sex as a biological variable in the search for novel antidepressants.

A roadblock to successful treatment for anxiety and depression is the high proportion of individuals that do not respond to existing treatments. Different underlying neurobiological mechanisms may drive similar symptoms, so a more personalized approach to treatment could be more successful. There is increasing evidence that sex is an important biological variable modulating efficacy of antidepressants and anxiolytics. We review evidence for sex-specific effects of traditional monoamine based antidepressants and newer pharmaceuticals targeting kappa opioid receptors (KOR), oxytocin receptors (OTR), and N-methyl-D-aspartate receptors (ketamine). In some cases, similar behavioral effects are observed in both sexes while in other cases strong sex-specific effects are observed. Most intriguing are cases such as ketamine which has similar behavioral effects in males and females, perhaps through sex-specific neurobiological mechanisms. These results show how essential it is to include both males and females in both clinical and preclinical evaluations of novel antidepressants and anxiolytics.

Determining the biological associates of acute cold pressor post-encoding stress effects on human memory: The role of salivary interleukin-1ß.

Stress generally hurts many aspects of memory, but an interesting finding to emerge from the stress and memory literature is that stress that occurs shortly after learning (i.e., post-encoding stress) usually benefits memory. Although this effect is well established, the biological mechanisms underpinning this effect are not-especially in humans. We addressed this gap in the present study by collecting saliva samples from 80 participants who were randomized to a post-encoding stress (i.e., cold pressor for 3 min) or control task (i.e., warm water for 3 min) and 48 h later completed a recognition memory task. Saliva was collected both prior to and 15 min after the offset of (18 min after the onset of) the stress/control manipulation. Drawing on animal and human work, we examined how five stress-responsive biomarkers-cortisol, salivary α-amylase, progesterone, estradiol, and the proinflammatory cytokine interleukin (IL)-1ß, all assessed in saliva-related to the effects of stress on memory. We found that stress enhanced recollection of negative images and that these effects were selectively related to salivary IL-1ß. Moreover, we found that the beneficial effects of stress on memory were statistically mediated by salivary IL-1ß. We found no robust associations-either linear or quadratic-between memory and any other biomarker, nor did we find significant interactions between biomarkers in predicting memory. These results suggest that immune system activity indexed by salivary IL-1ß may play an important role in contributing to post-encoding stress effects on human memory.

Impaired approach to novelty and striatal alterations in the oxytocin receptor deficient mouse model of autism.

Long-standing studies established a role for the oxytocin system in social behavior, social reward, pair bonding and affiliation. Oxytocin receptors, implicated in pathological conditions affecting the social sphere such as autism spectrum disorders, can also modulate cognitive processes, an aspect generally overlooked. Here we examined the effect of acute (pharmacological) or genetic (Oxtr-/-) inactivation of oxytocin receptor-mediated signaling, in male mice, in several cognitive tests. In the novel object recognition test, both oxytocin receptor antagonist treated wild type animals and Oxtr-/- mice lacked the typical preference for novelty. Oxtr-/- mice even preferred the familiar object; moreover, their performance in the Morris water maze did not differ from wild types, suggesting that oxytocin receptor inactivation did not disrupt learning. Because the preference for novel objects could be rescued in Oxtr-/- mice with longer habituation periods, we propose that the loss of novelty preferences following Oxtr inactivation is due to altered processing of novel contextual information. Finally, we observed an increased expression of excitatory synaptic markers in the striatum of Oxtr-/- mice and a greater arborization and higher number of spines/neuron in the dorsolateral area of this structure, which drives habit formation. Our data also indicate a specific reshaping of dorsolateral striatal spines in Oxtr-/- mice after exposure to a novel environment, which might subtend their altered approach to novelty, and support previous work pointing at this structure as an important substrate for autistic behaviors.

Effects of social defeat on paternal behavior and pair bonding behavior in male California mice (Peromyscus californicus).

Male parental care is an important social behavior for several mammalian species. Psychosocial stress is usually found to inhibit maternal behavior, but effects on paternal behavior have been less consistent. We tested the effects of social defeat stress on pair bond formation and paternal behavior in the monogamous California mouse (Peromyscus californicus). Social defeat reduced time spent in a chamber with a stranger female during a partner preference test conducted 24h after pairing, but increased latency to the first litter. In 10min partner preference tests conducted after the birth of pups, both control and stressed males exhibited selective aggression towards stranger females. Unlike prairie voles, side by side contact was not observed in either partner preference test. Stressed male California mice engaged in more paternal behavior than controls and had reduced anxiety-like responses in the open-field test. Defeat stress enhanced prodynorphin and KOR expression in the medial preoptic area (MPOA) but not PVN. Increased KOR signaling has been linked to increased selective aggression in prairie voles. Together the results show that defeat stress enhances behaviors related to parental care and pair bonding in male California mice.

Stress, sex, and motivated behaviors.

Stress is a major risk factor for development of psychiatric disorders such as depression and development of substance use disorder. Although there are important sex differences in the prevalence of these disorders, most preclinical models used to study stress-induced disorders have used males only. Social defeat stress is a commonly used method to induce stress in an ethologically relevant way but has only recently begun to be used in female rodents. Using these new female models, recent studies have examined how social defeat stress affects males and females differently at the behavioral, circuit, and molecular levels. This Mini-Review discusses sex differences in the effects of social defeat stress on social behavior and drug-seeking behavior as well as its impact on the mesolimbic dopamine system and the highly connected region of the bed nucleus of the stria terminalis. © 2016 Wiley Periodicals, Inc.

What can animal research tell us about the link between androgens and social competition in humans?

A contribution to a special issue on Hormones and Human Competition. The relationship between androgenic hormones, like testosterone (T), and aggression is extensively studied in human populations. Yet, while this work has illuminated a variety of principals regarding the behavioral and phenotypic effects of T, it is also hindered by inherent limitations of performing research on people. In these instances, animal research can be used to gain further insight into the complex mechanisms by which T influences aggression. Here, we explore recent studies on T and aggression in numerous vertebrate species, although we focus primarily on males and on a New World rodent called the California mouse (Peromyscus californicus). This species is highly territorial and monogamous, resembling the modern human social disposition. We review (i) how baseline and dynamic T levels predict and/or impact aggressive behavior and disposition; (ii) how factors related to social and physical context influence T and aggression; (iii) the reinforcing or "rewarding" aspects of aggressive behavior; and (iv) the function of T on aggression before and during a combative encounter. Included are areas that may need further research. We argue that animal studies investigating these topics fill in gaps to help paint a more complete picture of how androgenic steroids drive the output of aggressive behavior in all animals, including humans.

Acute stress impairs cognitive flexibility in men, not women.

Psychosocial stress influences cognitive abilities, such as long-term memory retrieval. However, less is known about the effects of stress on cognitive flexibility, which is mediated by different neurobiological circuits and could thus be regulated by different neuroendocrine pathways. In this study, we randomly assigned healthy adults to an acute stress induction or control condition and subsequently assessed participants' cognitive flexibility using an open-source version of the Wisconsin Card Sort task. Drawing on work in rodents, we hypothesized that stress would have stronger impairing effects on cognitive flexibility in men than women. As predicted, we found that stress impaired cognitive flexibility in men but did not significantly affect women. Our results thus indicate that stress exerts sex-specific effects on cognitive flexibility in humans and add to the growing body of research highlighting the need to consider sex differences in effects of stress.

Rapid effects of estrogens on behavior: environmental modulation and molecular mechanisms.

Estradiol can modulate neural activity and behavior via both genomic and nongenomic mechanisms. Environmental cues have a major impact on the relative importance of these signaling pathways with significant consequences for behavior. First we consider how photoperiod modulates nongenomic estrogen signaling on behavior. Intriguingly, short days permit rapid effects of estrogens on aggression in both rodents and song sparrows. This highlights the importance of considering photoperiod as a variable in laboratory research. Next we review evidence for rapid effects of estradiol on ecologically-relevant behaviors including aggression, copulation, communication, and learning. We also address the impact of endocrine disruptors on estrogen signaling, such as those found in corncob bedding used in rodent research. Finally, we examine the biochemical mechanisms that may mediate rapid estrogen action on behavior in males and females. A common theme across these topics is that the effects of estrogens on social behaviors vary across different environmental conditions.