A vasopressin circuit that modulates mouse social investigation and anxiety-like behavior in a sex-specific manner.

One of the largest sex differences in brain neurochemistry is the expression of the neuropeptide arginine vasopressin (AVP) within the vertebrate brain, with males having more AVP cells in the bed nucleus of the stria terminalis (BNST) than females. Despite the long-standing implication of AVP in social and anxiety-like behaviors, the circuitry underlying AVP's control of these behaviors is still not well defined. Using optogenetic approaches, we show that inhibiting AVP BNST cells reduces social investigation in males, but not in females, whereas stimulating these cells increases social investigation in both sexes, but more so in males. These cells may facilitate male social investigation through their projections to the lateral septum (LS), an area with the highest density of sexually differentiated AVP innervation in the brain, as optogenetic stimulation of BNST AVP → LS increased social investigation and anxiety-like behavior in males but not in females; the same stimulation also caused a biphasic response of LS cells ex vivo. Blocking the vasopressin 1a receptor (V1aR) in the LS eliminated all these responses. Together, these findings establish a sexually differentiated role for BNST AVP cells in the control of social investigation and anxiety-like behavior, likely mediated by their projections to the LS.

A vasopressin circuit that modulates sex-specific social interest and anxiety-like behavior in mice.

One of the largest sex differences in brain neurochemistry is the male-biased expression of the neuropeptide arginine vasopressin (AVP) within the vertebrate social brain. Despite the long-standing implication of AVP in social and anxiety-like behavior, the precise circuitry and anatomical substrate underlying its control are still poorly understood. By employing optogenetic manipulation of AVP cells within the bed nucleus of the stria terminalis (BNST), we have unveiled a central role for these cells in promoting social investigation, with a more pronounced role in males relative to females. These cells facilitate male social investigation and anxiety-like behavior through their projections to the lateral septum (LS), an area with the highest density of sexually-dimorphic AVP fibers. Blocking the vasopressin 1a receptor (V1aR) in the LS eliminated stimulation-mediated increases in these behaviors. Together, these findings establish a distinct BNST AVP → LS V1aR circuit that modulates sex-specific social interest and anxiety-like behavior.

Sex differences in afferents and efferents of vasopressin neurons of the bed nucleus of the stria terminalis and medial amygdala in mice.

Steroid-sensitive vasopressin (AVP) neurons in the bed nucleus of the stria terminalis (BNST) and medial amygdala (MeA) have been implicated in the control of social behavior, but the connectional architecture of these cells is not well understood. Here we used a modified rabies virus (RV) approach to identify cells that provide monosynaptic input to BNST and MeA AVP cells, and an adeno-associated viral (AAV) anterograde tracer strategy to map the outputs of these cells. Although the location of in- and outputs of these cells generally overlap, we observed several sex differences with differences in density of outputs typically favoring males, but the direction of sex differences in inputs vary based on their location. Moreover, the AVP cells located in both the BNST and MeA are in direct contact with each other suggesting that AVP cells in these two regions act in a coordinated manner, and possibly differently by sex. This study represents the first comprehensive mapping of the sexually dimorphic and steroid-sensitive AVP neurons in the mouse brain.

Modulation of social behavior by distinct vasopressin sources.

The neuropeptide arginine-vasopressin (AVP) is well known for its peripheral effects on blood pressure and antidiuresis. However, AVP also modulates various social and anxiety-related behaviors by its actions in the brain, often sex-specifically, with effects typically being stronger in males than in females. AVP in the nervous system originates from several distinct sources which are, in turn, regulated by different inputs and regulatory factors. Based on both direct and indirect evidence, we can begin to define the specific role of AVP cell populations in social behavior, such as, social recognition, affiliation, pair bonding, parental behavior, mate competition, aggression, and social stress. Sex differences in function may be apparent in both sexually-dimorphic structures as well as ones without prominent structural differences within the hypothalamus. The understanding of how AVP systems are organized and function may ultimately lead to better therapeutic interventions for psychiatric disorders characterized by social deficits.

Gut Barrier Dysfunction and Type 2 Immunity: Implications for Compulsive Behavior.

To date, much of the focus of gut-brain axis research has been on gut microbiota regulation of anxiety and stress-related behaviors. Much less attention has been directed to potential connections between gut microbiota and compulsive behavior. Here, we discuss a potential link between gut barrier dysfunction and compulsive behavior that is mediated through "type 2" rather than "type 1" inflammation. We examine connections between compulsive behavior and type 2 inflammation in Tourette syndrome, obsessive-compulsive disorder, autism, addiction, and post-traumatic stress disorder. Next, we discuss potential connections between gut barrier dysfunction, type 2 inflammation, and compulsive behavior. We posit a potential mechanism whereby gut barrier dysfunction-associated type 2 inflammation may drive compulsive behavior through histamine regulation of dopamine neurotransmission. Finally, we discuss the possibility of exploiting the greater accessibility of the gut relative to the brain in identifying targets to treat compulsive behavior disorders.

Oxytocin, Vasopressin, and Social Behavior: From Neural Circuits to Clinical Opportunities.

Oxytocin and vasopressin are peptide hormones secreted from the pituitary that are well known for their peripheral endocrine effects on childbirth/nursing and blood pressure/urine concentration, respectively. However, both peptides are also released in the brain, where they modulate several aspects of social behaviors. Oxytocin promotes maternal nurturing and bonding, enhances social reward, and increases the salience of social stimuli. Vasopressin modulates social communication, social investigation, territorial behavior, and aggression, predominantly in males. Both peptides facilitate social memory and pair bonding behaviors in monogamous species. Here we review the latest research delineating the neural circuitry of the brain oxytocin and vasopressin systems and summarize recent investigations into the circuit-based mechanisms modulating social behaviors. We highlight research using modern molecular genetic technologies to map, monitor activity of, or manipulate neuropeptide circuits. Species diversity in oxytocin and vasopressin effects on social behaviors are also discussed. We conclude with a discussion of the translational implications of oxytocin and vasopressin for improving social functioning in disorders with social impairments, such as autism spectrum disorder.

Knockdown of sexually differentiated vasopressin expression in the bed nucleus of the stria terminalis reduces social and sexual behaviour in male, but not female, mice.

The neuropeptide arginine-vasopressin (AVP) has long been implicated in the regulation of social behaviour and communication, but the sources of AVP release relevant for behaviour have not been precisely determined. Ablations of the sexually dimorphic AVP cells within the bed nucleus of the stria terminalis (BNST), which are more numerous in males, affect social behaviour differently in males and females. However, it is unknown whether these behavioural effects are caused by a reduction of AVP or of other factors associated with these cells. To test the role of AVP specifically, we used an shRNA viral construct to knock down AVP gene expression within the BNST of wild-type male and female mice, using scrambled sequence virus as a control, and evaluated subsequent changes in social behaviours (social investigation, ultrasonic vocalization (USV), scent marking, copulation, and aggression), or anxiety-like behaviours (elevated plus maze). We observed that, in males, knockdown of AVP expression in the BNST strongly reduced investigation of novel males, aggressive signalling towards other males (tail rattling, USV), and copulatory behaviour, but did not alter attack initiation, other measures of social communication, or anxiety-like behaviours. In females, however, BNST AVP knockdown did not alter any of these behaviours. These results point to differential involvement of AVP derived from the BNST in social behaviour.

Reduction in vasopressin cells in the suprachiasmatic nucleus in mice increases anxiety and alters fluid intake.

Central vasopressin (AVP) has been implicated in the control of multiple behaviors, including social behavior, anxiety-like behavior, and sickness behavior. The extent to which the different AVP-producing cell groups contribute to regulating these behaviors has not been extensively investigated. Here we test the role of AVP cells in the suprachiasmatic nucleus (SCN) in these behaviors by ablating these cells using viral-mediated, Cre-dependent caspase in male and female AVP-Cre + mice and Cre-controls. We compared anxiety and social behaviors, as well as sickness behaviors (lethargy, anhedonia (indexed by sucrose consumption), and changes in anxiety-like- and social behavior) induced via injection of bacterial lipopolysaccharide (LPS). We found that SCN AVP cell ablation increased anxiety-like behavior and sucrose consumption in both sexes, as well as increased urine marking by males in a non-social context, but did not alter behavioral responses to sickness. Our data suggest that SCN AVP does not strongly affect LPS-induced behavioral changes, but may contribute to anxiety-like behavior, and may play a role in ingestive reward/motivation and fluid intake.

Removal of vasopressin cells from the paraventricular nucleus of the hypothalamus enhances lipopolysaccharide-induced sickness behaviour in mice.

Vasopressin (AVP) cells in the paraventricular nucleus of the hypothalamus (PVN) are activated during sickness and project to multiple nuclei responsible for the anxiety, social and motivated behaviours affected during sickness, suggesting that these cells may play a role in sickness behaviours, typically expressed as reduced mobility, increased anxiety, anhedonia and social withdrawal. In the present study, we selectively ablated AVP neurones in the PVN of male and female mice (Mus musculus) and induced sickness behaviour via injection of bacterial lipopolysaccharide (LPS). We found that PVN AVP ablation increased the effects of LPS, specifically by further decreasing sucrose preference in males and females and decreasing the social preference of males, monitored within 24 hours of LPS injection. These results suggest that PVN AVP contributes to the change in motivated behaviours during sickness and may help promote recovery from infection..

Sex Differences in the Control of Social Investigation and Anxiety by Vasopressin Cells of the Paraventricular Nucleus of the Hypothalamus.

The neuropeptide arginine-vasopressin (AVP) has long been implicated in the regulation of social behavior and communication in diverse taxa, but the source of AVP release relevant for behavior has not been precisely determined. Potential sources include hypothalamic cell populations such as the paraventricular (PVN), supraoptic, and suprachiasmatic nuclei, as well as extrahypothalamic cell groups in the extended amygdala. To address if AVP-expressing cells in the PVN are important for mouse social communication, we deleted PVN AVP-expressing cells using viral-mediated delivery of Cre-dependent caspase-9 cell death construct into the PVN of AVP-Cre-positive mice (expressing Cre-recombinase under the control of the AVP promoter) or AVP-Cre-negative littermate controls, and assessed their levels of social investigation, social communication, anxiety, sex behavior, and aggressive behavior. We found that these lesions increased social investigation in females, but not in males. However, in males but not in females, these lesions increased non-social anxiety-related behaviors in the elevated-plus maze. These results therefore point at differential involvement of PVN AVP-expressing cells in the context of social and emotional behavior in the two sexes, which may contribute to sex differences in social communication and anxiety disorders.

Sex and gender: modifiers of health, disease, and medicine.

Clinicians can encounter sex and gender disparities in diagnostic and therapeutic responses. These disparities are noted in epidemiology, pathophysiology, clinical manifestations, disease progression, and response to treatment. This Review discusses the fundamental influences of sex and gender as modifiers of the major causes of death and morbidity. We articulate how the genetic, epigenetic, and hormonal influences of biological sex influence physiology and disease, and how the social constructs of gender affect the behaviour of the community, clinicians, and patients in the health-care system and interact with pathobiology. We aim to guide clinicians and researchers to consider sex and gender in their approach to diagnosis, prevention, and treatment of diseases as a necessary and fundamental step towards precision medicine, which will benefit men's and women's health.

Sexually dimorphic role of BNST vasopressin cells in sickness and social behavior in male and female mice.

Circumstantial evidence supports the hypothesis that the sexually dimorphic vasopressin (AVP) innervation of the brain tempers sickness behavior in males. Here we test this hypothesis directly, by comparing sickness behavior in animals with or without ablations of BNST AVP cells, a major source of sexually dimorphic AVP in the brain. We treated male and female AVP-iCre+ and AVP-iCre- mice that had been injected with viral Cre-dependent caspase-3 executioner construct into the BNST with lipopolysaccharide (LPS) or sterile saline, followed by behavioral analysis. In all groups, LPS treatment reliably reduced motor behavior, increased anxiety-related behavior, and reduced sucrose preference and consumption. Male mice, whose BNST AVP cells had been ablated (AVP-iCre+), displayed only minor reductions in LPS-induced sickness behavior, whereas their female counterparts displayed, if anything, an increase in sickness behaviors. All saline-treated mice with BNST AVP cell ablations consumed more sucrose than did control mice, and males, but not females, with BNST AVP cell ablations showed reduced preference for novel conspecifics compared to control mice. These data confirm that BNST AVP cells control social behavior in a sexually dimorphic way, but do not play a critical role in altering sickness behavior.

Associations of the Fecal Microbial Proteome Composition and Proneness to Diet-induced Obesity.

Consumption of refined high-fat, low-fiber diets promotes development of obesity and its associated consequences. Although genetics play an important role in dictating susceptibility to such obesogenic diets, mice with nearly uniform genetics exhibit marked heterogeneity in their extent of obesity in response to such diets. This suggests non-genetic determinants play a role in diet-induced obesity. Hence, we sought to identify parameters that predict, and/or correlate with, development of obesity in response to an obesogenic diet. We assayed behavior, metabolic parameters, inflammatory markers/cytokines, microbiota composition, and the fecal metaproteome, in a cohort of mice (n = 50) prior to, and the 8 weeks following, administration of an obesogenic high-fat low-fiber diet. Neither behavioral testing nor quantitation of inflammatory markers broadly predicted severity of diet-induced obesity. Although, the small subset of mice that exhibited basal elevations in serum IL-6 (n = 5) were among the more obese mice in the cohort. While fecal microbiota composition changed markedly in response to the obesogenic diet, it lacked the ability to predict which mice were relative prone or resistant to obesity. In contrast, fecal metaproteome analysis revealed functional and taxonomic differences among the proteins associated with proneness to obesity. Targeted interrogation of microbiota composition data successfully validated the taxonomic differences seen in the metaproteome. Although future work will be needed to determine the breadth of applicability of these associations to other cohorts of animals and humans, this study nonetheless highlights the potential power of gut microbial proteins to predict and perhaps impact development of obesity.

Dietary emulsifiers consumption alters anxiety-like and social-related behaviors in mice in a sex-dependent manner.

Dietary emulsifiers carboxylmethylcellulose (CMC) and polysorbate 80 (P80) alter the composition of the intestinal microbiota and induce chronic low-grade inflammation, ultimately leading to metabolic dysregulations in mice. As both gut microbiota and intestinal health can influence social and anxiety-like behaviors, we investigated whether emulsifier consumption would detrimentally influence behavior. We confirmed that emulsifier exposure induced chronic intestinal inflammation, increased adiposity, and altered gut microbiota composition in both male and female mice, although the specific microboal taxa altered following emulsifier consumption occurred in a sex-dependent manner. Importantly, emulsifier treatment altered anxiety-like behaviors in males and reduced social behavior in females. It also changed expression of neuropeptides implicated in the modulation of feeding as well as social and anxiety-related behaviors. Multivariate analyses revealed that CMC and P80 produced distinct clustering of physiological, neural, and behavioral effects in male and female mice, suggesting that emulsifier treatment leads to a syndrome of sex-dependent changes in microbiota, physiology, and behavior. This study reveals that these commonly used food additives may potentially negatively impact anxiety-related and social behaviors and may do so via different mechanisms in males and females.

Defining Dysbiosis in Disorders of Movement and Motivation.

The gut microbiota has emerged as a critical player in shaping and modulating brain function and has been shown to influence numerous behaviors, including anxiety and depression-like behaviors, sociability, and cognition. However, the effects of the gut microbiota on specific disorders associated with thalamo-cortico-basal ganglia circuits, ranging from compulsive behavior and addiction to altered sensation and motor output, are only recently being explored. Wholesale depletion and alteration of gut microbial communities in rodent models of disorders, such as Parkinson's disease, autism, and addiction, robustly affect movement and motivated behavior. A new frontier therefore lies in identifying specific microbial alterations that affect these behaviors and understanding the underlying mechanisms of action. Comparing alterations in gut microbiota across multiple basal-ganglia associated disease states allows for identification of common mechanistic pathways that may interact with distinct environmental and genetic risk factors to produce disease-specific outcomes.

Dissociation of Puberty and Adolescent Social Development in a Seasonally Breeding Species.

Alongside the development of sexual characteristics and reproductive competence, adolescents undergo marked cognitive, social, and emotional development [1]. A fundamental question is whether these changes are triggered by activation of the hypothalamic-pituitary-gonadal (HPG) axis at puberty (puberty dependent) or whether they occur independently of HPG activation (puberty independent). Disentangling puberty-dependent from puberty-independent mechanisms is difficult because puberty and adolescence typically proceed concurrently. Here, we test a new approach that leverages natural adaptations of a seasonally breeding species to dissociate pubertal status from chronological age. Siberian hamsters (Phodopus sungorus) reared in a long, summer-like day length (LD) exhibit rapid pubertal development, whereas those reared in a short, winter-like day length (SD) delay puberty by several months to synchronize breeding with the following spring [2, 3]. We tested whether the SD-induced delay in puberty delays the peri-adolescent decline in juvenile social play and the rise in aggression that characterizes adolescent social development in many species [4-6] and compared the results to those obtained after prepubertal gonadectomy. Neither SD rearing nor prepubertal gonadectomy altered the age at which hamsters transitioned from play to aggression; SD-reared hamsters completed this transition prior to puberty. SD rearing and prepubertal gonadectomy, however, increased levels of play in male and female juveniles, implicating a previously unknown role for prepubertal gonadal hormones in juvenile social behavior. Levels of aggression were also impacted (decreased) in SD-reared and gonadectomized males. These data demonstrate that puberty-independent mechanisms regulate the timing of adolescent social development, while prepubertal and adult gonadal hormones modulate levels of age-appropriate social behaviors.

Effects of gut-derived endotoxin on anxiety-like and repetitive behaviors in male and female mice.

BACKGROUND: Gut dysbiosis is observed in several neuropsychiatric disorders exhibiting increases in anxiety behavior, and recent work suggests links between gut inflammation and such disorders. One source of this inflammation may be lipopolysaccharide (LPS), a toxic component of gram-negative bacteria. Here, we (1) determine whether oral gavage of LPS, as a model of gut-derived endotoxemia, affects anxiety-like and/or repetitive behaviors; (2) test whether these changes depend on TLR4 signaling; and (3) test the extent to which gut-derived endotoxin and TLR4 antagonism affects males and females differently. METHODS: In experiment 1, male wild-type (WT) and Tlr4-/- mice were tested for locomotor, anxiety-like, and repetitive behaviors in an automated open field test apparatus, 2 h after oral gavage of LPS or saline. In experiment 2, male and female WT mice received an oral gavage of LPS and an injection of one or two TLR4 antagonists that target different TLR4 signaling pathways ((+)-naloxone and LPS derived from R. sphaeroides (LPS-RS)). Univariate and multivariate analyses were used to identify effects of treatment, sex, and genotype and their interaction. RESULTS: In experiment 1, oral gavage of LPS increased anxiety-like behavior in male WT mice but not in Tlr4-/- mice. In experiment 2, oral gavage of LPS increased anxiety-like and decreased repetitive behaviors in WT mice of both sexes. Neither antagonist directly blocked the effects of orally administered LPS. However, treatment with (+)-naloxone, which blocks the TRIF pathway of TLR4, had opposing behavioral effects in males and females (independent of LPS treatment). We also identified sex differences in the expression of interleukin-6, a pro-inflammatory cytokine, in the gut both in basal conditions and in response to LPS. CONCLUSION: In spite of the ubiquitous nature of LPS in the gut lumen, this is the first study to demonstrate that intestinally derived LPS can initiate behavioral aspects of the sickness response. While an increased enteric load of LPS increases anxiety-like behavior in both sexes, it likely does so via sex-specific mechanisms. Similarly, TLR4 signaling may promote baseline expression of repetitive behavior differently in males and females. This study lays the groundwork for future interrogations into connections between gut-derived endotoxin and behavioral pathology in males and females.

Vasopressin deletion is associated with sex-specific shifts in the gut microbiome.

Brattleboro rats harbor a spontaneous deletion of the arginine-vasopressin (Avp) gene. In addition to diabetes insipidus, these rats exhibit low levels of anxiety and depressive behaviors. Recent work on the gut-brain axis has revealed that gut microbiota can influence anxiety behaviors. Therefore, we studied the effects of Avp gene deletion on gut microbiota. Since Avp gene expression is sexually different, we also examined how Avp deletion affects sex differences in gut microbiota. Males and females show modest but differentiated shifts in taxa abundance across 3 separate Avp deletion genotypes: wildtype (WT), heterozygous (Het) and AVP-deficient Brattleboro (KO) rats. For each sex, we found examples of taxa that have been shown to modulate anxiety behavior, in a manner that correlates with anxiety behavior observed in homozygous knockout Brattleboro rats. One prominent example is Lactobacillus, which has been reported to be anxiolytic: Lactobacillus was found to increase in abundance in inverse proportion to increasing gene dosage (most abundant in KO rats). This genotype effect of Lactobacillus abundance was not found when females were analyzed independently. Therefore, Avp deletion appears to affect microbiota composition in a sexually differentiated manner.

Atypical Social Development in Vasopressin-Deficient Brattleboro Rats.

Over the past 3 decades, a large body of evidence has accumulated demonstrating that the neuropeptide arginine vasopressin (AVP) plays a critical role in regulating social behavior. The overwhelming majority of this evidence comes from adults, leaving a gap in our understanding of the role of AVP during development. Here, we investigated the effect of chronic AVP deficiency on a suite of juvenile social behaviors using Brattleboro rats, which lack AVP due to a mutation in the Avp gene. Social play behavior, huddling, social investigation & allogrooming, and ultrasonic vocalizations (USVs) of male and female rats homozygous for the Brattleboro mutation (Hom) were compared with their wild-type (WT) and heterozygous (Het) littermates during same-sex, same-genotype social interactions. Male and female Hom juveniles exhibited less social play than their Het and WT littermates throughout the rise, peak, and decline of the developmental profile of play. Hom juveniles also emitted fewer prosocial 50 kHz USVs, and spectrotemporal characteristics (call frequency and call duration) of individual call types differed from those of WT and Het juveniles. However, huddling behavior was increased in Hom juveniles, and social investigation and 22 kHz USVs did not differ across genotypes, demonstrating that not all social interactions were affected in the same manner. Collectively, these data suggest that the Avp gene plays a critical role in juvenile social development.

Evaluating sex as a biological variable in preclinical research: the devil in the details.

Translating policy into action is a complex task, with much debate surrounding the process whereby US and Canadian health funding agencies intend to integrate sex and gender science as an integral component of methodological rigor and reporting in health research. Effective January 25, 2016, the US National Institutes of Health implemented a policy that expects scientists to account for the possible role of sex as a biological variable (SABV) in vertebrate animal and human studies. Applicants for NIH-funded research and career development awards will be asked to explain how they plan to factor consideration of SABV into their research design, analysis, and reporting; strong justification will be required for proposing single-sex studies. The Canadian Institutes of Health Research is revising their peer review accreditation process to ensure that peer reviewers are skilled in applying a critical lens to protocols that should be incorporating sex and gender science. The current paper outlines the components that peer reviewers in North America will be asked to assess when considering whether SABV is appropriately integrated into research designs, analyses, and reporting. Consensus argues against narrowly defining rules of engagement in applying SABV, with criteria provided for reviewers as guidance only. Scores will not be given for each criterion; applications will be judged on the overall merit of scientific innovation, rigor, reproducibility, and potential impact.