Brian C. Trainor - US grants

DESCRIPTION (provided by applicant): Many of the behavioral effects of testosterone occur after conversion to estradiol by the aromatase enzyme. Although there has been interest in the role of aromatase as a regulator of male mating behavior, relatively little research has considered whether aromatase regulates other social behaviors. This application proposes to examine how aromatase mediates the effects of testosterone on paternal behavior and inter-male aggression in the monogamous California mouse, a species in which males show high levels of both paternal behavior and aggression. First, the proposed research will test whether the positive effect of testosterone on paternal behavior in this species occurs through conversion to estradiol. Second, this research will examine whether increased testosterone after winning an aggressive encounter increases aggression, and whether aromatase mediates such an effect. Finally, this research will determine if central aromatase activity is correlated with paternal and aggressive experience. Understanding how aromatase modulates behavioral effects of T has potentially important implications both for our understanding of the neuroendocrine control of mammalian social behavior and for human health. Testosterone replacement therapy in humans is becoming increasingly common, and most studies restrict behavioral analyses to sexual functions, mood, and cognition. In summary, this proposal examines how aromatase mediates the effects of testosterone and social behavior.

DESCRIPTION (provided by applicant): One of the most important scientific advances in the post-genomic age has been the elucidation of gene and environment interactions. The existence of these interactions has been well-established, but only within the past decade could the details of these interactions be examined at a molecular level. This application proposes to examine the effects of day length on estrogen receptor (ER) expression, aggression, and mating behavior in beach mice. First, the effects of long days and short days on ERalpha and ERbeta protein and mRNA will be examined. Second, the functional consequences of ER regulation will be examined with hormone manipulation experiments that will test the effects of ERalpha and ERbeta on aggression and mating behavior in long days and short days. Finally, this research will examine how ER positive cells respond in different social contexts. Importantly, the behavioral responses of the beach mouse resemble those of humans in that behavioral changes are observed across seasons, but reproductive activity is not suppressed by short days. There is a growing body of clinical research which indicates that estrogenic function has important effects on aggressive behavior and mental disorders. The proposed research should provide a novel and important framework to understand how the environment interacts with ER function to modulate behavior.

DESCRIPTION (provided by applicant): Estrogens affect a wide variety of processes in the brain and many aspects of behavior including social interactions, reproduction, and memory. A variety of estrogen receptors (ER) and signaling pathways make up a complex regulatory network that produces intriguing examples of functional plasticity. For example, at ovulation estrogens switch from exerting negative feedback on gonadotropin releasing hormone to exerting positive feedback. Negative feedback is mediated in part by rapid acting nongenomic effects of estrogens whereas positive feedback relies on a sustained increase in estrogens that apparently drives changes in gene expression. Recent studies on Peromyscus identified a similar pattern for male behavior. Estrogens decrease aggressive behavior in mice housed in long days (16L:8D) but increase aggressive behavior in mice housed in short days (8L:16D). This functional plasticity appears to be mediated by a fundamental change in the down-stream effects of ERs. In long days, hormone manipulations affect behavior only after 10 days. Microarray analyses showed increased estrogen-regulated transcription in the brain under long days compared to short days. This suggests that estrogens may decrease aggression by driving transcription. In short day's estradiol injections increase aggression within 15 minutes, suggesting these effects are mediated by nongenomic pathways. This proposal, submitted by a new investigator, examines how a photoperiod modulates the effects of estrogens on aggression. Using behavioral, cellular, and molecular analyses of aggressive behavior we outline how the different environments can induce functional plasticity in estrogen regulated behavior. Dysregulated aggression is a component of mental disorders including bipolar disorder, schizophrenia, and borderline personality disorder. Correlational and clinical trial data suggest that estrogens affect aggressive behavior in men and women. However, virtually all studies examining the effects of estrogens on human behavior utilize either peripheral hormone manipulations or measurements. There is growing appreciation that steroid hormones are synthesized de novo in brain regions such as the hypothalamus and hippocampus. Recent data show estrogen synthesis in the brain is modulated by social interactions on a moment-to- moment basis. This strongly suggests that rapid actions of estrogens synthesized in the brain may be of critical important for behavior. These observations require us to reassess how we view the relationships between estrogens and behavior in humans, because the vast majority of human studies only consider estrogens derived from gonadal hormones. In our studies we can investigate both slow (genomic) and rapid (nongenomic) mechanisms of aggression simply by manipulating photoperiod, giving us a unique opportunity to examine how estrogens interact with the environment to affect behavior. We hypothesize that the differential effects of estrogens on aggression are mediated by differences in genomic and nongenomic activation. In the first specific aim we will confirm whether estrogens increase aggression by acting nongenomically. In the second specific aim we will use immunohistochemistry and western blots to identify intracellular signaling pathways that could mediate the rapid effects of estrogens on aggression. Finally we will use hormone manipulations and real-time PCR to test whether melatonin inhibits estrogen-dependent gene expression in the brain, thereby blocking genomic action in short-day mice. The proposed research will identify intracellular signaling pathways involved in regulating aggression and should provide insights for developing new strategies for managing exaggerated aggressive behaviors. PUBLIC HEALTH RELEVANCE: Estrogens affect a wide variety of processes in the brain and many aspects of behavior including social interactions, reproduction, and memory. The amount of light a male California mouse is exposed to each day determines whether estrogens increase or decrease aggression. The proposed research will investigate the cellular mechanisms that underlie this gene-environment interaction that affects a behavior associated with many mental disorders.

DESCRIPTION (provided by applicant): Major depression affects more than 10 percent of the US population. There are several treatment options available, but many individuals do not respond to therapy, medication or both. Despite agreement that neurotransmitters such as serotonin are important, it is clear that the mechanisms contributing to depression are considerably more complex than a simple deficit of serotonin function. One important feature of depression is that women are almost twice as likely as men to be affected by depression. Anxiety disorders are also more prevalent in women. For a variety of reasons however, most animal models examining neurobiological mechanisms related to depression focus on males. Indeed, there is an urgent need for the development of model systems in which behaviors related to depression and anxiety can be studied in both sexes (Wizemann and Pardue 2001). The chronic mild stress procedure can be applied in both male and female rodents, and this paradigm induces anhedonia (loss of interest in a rewarding stimulus). However, some laboratories have reported difficulty in replicating the effects of chronic mild stress on behavior. In contrast, the social stress (subordination) paradigm produces repeatable results in laboratory groups around the world. Social stress induces pronounced behavioral changes including anhedonia and a marked increase in social avoidance (or withdrawal). The behavioral effects of social stress are reversed by chronic, but not acute, antidepressant treatment. This is relevant because chronic, but not acute antidepressant treatment is effective in treating affective disorders in humans. The overwhelming majority of studies using social stress have focused on males. This is because in most species of rodents, female aggression is minimal, so it is difficult to create social stress using intra-female aggression. In contrast, female California mice (Peromyscus californicus) are aggressive, as males and females defend territories. In addition, preliminary data show that females have larger corticosterone responses than males during resident-intruder aggression tests. Insights into idiopathic depression, we expect it will be useful for testing mechanistic. Although our model may not provide hypotheses related to stress-induced depression. This application proposes to use the unique biology of the California mouse to examine sex differences in neurobiological mechanisms that mediate the effects of social stress on affective behaviors. PUBLIC HEALTH RELEVANCE: Affective disorders are more likely to occur in women, yet most mouse models focus on males in part due to logistical issues. We propose to use the social stress paradigm to examine social withdrawal and anhedonia in male and female California mice. We will examine the effects of social stress on behavior and expression of brain derived neurotrophic factor.

Psychosocial stress is an important factor contributing to the onset of mood and anxiety disorders. Depression and anxiety disorders are more common in women than men, and there are important sex differences in responses to psychosocial stress. These data suggest that sex differences in responses to stress could mediate population-level sex differences in mental disorders. The social defeat paradigm reliably induces social withdrawal responses in animal models, a symptom that is present in both mood and anxiety disorders. However, a stumbling block to studying sex differences using this approach comes down to a simple problem. The most widely used rodent species have social organizations in which females are not aggressive towards each other. The basis for social defeat is physical confrontation. Thus, despite a steady drumbeat of ground breaking discoveries based on male mice and rats, there is a paucity of research on females. We study monogamous California mice, in which both males and females are aggressive. On average, females exposed to three episodes of social defeat show social withdrawal behavior whereas males do not. Kappa opioid receptors (KOR) function at the intersection of stress and depression-like behavior because they are activated during stress and can induce dysphoria. Studies in male mice suggest that the dysphoric effects of defeat stress are mediated in part by KOR activity. However, no study has ever tested this hypothesis in females. Preliminary data show that female California mice are, on average, more sensitive to the aversive properties of KOR than males. The proposed experiments test the hypothesis that sex differences in KOR activity contribute to sex differences in stress-induced social withdrawal. We also examine sex differences in KOR-dependent activation of p38 MAP kinase, which has been shown to mediate the aversive behavioral effects of KOR. The results of these experiments will provide novel insights into sex differences in the neurobiological and behavioral responses to social stress. Just in Time Information for 1R01MH097714-01A1 PI: Trainor, Brian C.

DESCRIPTION (provided by applicant): Psychosocial stress is an important risk factor for psychiatric disorders such as depression and anxiety. There has been increasing interest in targeting kappa opioid receptors (KOR) as a novel therapeutic target. Agonists for KOR have been reported to induce dysphoria and regulate the hypothalamic-pituitary-adrenal axis (HPA). New evidence suggests that there is a major gap in our understanding of the aversive properties of KOR. Studies showing that KOR mediates effects of stress on behavior primarily focus on short term effects of stress (15 min). However, after two days of psychosocial stress KOR loses its aversive properties. We hypothesize that this long term effect of psychosocial stress induces a neuroadaptation that fundamentally alters the effects of KOR. This is a critical idea because the field is working on the assumption that KOR antagonists have antidepressant properties. Our hypothesis suggests that KOR agonists will have stronger antidepressant properties for individuals exposed to long term psychosocial stress. Kappa opioid receptors inhibit serotonergic tone by inhibiting the activity of dorsal raphe nucleus (DRN) serotonin neurons. Increased serotonergic tone is linked to increased sensitivity to threat and increased inhibition o the HPA axis, and psychosocial stress can increase baseline activity of DRN serotonin neurons. By studying monogamous California mice, we are one of the only lab groups with the capability to study the effects of social defeat in both males and females. Females exposed to defeat exhibit social avoidance to non- threatening social stimuli. We hypothesize that defeat stress results in desensitization of the inhibitory effects of KOR on the DRN, which facilitates social avoidance. We predict that this effect is greater in females than males. If KOR inhibition of the DRN is stronger in stressed males, then social avoidance show be diminished and baseline corticosterone levels should be high. This is exactly what we have observed. Intriguingly, women diagnosed with depression have been reported to show stronger avoidance of social cues while recent work suggests that elevated baseline cortisol levels are more likely to occur in men with depression. First we use place preference studies to examine how defeat stress affects the aversive and rewarding properties of KOR. Second, we use multilabel immunohistochemistry to examine KOR induced changes in extracellular signal regulated kinase (ERK) and p38 MAP kinase (p38) in serotonin neurons in the dorsal raphe nucleus (DRN). These pathways are activated by KOR. Finally, we use site specific manipulations to test whether changes in serotonergic signaling mediate the effects of KOR on aversion, social interaction, and corticosterone.

Project summary Social anxiety disorder (or social phobia) is the most common form of anxiety disorder in the United States. Affected individuals avoid social contexts, which disrupts social relationships and impairs performance at school or work. New therapeutic approaches are needed because ~40% of affected individuals who seek existing treatments do not respond. Oxytocin is a well-known modulator of social behaviors, and has been put forth as a possible therapeutic. In some studies using human participants, intranasal oxytocin enhances social approach related behaviors. However, other studies (especially in women) report that intranasal oxytocin increases social anxiety. How can the same neuropeptide exert such different effects on behavior? Our central hypothesis is that oxytocin acts in the mesolimbic dopamine system to promote social approach, whereas oxytocin acts in the bed nucleus of the stria terminalis (BNST) to enhance social anxiety. This hypothesis is conceptually innovative because it can reconcile apparently contradictory findings in both human and animal studies of oxytocin function. The proposed studies will test this hypothesis in both males and females because social anxiety disorder is more prevalent in women than men. Our studies will use the California mouse social defeat model, which induces a stronger social anxiety phenotype in females versus males. First, we will use antisense morpholinos to selectively inhibit oxytocin synthesis in neurons within the BNST or hypothalamus (which project to the nucleus accumbens, NAc) to determine how these cells modulate social anxiety and social approach. Next, we will use viral vectors to visualize oxytocin producing cells in the BNST and hypothalamus. We will isolate oxytocin neurons for single-cell RNAseq analyses, and we will also conduct electrophysiological analyses. We will determine the extent to which social stress induces molecular and physiological responses that increase excitability. Finally, we will study the behavioral effects of oxytocin receptor (OTR) in the NAc and BNST using biased agonists that selectively induce OTR coupling of either Gq or Gi pathways. Our research team is ideally suited to execute these studies. Dr. Trainor's lab developed the California mouse social defeat model and collected most of the preliminary data. Dr. Robison is a behavioral neuroscientist with strong molecular and electrophysiology skills. Dr. Settles is an expert in bioinformatics and performed analyses of single-cell RNAseq data. Dr. Chini is a leading authority on OTR G-protein coupling and provides expertise for pharmacology studies. Dr. Grinevich developed viral tools for targeting oxytocin neurons and provides viruses and advice. Our analyses of how stress alters the physiological and molecular phenotypes of distinct populations of oxytocin neurons from males and females are unprecedented, and could lead to novel insights into how to selectively target these cells.