Larry J. Young - US grants

DESCRIPTION (Adapted from applicant's abstract): Microtine rodents provide an excellent opportunity for investigating the hormonal and neuronal substrates underlying complex social behaviors such as affiliation. Microtus orchogaster typically inhabit the prairie grasslands of North America and exhibit high levels of affiliative behavior. M. orchogaster tend to nest in groups composed either of mating pairs or extended families and form long lasting social bonds with their mates. In contrast, a related species from the Rocky Mountain area, M. montanus, are asocial and tend not to nest in male-female pairs. Arginine vasopressin (AVP) appears to play a central role in the control of some aspects of affiliative behaviors in male M. orchogaster. The distribution of AVP receptor (subtype V1a) gene expression in the brain differs strikingly between these two species. It has been hypothesized that this species difference in vasopressin receptor gene expression may be related to the species differences in social behaviors. This application is designed to investigate the molelcular mechanisms which control species-specific gene expression by analyzing the structure of the V1a receptor gene in the two vole species. Further experiments will use transgenic technology to understand how species-specific vasopressin receptor gene expression is achieved. Finally, the relationship between vasopressin receptor gene expression and social behavior will be investigated using a novel transgenic approach; vasopression receptor gene expression will be manipulated in the asocial M. Montanus brain and the resulting changes in social behavior will be characterized. Investigating the molecular nature of species-specific gene expression will lead not only to a better understanding of the neuroendocrine bases for social behavior, but also may provide insight into the complex control of region specific gene expression in the mammalian brain which is of great clinical importance.

Although a considerable body of research has focused on the neural substrates of social separation, relatively little is known about the neurobiology of social attachment. This proposal focuses on a monogamous rodent, the prairie vole, which forms selective, enduring social attachments or pair bonds. Several lines of evidence implicate central oxytocin (OT) pathways in prairie vole formation. OT increases and a selective OT antagonist decreases mating-induced pair bonding in female prairie voles as measured by the development of a partner preference. Blockade of OT receptors specifically in the nucleus accumbens inhibits development of a partner preference in female prairie voles. OT receptors are not found in this region in related vole species that do not form pair bonds. In prairie voles, OT receptors in the nucleus accumbens show a surprising individual variability. In approximately 20% of prairie voles, OT receptor mRNA or binding is virtually absent in this region, while OT receptors in other brain regions remain unaffected. We propose four new studies to investigate the importance of OT receptors in the nucleus accumbens for the development social attachments. The first study increases regional OT receptor expression in both prairie voles and non-monagamous montane voles, which do not form pair bonds. The second study explores the individual variation in nucleus accumbens OT receptors in prairie voles by comparing females with high and low levels of binding for regional gene induction and partner preference formation in response to OT. A third study investigates potential development mechanisms for the individual variation, based on a recent discovery that rats raised under different rearing conditions have different levels of OT receptor binding in adulthood. Finally, a fourth study explores molecular mechanisms for the individual differences in OT receptor expression. Taken together, these studies not only define a key step in the neurobiology of pair bonding, they may provide a molecular, cellular, and behavioral understanding of individual variation in a natural animal model system. The ultimate goal of this research is to provide new approaches to clinical disorders characterized by deficits in attachment, including autism and schizophrenia.

DESCRIPTION (provided by applicant): This proposal seeks support for the continued career development of the Candidate, Larry J. Young, PhD. Dr. Young is an Associate Professor in the Department of Psychiatry and Behavioral Sciences and a faculty member in the Center for Behavioral Neuroscience (CBN) at Emory University. Emory and the CBN provide an ideal environment for the continued development of the Candidate's research program. Dr. Young's work focuses on the molecular, cellular and neurobiological mechanisms underlying complex social behaviors. The long-term goal of the Candidate is to incorporate state-of-the-art technologies to understand the molecular mechanisms underlying individual variation in social behaviors. Using the highly social and monogamous prairie vole as an animal model, the Candidate's research suggests that the vasopressin receptor (V1aR) plays a critical role in modulating affiliative behaviors. Molecular studies suggest that variation in the gene encoding V1aR (avprla) contributes to individual variation in social behavior. The Aims in this proposal are designed to test four specific hypotheses. The first Aim will test the hypothesis that polymorphisms in an avprla microsatellite directly contribute to variation in V1aR distribution in the brain using a knock-in mouse approach. The second Aim uses a viral vector-based siRNA approach to directly test the hypothesis that variation in avprla expression contributes to variation in social behavior in prairie voles. Studies in the third Aim will test the hypothesis that diversity in avprla expression in the vole is a primary source of heritable variation in social behavior using a selective breeding strategy. The fourth Aim will directly test the hypothesis that polymorphisms in the human AVPR1A contribute to variation in human social cognition. Each of these Aims involves the implementation of new experimental approaches into the Candidate's research program. The Career Development Plan will entail collaborations with colleagues at Emory and abroad to develop the Candidate's expertise in creating knock-in mice using homologous recombination, modulating gene expression using siRNA technology, genomics and human genetic analyses. The research outlined in this proposal has important implications for understanding the etiology of the social impairments associated with Autism Spectrum Disorders (ASD). Ultimately, these findings may lead to novel therapeutic targets for the treatment of the social impairments which characterize this devastating disorder.

gender difference; neurophysiology; brain; Primates; animal colony;

DESCRIPTION (provided by applicant): We propose to develop genomic resources for the prairie vole (Microtus ochrogaster) that will significantly enhance the value of this species as a model for understanding the genetic and neurobiological mechanisms governing social behavior. Microtine rodents are ideally suited for investigating the mechanisms underlying variation in social behavior. The monogamous prairie voles (M. ochrogaster) are highly affiliative, form life-long social attachments and display high levels of biparental care. In contrast, montane (M. montanus) and meadow voles (M. pennsylvanicus) are relatively asocial, do not form social attachments, and are minimally parental. In addition to this species diversity, there is remarkable intra-species variation in social behavior among prairie voles, both within a population and among populations of different geographical ranges. This behavioral diversity provides and exciting opportunity to discover novel genes contributing to the regulation and diversity of social behavior. Over the past 15 years, there has been remarkable progress in understanding the neural and molecular mechanisms regulating social bonding and parental care in prairie voles. These studies have focused on a few candidate neuropeptides and neurotransmitter systems (e.g. oxytocin, vasopressin, CRF, dopamine and their respective receptors). The discoveries made in prairie voles have important implications for psychiatric disorders associated with impairments in social behavior, including autism, schizophrenia and depression. In fact, principles discovered thus far using the prairie vole model have already been applied to autism research with promising results. However, the development of this model to its full potential is limited by the paucity of molecular and genomic resources available for the prairie vole. We propose to generate two genomic resources that will greatly accelerate investigations of the basic genetic mechanisms regulating social behavior. First, we will construct a microsatellite-based genetic linkage map of the prairie vole genome that can be used in Quantitative Trait Locus (QTL) studies. We will also construct a 10X coverage BAC library of the prairie vole genome. We will use this resource to isolate clones containing several genes of interest to the prairie vole research community. Used together, these resources will facilitate the discovery of novel genes involved in the regulation of social behavior. These resources will be made freely available to the rapidly growing prairie vole research community. PUBLIC HEALTH RELEVANCE Psychiatric disorders such as autism spectrum disorder, schizophrenia, and depression are associated with severe impairments in the social domain. Understanding the neurobiological and genetic mechanisms underlying the regulation of normal social behavior will provide insights into the systems that may be disrupted in these devastating disorders. The resources developed through this proposal will accelerate the discovery of novel genes contributing to variation in social behavior, potentially leading to novel therapeutic targets for treating these disorders.

Animal Model; Animal Models and Related Studies; Autism; Autism, Early Infantile; Autism, Infantile; Autistic Disorder; Bonding; Bonding (Psychology); Bonding, Psychological; Brain; CRISP; Cell Communication and Signaling; Cell Signaling; Clinical Trials; Clinical Trials, Unspecified; Computer Retrieval of Information on Scientific Projects Database; Cycloserine; Data; Disruption; Drug Design; Drugs; Encephalon; Encephalons; Funding; Glutamate Agonist; Glutamates; Goals; Grant; Institution; Intracellular Communication and Signaling; Investigators; Kanner's Syndrome; L-Glutamate; Learning; Medication; Mental disorders; Mental health disorders; NIH; National Institutes of Health; National Institutes of Health (U.S.); Nerve Transmitter Substances; Nervous System, Brain; Neurotransmitters; OXT; Object Attachment; Object Relationship; Ocytocin; Oxytocin; Pharmaceutic Preparations; Pharmaceutical Preparations; Psychiatric Disease; Psychiatric Disorder; Pyschological Bonding; R-4-Amino-3-isoxazolidinone; Recombinant Oxytocin; Reporting; Research; Research Personnel; Research Resources; Researchers; Resources; Signal Transduction; Signal Transduction Systems; Signaling; Source; System; System, LOINC Axis 4; United States National Institutes of Health; Unspecified Mental Disorder; affiliative behavior; biological signal transduction; clinical investigation; drug/agent; memory process; mental illness; model organism; neural mechanism; neuromechanism; prairie vole; psychological disorder; social attachment; social bonding; social cognition

DESCRIPTION: Major Depressive Disorder (MDD) has become one of the most pressing health-related problems in the United States. While there appears to be numerous etiologies for MDD, social bond disruption and early-life stress are two of the most commonly cited risk factors. Unfortunately, the consequences of social bond disruption on the mental state are difficult to study in traditional laboratory animal models. Furthermore, current animal models provide little insight into how early-life stress influences the prosocial brain. Monogamous prairie voles offer a novel animal model for investigating the convergence of these two factors in the development of depression at both the behavioral and neurobiological levels. Male prairie voles separated from their female partner display depressive-like behavior and an activation of the stress axis. The increase in depressive-like behavior following social loss appears to be mediated by the corticotropin-releasing factor (CRF) system, as blocking either of the CRF receptors (CRF1 or CRF2) prevents the onset of depressive-like behavior following social loss. Interestingly, monogamous prairie voles and non-monogamous meadow voles exhibit species differences in CRF1 and CRF2 in the brain, particularly in the nucleus accumbens (NAcc) Activation of CRF1 and CRF2 in the NAcc facilitates pair bond formation, and pair bonding results in increased CRF mRNA in the bed nucleus of the stria terminalis (BnST). These data have led to the hypothesis that activation of CRF receptors in the NAcc of male prairie voles also plays an important role in the emergence of depressive-like behavior following separation from the partner. In this proposal, a pharmacological approach will be used to determine whether CRF receptors in the NAcc are responsible for social loss-induced depressive-like behavior. Viral vector mediated siRNA silencing will be used to determine whether CRF expressing neurons in the BnST regulate the onset of depressive-like behavior following social loss. Finally the influence of early-life social experience and adversity on the CRF system and the behavioral response to social loss will be examined in prairie voles. These studies will provide a deeper understanding of how social loss and early life stress impact the brain mechanisms underlying major depression. PUBLIC HEALTH RELEVANCE Major depression is one of the most debilitating psychiatric disorders and most burdensome diseases to society, with as many as 30 million Americans affected. Social bond disruption and early-life stressors have a major impact on the likelihood of developing depression. The studies in this proposal will investigate the interaction of social bond disruption and early-life stressors on the brain mechanism underlying depressive-like behavior in an animal model.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This project examines the behavioral and physiological consequences of the loss of a bonded partner in monogamous prairie voles. Male prairie voles bond with their mate through mechanisms involving vasopressin and dopamine. However little is known about the consequences of social loss. In the past year we have found that loss of a partner (but not of a sibling) results in an increase in basal stress hormones as well as passive behavior in the forced swim test and tail suspension test. These changes are reminiscent of the behavioral changes associated with depression. We hypothesized that the peptide CRF might be involved in these behavioral changes. Indeed we found that blocking CRF receptors at the time of losing the partner completely blocks the emergence of depressive-like behavior. These studies provide an important animal model for understanding the consequences of social loss, grieving, and depression.

This award supports PIs Loren Hayes and Larry Young to undertake a ten-day planning visit to National Taiwan University in Taipei in March 2010. The planning visit will bring together scientists interested in the behavioral ecology of social rodents with scientists interested in neurobiology and gene expression to discuss a proposed evolutionary-mechanistic model for sociality and to develop a long-term collaboration to test hypotheses linking behavioral ecology, genomics and neuroanatomy and to generate international research opportunities for U.S. graduate students and junior researchers. Hayes, a behavioral ecologist, and Young, a neuroscientist, will collaborate with behavioral scientist Kirk Lin of the Institute of Ecology and Evolutionary Biology and Alex Hon-Tsen Yu of the Institute of Zoology, both at National Taiwan University. The collaborators will each be joined by a graduate student or postdoc.

The proposed study species (Microtus kikuchii) is thought to be socially monogamous. Understanding the sociality and mating systems of this species could lead to comparisons with other socially monogamous mammals (e.g. Microtus ochrogaster), increasing understanding of convergent evolution.

The PIs will assess the long-term potential for collaboration, including assessing potential field sites, evaluating laboratory capabilities in Taiwan, developing preliminary projects for affiliated graduate students/postdocs to generate data for long-term study, and discussing strategies for collaborative and independent grant-writing to support the long-term goals of the project.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This project is to advance the development of the prairie vole as an extraordinary model organism for understanding the biological underpinnings of social behaviors by developing a key genomic resource, a catalog of vole gene sequences. Specifically we will 1) generate partial or complete gene sequences for e 18,000 vole genes that encompass 50% of the transcriptome, and 2) for the first time examine the nature of social bond formation on a genome-wide scale. In summary, this research project will generate a critical resource for an emerging model of human social behavior and will likely lead the discovery of novel gene pathways involved in the regulation of complex social behavior such as social bonding. We have just begun this project and already have prairie vole embryo RNAs and plan to now continue charactering the transcriptome in the brain.

DESCRIPTION (provided by applicant): Oxytocin (OT) is a neuropeptide that has been implicated in the regulation of social cognition and social behavior both in animal models and in humans. In rodents, OT is involved in social information processing, parental nurturing, and social bonding. In humans, intranasal OT enhances interpersonal trust, eye-to-eye contact, memory of faces, and the ability to infer the emotions of others. These observations as well as genetic association studies focusing on polymorphisms of OT receptor (OTR) gene suggest that dysregulation of the OT system may contribute to the social cognitive deficits associated with several psychiatric disorders, including autism spectral disorders, schizophrenia, and depression. Despite its evident role in regulating social cognition in humans, very little is known regarding the localization of OTR in the human brain or its dysregulation in psychiatric disorders. The development of a PET imaging technology for the in vivo imaging of the OTR would greatly enhance basic and clinical research investigating the relationship between OTR and human social cognition. Furthermore, its application may lead to the development of a potential diagnostic tool for disorders such as autism spectrum disorders, which are currently diagnosed solely based on behavioral observations. In this application we proposed to synthesize and characterize candidate selective, small molecule OTR PET radioligands with the ultimate goal of generating tools for investingating the relationship between OTR distribution in the brain and human social cognition and psychopatholgy. Radiochemistry will focus on C-14 and I-125 derivatives for preliminary in vitro and in vivo evaluations followed by C-11 and F-18 derivatives for biodistribution and PET imaging studies. The affinity and selectivity of the canditate compounds for the human OTR will be examined using standard receptor binding assays. The ability of the candidate compounds to penetrate the blood brain barrier and label OTR will be evaluated using transgenic mice expressing the human OTR. Biodistribution studies will also be performed in these transgenic mice as well as rats. Finally, in vivo PET imaging of OTR density within the brain of the rhesus monkey will be performed. Thus, the experiments in this proposal may ultimately provide an excellent tool to investigate the relationship of OTR distribution, social cognition, and psychopathology. The proposed research plan presents the working hypothesis that a suitable small molecule oxytocin receptor PET ligand can be developed through minor modifications of known selective small molecule antagonists. PUBLIC HEALTH RELEVANCE: Oxytocin modulates several aspects social cognition in both animal models and man. There is evidence that dysregulation in the oxytocin system, including oxytocin receptors, may contribute to the social deficits in psychiatric disorders such as autism spectrum disorder, depression and schizophrenia. The development of a PET ligand to detect and quantify oxytocin receptor in the living brain may therefore prove to be a useful tool for examining the relationship between oxytocin receptor distribution and social cognition in psychiatric patients and may lead to a better understanding of the etiology of social deficits in these disorders.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The goal of this project is to use two different approaches to generate prairie voles with disrupted vasopressin receptor signaling. The first approach involves creating transgenic prairie vole expressing shRNA's targeting the vasopressin receptor gene. During this reporting period, we have screened several potential shRNA's in vitro and have identified the optimal sequences. We have also put these shRNA sequences into a lentiviral vector and shown that they decrease vasopressin receptor binding in the brain. We are now ready to inject these viruses into embryos. The second approach is to create Zinc Finger Nuclease proteins that target the vasopressin receptor sequence to cause deletions. Working with the company that produces these nucleases, we have identified several candidates and will begin screening these in an invitro system before we inject into vole embryos.

DESCRIPTION (provided by applicant): Oxytocin (OT) is a neuropeptide that plays an important role in regulating many aspects of social behavior, including maternal nurturing, social information processing, and social attachment. Intranasal administration of OT in humans increases attention to social cues, gazing into the eyes, inferring the emotions of others, trust and socially reinforced learning. Several studies have demonstrated that OT enhances some aspects of social functioning in individuals with autism spectrum disorder (ASD), and the OT system is a potential pharmacological target for enhancing social function in ASD. Furthermore, there is evidence of altered OT systems in ASD, including decreased concentrations of OT in plasma, genetic association between ASD and polymorphisms in the OT receptor gene (OXTR), and reduced OXTR mRNA in the brains of subjects with ASD. Genetic polymorphisms in the OXTR gene have been associated with variation in social cognition in both ASD and healthy subjects. The socially monogamous prairie vole has provided great insights into the role of OT in regulating social behavior. OT acts in the nucleus accumbens (NAcc) to promote alloparental nurturing and pair bonding between mates. Variation in OXTR density in the NAcc is correlated with variation in alloparental behavior and pair bonding. In this project we will explore the contribution of a natural genetic variation in the OXTR gene to social behavior and susceptibility to early-life social stressors. The first aim will determine whether a single nucleotide polymorphism in the prairie vole oxtr gene that predicts OXTR expression in the striatum (e.g. NAcc and caudate putamen) is associated with variation in social behavior in male and female prairie voles at multiple developmental epochs. In the second Aim, we will infuse an shRNA viral vector targeting the Oxtr in the NAcc of high OXTR expressing genotype voles early in development to determine whether OXTR knockdown the NAcc recapitulates the phenotype-genotype relationships observed in Aim 1. The third aim will test the hypothesis that animals with low levels of OXTR in the NAcc are more severely impacted by early-life social deprivation, modeling gene x environment interactions. Finally we will explore the possibility that a pharmacological approach to stimulate OT release can rescue the social deficits generated by the OXTR polymorphism and early-life social deprivation. We will examine three different developmental windows for chronic OT based therapy as well as an acute treatment in adults on partner preference formation. These studies will provide detailed insight into the acute and developmental impact of OXTR signaling on a suite of social behaviors, determine the effect of variation in OXTR expression in brain regions known to regulate social behavior, and begin to explore a potential pharmacological intervention to enhance OXTR signaling in individuals with compromised OXTR function. This work will inform future development of novel therapeutic strategies to enhance social function in ASD and other psychiatric disorders.

PROJECT SUMMARY The oxytocin (OT) system is perhaps the most viable neurobiological target for enhancing social cognition in psychiatric disorders with compromised social function, including autism spectrum disorder (ASD). In animals, OT enhances neural responsiveness to social cues, thereby facilitating social recognition, attachment and reward. In humans with ASD, intranasal OT enhances brain reward system responses to social stimuli, increases gaze to the eyes of others, and improves social reciprocity. The efficacy of intranasal OT as a therapy for social disorders is limited by poor penetrance into the brain. In order to inform future translational applications, our goal is to gain a better understanding of the precise mechanisms by which OT influence social information processing in the context of intrinsic or extrinsic reward. Social learning involves social information processing in the context of reward and is paramount to normative social skill development, and the role of OT in this process is a central theme of the Center. We will sustain a highly coordinated, interdisciplinary research program involving an outstanding team of investigators to test the hypothesis that OT facilitates social learning by enhancing the flow of information across neural networks involved in salience and reward. Project scientists will use cutting-edge techniques to manipulate OT signaling in specific circuits during social engagement to understand how OT influences socially relevant neural communication. Project 1 will examine the effect of pharmacologically-evoked endogenous OT release during a social encounter on a social salience brain network in monogamous prairie voles that have high or low densities of OT receptors. Project 2 will perform simultaneous electrophysiological recordings in three brain regions that process social information and reward during social bond formation in prairie voles. Optogenetic manipulations of the circuit will be used to test causal relationships between OT- dependent neural activity and social attachment. Project 3 will analyze the influence of optogenetic stimulation of OT release and targeted genetic mutations on neural communication in rats in the context of a social learning paradigm using extrinsic reinforcers. A potential interaction of OT and cholinergic systems will be examined. Project 4 will record neural activity in these same brain regions following local OT infusion in rhesus macaques while performing an extrinsic reward-based social discrimination visual task. A Bioanalytic Core will provide vital histological, genotyping and neural molecular phenotyping services for the research Projects. An Administrative Core will manage all Center related activities (seminar series, meetings, Pilot Project grants), provide statistical consultation and coordinate outreach and training activities of Center personnel by strengthening existing relationships and forging new ones with numerous organizations. As a result, the Center will create a vibrant collaborative research, training and outreach environment that will have a national impact on mental health research. The data collected by Center faculty will have important translational implications that will inform novel strategies for treating social deficits in psychiatric disorders.

Project Summary Oxytocin (OT) regulates many aspects of social behavior, including parental nurturing, individual discrimination and social attachment. OT is hypothesized to influence social behaviors by enhancing the salience and reinforcing value of social stimuli. Building on this hypothesis, rodent research has provided a compelling rationale for targeting the OT system to improve social cognition in psychiatric disorders such as autism spectrum disorders (ASD). Indeed, intranasal administration of OT enhances some aspects of social functioning in individuals with autism spectrum disorder (ASD), and the OT system is a leading pharmacological target for enhancing social function in ASD. Single nucleotide polymorphisms (SNPs) in noncoding regions of the human OT receptor gene (OXTR) are associated with core symptoms of ASD, altered brain activity patterns, and ASD diagnosis. However, human gene association studies do not provide insights about the exact mechanisms by which polymorphisms in OXTR lead to variation in brain function or social behavior. The socially monogamous prairie vole is an ideal model organism to explore the molecular processes by which variation in the OT system can lead to alterations in brain phenotype, and downstream social behaviors. OT receptor (OXTR) signaling in the nucleus accumbens (NAcc) is critically involved in alloparental nurturing and social bond formation in prairie voles. There is remarkable individual variation in the density of OXTR in the NAcc of prairie voles that is associated with variation in social behavior and resilience to early life social neglect. A set of 9 SNPs in the prairie vole OXTR gene (Oxtr) explains more than 70% of the variation in OXTR density in the NAcc, but not in other brain areas. These 9 SNPs are all equally strongly associated with OXTR density in the NAcc, due to fact that they are all perfectly correlated with each other in our colony. In this case, when multiple correlated SNPs are driving the association, genotype-phenotype association study design is not useful to disentangle the individual SNP?s contribution to variation in the phenotype. The goal of this proposal is to develop a state-of-the- art genome editing technique combining viral vector procedures with the novel CRIPSR-Cas9 genome editing method to enable targeting of Oxtr SNPs in the NAcc in adult prairie voles. The viral vector mediated CRISPR- Cas9 method will introduce small mutations hypothesized to alter the function of the genomic region including the targeted SNP and will result in changed NAcc OXTR expression when functional SNPs are investigated. Hence, the functional role of the individual SNPs in the prairie vole Oxtr can be explored using this method. In addition, this approach, for the first time developed in prairie voles, will be useful for future experiments for example identifying the functional roles of specific OXTR neuronal populations in social behavior and will give insights with important implications for human genetic studies investigating the potential influence of OXTR SNPs on brain phenotype, social cognition and psychopathology.

PROJECT SUMMARY (Project 1, Young) Evidence from animal studies suggests that oxytocin (OT) plays an important role in facilitating social behavior. It is hypothesized that the influence of OT on social cognition is facilitated by increasing the salience and rewarding value of social stimuli. Human studies, inspired by the work in animal models, have investigated the effects of intranasal (IN) administration of OT on a wide range of human social behaviors, including psychiatric outcomes such as social functioning in individuals with autism spectrum disorder (ASD). The clinical efficacy of IN-OT is however questionable because of limited blood-brain-barrier penetration and diffusion within the brain. Stimulating endogenous OT release would circumvent these issues. Studies investigating the effects of targeting the melanocortin system have shown that melanocortin 4 receptors (MC4Rs) interact with several neurochemical systems known to modulate social behavior including OT. Work in our laboratory has shown that peripheral injection of melanotan II (MTII), a brain penetrant small-molecule selective MC4R agonist, facilitates partner preference formation in monogamous prairie voles. This effect is blocked by central infusion of an OT receptor (OXTR) antagonist (OTA). Further, MTII selectively activates hypothalamic OT neurons and potentiates OT release in the nucleus accumbens (NAc). MTII does not have a direct effect on OT release at projection terminals but is thought to result in local release of OT in the paraventricular nucleus of the hypothalamus (PVN) and thereby priming OT neurons in this region to be more responsive to stimuli. We have shown that MTII alone does not increase OT release outside of the PVN, but when combined with a hypertonic osmotic challenge, a potent stimulus for terminal OT release, results in approximately a 2-fold increase in OT release in the NAc. In line with this finding, preliminary data from our laboratory suggests that MTII combined with social exposure, also known to stimulate OT release, results in robust brain activation. This effect is absent under non-social conditions and blocked by central injection of an OTA. The aim of this project is to further investigate the mechanisms through which MTII affects brain circuitry by studying how this compound influences activation across multiple nodes in a brain network involved in social salience processing. Our hypothesis is that MTII paired with social interaction will result in increased activation of nodes in the network and coordinated activity across nodes. We further hypothesize that the effect of MTII on brain activity is mediated through the OT system and will therefore be attenuated by administration of an OTA, as well as in animals carrying alleles associated with low brain OXTR density. Finally, we will directly investigate if the effect of MTII is mediated through OXTRs in the PVN, through autoregulation on OT neurons, by using a viral vector mediated CRISPR-Cas9 approach to site specifically delete OXTRs in OT neurons. These experiments will give important insights into the neural mechanisms of a translationally relevant approach to manipulate the OT circuitry and will lay the ground for further non-invasive stimulation of the OT system.

? DESCRIPTION (provided by applicant): Anxiety disorders are the most prevalent psychiatric disorders and affect over 40 million American adults over the age of 18 every year. Significantly, ~ 20% of individuals who seek treatment for independent anxiety disorders also have a current drug use disorder. The extended amygdala is thought to play a critical role in adaptive motivational behavior, and has been implicated in the pathophysiology of maladaptive fear, anxiety, and addiction. Two key elements of the extended amygdala are the central nucleus of the amygdala (CeA) and the bed nucleus of the stria terminalis (BNST). Notably, evidence from clinical and preclinical studies suggests that differences in BNST activity determine individual differences in trait anxiety levels and also anticipatory anxiety (AA). Activity of corticotropin release factor (CRF) containing neurons in the BNST plays a central role in the normal adaptive response to stress. However, chronic release of CRF also plays a critical role in several psychopathologies, including anxiety disorders, posttraumatic stress disorder (PTSD), stress-induced drug recidivism, all of which have excessive AA as a core symptom. To date the cellular mechanisms underlying the switch from a normal adaptive response to a psychopathological state remain unknown. However, evidence from imaging studies suggests that activation of a circuit comprising the insular cortex (IC), amygdala, BNST, and ventrolateral periaqueductal grey (vlPAG) plays a key role in regulating the expression of AA. Using a transgenic CRF-Cre mouse line, we now have exciting pilot data showing that cell type-selective inhibition of CRF neurons in the BNST blocks the development of AA. The proposed work will use a multidisciplinary approach to define a functional circuit by testing the hypothesis that BNSTov CRF neurons act to integrate viscerosensory and emotional information from the IC and amygdala and relay this information to the vlPAG to regulate the expression of AA. The long-term objectives of this proposal are to delineate the cellular mechanisms contributing to the pathological switch in BNST function, with the hope of identifying novel targets for clinical intervention. Three Specific Aims will test the hypothesis: Aim 1 will examine the necessity / sufficiency of BNSTov CRF neuron activation in the expression of AA. Aim 2 will examine the role of the insular cortex (IC) and posterior basolateral amygdala (BLAp) in activating BNSTov CRF neurons during AA and their downstream connection with the vlPAG, and Aim 3 will examine the effects of chronic stress on the functioning of the proposed pathway.