Nirao M. Shah - US grants

DESCRIPTION (provided by applicant): All animals have evolved behaviors that result in innate responses to the external world. These responses can often be observed without prior learning or experience, suggesting that the neural circuits that generate them are developmentally programmed. In mice sexually dimorphic behaviors represent a set of innate behaviors that are controlled by odorant cues and by internal regulators such as gonadal hormones. Sexually dimorphic behaviors are qualitative or quantitative differences in behavior between the sexes, and much work remains to be done to characterize the neural circuits that mediates these behavioral responses. Such innate behavioral differences between the sexes result from sexually differentiated neural circuits. Testosterone and its receptor, the androgen receptor (AR), are required for male-specific behaviors. We and others observe sexual dimorphism in AR expression in a pool of neurons within the bed nucleus of the stria terminalis. This research proposal takes a genetic approach to characterize the role of this AR+ dimorphic subpopulation of neurons in sexually dimorphic behaviors in mice. This project will examine whether the AR+ BNST neurons are activated during mating and aggression, whether odorant cues are sufficient to activate them, and whether vomeronasal odorant detection is utilized to relay these odorant cues. Using gene targeting this project examines the behavioral consequences of ablating the dimorphic BNST neurons in the adult animal. Finally, using a genetic strategy this project will examine the function of AR in the dimorphic BNST by deleting the AR gene in the adult animal. An inherited loss of function of AR in humans manifests as physical and behavioral feminization (androgen insensitivity syndrome). In adult humans anti-androgen therapy or low levels of testosterone may be associated with a loss of libido and emotional well-being. Our examination of animals with a deletion of AR in BNST neurons should shed some light, in principle, on how neurons respond to a loss of testosterone signaling. More generally, our studies should further our understanding of how discrete brain regions integrate external sensory cues with internal physiological states to generate meaningful behavior.

We are interested in understanding how the brain generates social attachments. Humans form enduring social relationships that shape the rules for interacting with other individuals. Ruptured social ties are often the first sign of mental illness, and are usually the most difficult to heal. Even in healthy individuals, breakdown of a social relationship such as marriage leads to a dramatic increase in stress and anxiety. In humans, the neuropeptides vasopressin and oxytocin play critical roles in the formation of social attachments. Moreover, altered signaling of these neuropeptide pathways is associated with a decline in the quality of social relationships and with serious illnesses such as autism. Mice, zebrafish, worms and fruitflies do not display social attachments, precluding the use of genetic tools to dissect the neural and molecular networks that mediate these behaviors. Voles, which are small rodents, display striking social bonds such that a mated pair displays enduring co-habitation and sexual fidelity. As in humans, vasopressin and oxytocin are critical for the formation of social ties in voles. Progress in dissecting the neural circuits that mediate social attachment is stymied due to the lack of gene targeting approaches in voles. We propose to develop the reverse genetic strategies in voles that have revolutionized experimental manipulations in the mouse. We propose to develop embryonic stem cells to enable targeted gene knock-out and knock-in experiments in vivo. We will combine these genetic tools with behavioral analysis, high resolution anatomic and functional neural circuit mapping, and systems analysis of signaling to understand how the brain normally generates social attachments. These insights will be applied to understanding how neural circuits malfunction in autism and other mental disorders. Our studies should eventually lead to effective therapies to restore the ability to form enduring social attachments.

DESCRIPTION (provided by applicant): How neural developmental programs are linked to the establishment of mature brain circuits and related behavior remains a central question of neuroscience. Our previous studies revealed that a class of neural progenitors defined by the expression of the homeodomain encoding transcription factor, Dbx1, are dedicated for the generation of subsets of neurons of the limbic system including the olfactory bulb. Based on these findings, we hypothesize that Dbx1+ progenitors located in a distinct embryonic niche generate a functionally distinct interconnected subset of olfactory bulb output neurons that are dedicated to the processing of subsets of innate behaviors. Testing of this hypothesis will be accomplished using a combination of multidisciplinary approaches including genetic fate mapping, optogenetics and innate behavioral testing.

DESCRIPTION (provided by applicant): This grant application seeks to define the genetic and neural circuit basis of the functional role of the ventromedial hypothalamus (VMH). The VMH is molecularly heterogeneous and correspondingly, it has been implicated in the regulation of diverse behaviors and physiological function: motor and visceromotor functions, neuroendocrine function, feeding, and sexually dimorphic social and emotional behaviors. One appealing possibility is that these diverse functions are controlled by discrete subsets of VMH neurons. We and others have identified a small cluster of sexually dimorphic neurons within the VMH. We hypothesize that these neurons control sexually dimorphic social and emotional behaviors. In Aim 1, we will use a novel Cre recombinase mouse strain we have generated to genetically trace the connections of these dimorphic VMH neurons. We will also test the hypothesis that projections to different areas emanating from these dimorphic neurons are activated during distinct behaviors. In Aim 2, we will utilize a novel Cre-dependent, pro-apoptotic gene to genetically ablate these dimorphic VMH neurons in adult males and females. These mice will subsequently be tested for deficits in dimorphic social and emotional behaviors. In Aim 3, we will utilize a Cre- dependent heterologous receptor (DREADD) activated by a heterologous ligand (clozapine-N-oxide) to switch on activity in these VMH neurons in vivo in males and females. This experiment will test whether activity in these neurons is sufficient to elicit dimorphic socia and emotional behavior. Taken together, our molecular genetic approaches will uncover the connectivity and functional relevance of a sexually dimorphic neuronal cluster in the mammalian forebrain. Health Relatedness: The devastating clinical manifestations of common neurodegenerative conditions and psychiatric conditions often reflect dysfunction of specific neural circuits. The VMH has been implicated in the regulation of social and emotional behaviors, neuroendocrine function, feeding, and motor and visceromotor function. VMH-localized lesions such as tumors result in altered cognition, abnormal social behaviors, and metabolic changes. Our studies will provide novel mechanistic insight into the functional relevance of the VMH and the circuits in which it participates in health. These findings may ultimately allow development of more rationally-targeted diagnostic and therapeutic options for VMH dysfunction in neurological disorders. Finally, the Cre-dependent, pro-apoptotic genetically encoded reagent that we use in this proposal will be useful in developing models of neurodegeneration in any neuronal (or other cell) type.

DESCRIPTION (provided by applicant): Alcohol use disorders (AUDs) are a major problem in medicine and society, and the few available treatment strategies have so far met with limited success. It is well established that genetic, psychosocial, and environmental factors contribute to an increased risk for AUDs. Our laboratory has pioneered the development of Drosophila as a model system for the identification of genes and molecular pathways that regulate simple ethanol-induced behaviors, such as intoxication and the development of tolerance, and many of the underlying molecular mechanisms have been found to be conserved between flies and mammals. Here, we propose to investigate the mechanisms underlying more complex behaviors - ethanol consumption, preference, relapse, and reward - in Drosophila. The Specific aims of this proposal are: 1) to characterize in detail, using newly developed software, the behavior of flies during ethanol self-administration and relapse, and study the influences of experience as well as social and environmental context; 2) to define the neuromodulators and neural circuits engaged during these behaviors; 3) to establish whether genes that regulate acute ethanol intoxication also mediate ethanol consumption, relapse and reward, and identify novel genes that regulate these behaviors; and 4) to identify potential pharmacotherapies for AUDs by screening a large collection of FDA-approved drugs for their efficacy in curbing ethanol consumption and relapse. The ease, potential for high throughput, and relatively low expense of Drosophila studies make the fly a powerful system to investigate the molecular and neural mechanisms underlying ethanol preference, relapse and reward. These mechanisms will provide novel insights into these complex behaviors, which can be translated to rodent models, provide candidate genes for the genetics of AUDs in humans, and ultimately, suggest potential targets for pharmacotherapy.

? DESCRIPTION (provided by applicant): Developing and validating a toolkit for cell type specific gene manipulation PIs: Clandinin and Shah Neurons express complex arrays of genes that play crucial roles in determining neuronal function. As such, single-gene mutations can lead to neurodevelopmental, neurophysiological, and neurodegenerative diseases. However, the nervous system is made up of many different types of neurons, which differ both in the genes they express and the function those genes perform. The ability to inactivate targeted genes only in the cell type of interest is therefore critical for our understanding of neural circuit function This application will generate a generalizable, validated set of transgenic flies and mice for cell type specifically manipulating genes that control the inputs, outputs, and activity patterns of neurons in physiological and behavioral studies of a wide array of circuits. This application describes three goals for developing and validating this toolkit. First, we will generate conditional allelesof 24 genes important for neuronal excitability and signaling in Drosophila. Second, we will validate our cell type specific gene disruption in vivo using a combination of voltage imaging, calcium imaging and behavioral assays, providing strong evidence of the broad utility of this toolkit. Third, since mice are critical model organisms for studying vertebrate nervous systems in health and in disease, we will adapt the same tool we use in flies to mice, targeting a key set of genes controlling excitation, inhibition and neuromodulation. Development of this toolkit will provide th neuroscience community with the means to manipulate essential neural genes with unprecedented precision in cell types of interest, thereby allowing fundamental questions about how genes shape neuronal circuit function to be addressed. As mutations in these classes of genes lead to devastating neurological disorders, this tool will facilitate studies that expand our understanding of these diseases and the treatment possibilities. As pharmacological approaches to treating brain dysfunction are ultimately limited by molecular specificity, understanding cell-type specific gene function is critical to the development of new treatment strategies. Finally, as the tool we will develop can be generalized to virtually any gene, future studies can extend the use of this tool to any gene of interest, in either flies or mice.

? DESCRIPTION (provided by applicant): This grant application seeks to uncover the molecular and neural networks underlying social attachment behaviors in prairie voles. Humans form attachments at many levels of social interactions, including with spouses, family members, friends, and other members of the community. The neurobiological mechanisms that control the formation and maintenance of social attachment remain poorly understood. This is in part because traditional genetic model systems such as mice, fish, flies, and worms do not exhibit social attachment behavior as adults, precluding the use of powerful molecular genetic approaches to dissect mechanisms underlying social attachment behavior. Prairie voles are small rodents that form an enduring social bond between adults, and they also display other related afflictive behaviors. Pharmacologic studies in prairie voles have implicated vasopressin and oxytocin signaling in the control of social attachment behaviors. However, there are significant limitations of these pharmacological manipulations such that the genetic requirement of specific neuropeptide signaling pathways remains unclear. Progress in uncovering the molecular and neural circuit basis of social attachments in prairie voles has been slowed by the absence of gene targeting techniques as well as by the absence of identification of behaviorally-salient neurons. In this grant application, we propose to develop gene targeting technology in prairie voles, focusing on vasopressin and oxytocin signaling pathways (Aim 1). In Aim 2, we propose to analyze behavioral deficits in social attachment in prairie voles genetically mutant for these signaling pathways. Finally, in Aim 3 we propose developing molecular tools to uncover genetic and neural pathways underlying social attachment in prairie voles. Taken together, our studies will enable gene targeting in prairie voles and elucidate neural mechanisms that control social attachment behavior. Health relatedness: Social attachments are thought to be critical for our mental health and for success in personal and professional interactions. Failure to form or maintain social attachments is often an early indicator of a serious mental illness such as autism spectrum disorder or schizophrenia. Strikingly, vasopressin and oxytocin are also thought to play a critical role in human social attachments, and dysregulated signaling via these neuropeptide pathways has been implicated in autism spectrum disorders. Our proposal seeks to establish the prairie vole as a new mammalian genetic model system and to uncover mechanisms underlying social attachment behavior. These advances may therefore provide a useful model system to study social behaviors relevant to human health and mental illnesses.

The Stanford Neuroscience Training Program remains the only doctoral degree granting entity for Neuroscience at Stanford. This interdisciplinary program consists of 91 students and 99 faculty from 28 departments (11 clinical, 17 basic scientists) and 4 schools. The breadth of departments illustrates the breadth of research areas which span molecular/cellular to systems and behavior, from human cognition to translational work. The training grant and its implementation is the central funding source, with 14 slots, and the foundation of the program. Our mission is to identify, recruit and train predoctoral PhD students to become the next generation of leaders in neuroscience at all societal levels. This training plan has four components: curriculum, research, mentoring and leadership, typically accomplished in under 6 years. The curriculum uses best practices in teaching to provide a foundation in neuroscience that allows for the rigorous identification of a scientific question, design, implementation and analysis of a research project culminating in an independent publication. A core module system challenges students to learn how different fields approach scientific problems. Students participate in journal clubs, a recurring responsible conduct and ethics course, and higher- level courses specifically governed by the interests and needs of each individual student. Research begins with rigorous and challenging rotations that allow students to explore new research areas and new technologies. From these rotations, students select a research laboratory to do their thesis work that is supervised by a faculty mentor and committee of experts. Training in experimental design, rigorous data collection and statistical analysis, is achieved through both didactic course work and direct application to their own work. Our mentoring approach is that it takes a village. From day 1 students have a First Year advisor and a senior student advisor. After selecting a laboratory students have a personal faculty mentor as well as a committee of advocates. They also have access to a senior advisory panel of faculty and both individual and group focused peer-mentoring groups. And finally, leadership includes involvement in directing all aspects of the program as student representatives on all committees as well as TAs for major courses, designing and teaching neuroscience courses and engaging in university and community outreach such as Brain Day and Stanford Summer Research Program. Within each component, we include elements for professional development like reading, critically evaluating and writing scientific papers, preparing and presenting scientific presentations to both professional and lay audiences, networking both academically and in industry as well as professional interactions related to biases such as gender, socioeconomic and race. The program's infrastructure combines faculty, student and administrative feedback on all major levels. There is a program committee, chaired by the director that oversees all infrastructure, an admissions and curriculum committee, used to oversee these two major undertakings and an informal advisory committee that includes other university leadership.

Project Summary/Abstract We propose to understand at cellular and circuit levels how Kiss1-expressing neurons in the anteroventral periventricular hypothalamus (AVPV) regulate female mating behavior. The ventromedial hypothalamus ventrolateralis (VMHvl) and AVPV have been shown to influence diverse female reproductive behaviors and physiology. We recently showed that presynaptic termini of progesterone receptor (PR)-expressing neurons of the VMHvl (Pvl) exhibit significant plasticity in the AVPV across the ovarian cycle. Optogenetic inhibition of this projection of Pvl neurons to the AVPV essentially eliminates female sexual behavior. In preliminary studies, we find that the subset of AVPV neurons expressing the neuropeptide Kisspeptin (Kiss1) are innervated by Pvl neurons, and that Kiss1+ AVPV (Kavpv) neurons are important for regulating female sexual behavior in vivo. Our proposed work is distinct from previous AVPV studies in that we will perform our unbiased circuit mapping, imaging, and functional studies focusing exclusively on Kavpv neurons. The AVPV is heterogeneous not only molecularly but also functionally, and brain-wide connections and behavioral contributions of distinct AVPV neuronal subtypes remain poorly understood. Moreover, and in contrast to prior work in this region, our studies will assess Kavpv neuronal connectivity and function across distinct phases of the female cycle, thereby shedding new light into how physiologically distinct hormonal states influence Kavpv neurons and behavior. In Aim 1, we will map the presynaptic inputs and postsynaptic projections of Kavpv neurons in an unbiased, brain- wide manner and validate the synaptic connectivity across the estrus cycle using electrophysiology and in vivo 2-photon imaging. In Aim 2, we will determine the activity patterns of Kavpv neurons in female during sexual and other social behaviors in freely moving animals. In Aim 3, we will test whether acute manipulation of Kavpv neurons is essential for and, even when females are in a hormonal state that renders them unreceptive, sufficient to induce female sexual behavior. The two PIs have complementary expertise for the proposed studies, and the team is therefore well suited for this project. In summary, if successful our studies will uncover mechanisms whereby an ovarian hormone sensitive hypothalamic circuit regulates female sexual and reproductive behaviors. Health Relatedness: It is well known that ovarian sex hormones can influence behavioral, cognitive, and emotive states in women. How these hormones regulate distinct behaviors and other states at the level of specific neurons and synapses is poorly understood. In addition, translational research has identified diverse neuro- psychiatric illnesses that are influenced by these hormones. Our basic research proposal, if successful, will provide new insights into how ovarian hormone sensitive hypothalamic pathways regulate social interactions in healthy animal models, and they have the potential to suggest new research avenues in translational work focused on ovarian sex hormone influenced neural circuits in disease states.