Joanna Dabrowska - US grants

DESCRIPTION (provided by applicant): According to the World Health Organization depression affects 121 million people worldwide. Chronic stress and thus increased corticotrophin releasing factor (CRF) signaling is indicated as a potential risk factor for depression. Conversely, depression is linked to decreased function of the central oxytocin (OT) system, which has been also shown to attenuate stress response. Therefore this proposal is designed to explore a complex interaction between the CRF and OT systems in two key brain regions involved in the response to stress and affective behavior. We have recently shown that an intimate neuroanatomical circuit exists between neurons expressing these neurotransmitters in the paraventricular nucleus of the hypothalamus (PVN), a central hub of the stress axis, and in the bed nucleus of the stria terminalis (BNST), a brain region responsible for the affective component of the stress response. Hence, OT neurons in the PVN selectively express CRF type 2 receptors (CRFR2) and send oxytocinergic input to the BNST, where OT axons and terminals also express CRFR2. Notably, these OT-positive axons make perisomatic contacts with CRF neurons of the BNST, which in turn express high levels of the OT receptor (OTR) (Dabrowska et al., 2011). Here we propose a novel concept of a feedback loop between the CRF and OT systems, which could modulate the stress response and affective behavior. In this model, local CRF release following stress would activate CRFR2 on OT neurons in the PVN to induce somatodendritic OT release in the PVN and terminal OT release in the BNST, which would consequently influence emotional behavior. Therefore, the reciprocal interaction between the CRF and OT systems might play a critical role in regulating affective behavior, and I hypothesize that CRFR2 is a key component of this relationship. Thus with this proposal I will determine the role of CRFR2 in the CRF-OT relationship using integrative neuroanatomical, neurophysiological, in vivo microdialysis, and behavioral approaches. I will answer the following critical questions: Specific Aim 1) what is the origin of CRF terminals, which make synaptic contacts with magnocellular OT neurons in the PVN, SA2) whether and how excitability of these OT neurons in the PVN can be modulated by activation of CRFR2, SA3) whether and how activation of CRFR2 impacts OT release in the BNST and PVN, and SA4) whether and how activation of CRFR2 and OTR in the BNST impact affective behavior in control and chronically stressed rats? Four analytical approaches will be used to directly address these questions: SA1) adenoviral-based neuronal tracing studies combined with confocal microscopy, SA2) in vitro whole-cell patch-clamp electrophysiological recordings from OT neurons in the PVN, SA3) in vivo microdialysis of the OT in the BNST and PVN, and SA4) behavioral experiments to define the role of CRFR2 in the CRF-OT interaction in regulating affective behavior following chronic stress. Understanding the nature of the feedback loop between the PVN and the BNST at the functional level would represent a milestone in our understanding of the etiology of the stress-induced affective disorders and could ultimately provide novel directions for the treatment strategies. I am pursuing a career path to become an NIH-funded independent investigator and I already possess the motivation and expertise to independently carry out specific aims 1 and 4. However, to successfully carry out work proposed in specific aims 2 and 3 I will require additional mentoring. I have prepared a detailed mentoring plan and will receive specific training from world-renowned scientists during the mentored (K99) phase of the award. My primary mentor, Dr. Donald Rainnie and my co-mentor, Dr. Larry Young, are both senior, NIH- funded faculty members in the Department of Psychiatry and Behavioral Sciences at Emory University. Their labs are located at the Yerkes National Primate Research Center. Dr. Rainnie is an acknowledged expert in in vitro patch-clamp recording techniques, who has over 20 years experience in electrophysiological recordings from neurons of the extended amygdala. Dr. Rainnie has published key papers on the regulation of these neurons by neurotransmitters such as CRF, and he will personally train me in the whole cell patch-clamp technique as well as teach me the foundations of neurophysiology. Dr. Young, a world-renowned expert in the neurobiology of oxytocin and social behavior, will provide training in the pharmacology of oxytocin and the oxytocin receptor, and he will guide me in the research pertaining to the oxytocin side of the project. Furthermore, Dr. Young will facilitate my training in in vivo microdialysis for oxytocin wth his current collaborators from Germany. Dr. Rainer Landgraf from Max Planck Institute of Psychiatry will train me in the radioimmunoassay technique developed in his laboratory, which has the necessary sensitivity that is needed to measure extracellular oxytocin levels in the PVN and BNST. Dr. Oliver Bosch from the University of Regensburg will train me in the construction of custom-made microdialysis probes with the pore size suitable for neuropeptides, as well as probes validation and optimal implantation into the PVN and BNST. My mentoring team will work in a collaborative and organized manner to foster my transition to an independent research career towards the end of the mentored phase of the award. This K99/R00 Career Development Award will enable me to pursue specialized, multi-disciplinary training in the research on the neuropeptide modulation of the affective behavior with great promise for translating my discoveries into novel and better treatments for psychiatric disorders. The mentored phase will accelerate my development towards becoming an independent investigator in an academic setting. In the end of mentored (K99) phase of the award, I will be a highly competitive applicant for academic junior faculty positions. My career objective is to conduct independent research to understand how stress and affect interact and elucidate their respective roles in the etiology of mental disorders like depression and anxiety. PUBLIC HEALTH RELEVANCE: Corticotrophin releasing factor and oxytocin are two powerful neuropeptides that regulate the stress response and emotional behavior. We have shown that these two neurotransmitters are linked in a reciprocal anatomical circuit. In this proposal we will use a multidisciplinary approach to elucidate how they functionally interact with each other, and what role this interaction plays in the etiology of mental disorders like depression and anxiety. Understanding the functional interaction between these two neuropeptide systems could ultimately provide novel directions for the development of pharmacotherapy for stress-related affective disorders.

SUMMARY: Stress can precipitate the onset of psychiatric disorders, including generalized anxiety disorder (GAD) and posttraumatic stress disorder. Currently available pharmacotherapies have limited efficacy in anxiety disorders - hence there is an urgent need to identify new approaches that can ameliorate anxiety. Stress-induced abnormalities in neuronal activity lead to long-lasting changes in anxiety, hence targeting the mechanisms underlying these effects in stress-sensitive brain regions may provide new insight for treatment. In rodent and human studies, the bed nucleus of the stria terminalis (BNST) has emerged as a key brain region translating stress into sustained changes in anxiety. Repeated stress increases the activity of BNST output neurons, which produce the stress hormone corticotropin-releasing factor (CRF). This parallels a state of anxiety, fear and hypervigilance, hallmarks of stress-induced mental disorders. Accordingly, activity of the BNST is elevated in patients suffering from GAD. Therefore, the factors that regulate BNST output have untapped potential as novel therapeutic targets. We propose that one such target may be engaging local inhibitory interneurons to strengthen inhibition of BNST output neurons. Oxytocin (OT) has been shown to inhibit the stress response and reduce anxiety, but the sites or cellular targets of these OT actions remain unclear. BNST neurons express OT receptors and receive OT input from the paraventricular nucleus of the hypothalamus (PVN). Our preliminary data indicate that OT activates a subset of inhibitory interneurons in the BNST. Based on this premise, the main objective of this proposal is to establish whether by activating the inhibitory interneurons, OT inhibits BNST output and thus reduces anxiety. The central hypothesis of this proposal is that OT ameliorates stress-induced anxiety-like behavior by engaging interneurons that provide inhibitory control over BNST output (CRF) neurons. This innovative concept will be investigated using cutting- edge experimental approaches, including OT promoter-driven expression of inhibitory DREADDs (Designer Receptors Exclusively Activated by Designer Drugs) to selectively suppress the release of OT in the projection from the PVN to the BNST (OT PVNèBNST pathway). Rigorously designed experiments combining behavioral and electrophysiological measures will test the central hypothesis with the following specific aims: 1) Determine the role of OT in the BNST in the regulation of anxiety and fear in male and female rats and 2) Determine the role of OT in regulating the activity of BNST neurons in male and female rats. Accomplishing these Aims will fill a significant knowledge gap by establishing the role of OT in the BNST in ameliorating the effects of stress. We expect that by refining our understanding of central OT effects this proposal will have a positive impact on uncovering the mechanisms underlying the neurobiology of stress-induced psychiatric disorders.

Contact PD/PI: Dabrowska, Joanna SUMMARY: Stress can precipitate the onset of psychiatric disorders, including generalized anxiety disorder (GAD) and posttraumatic stress disorder. Currently available pharmacotherapies have limited efficacy in anxiety disorders - hence there is an urgent need to identify new approaches that can ameliorate anxiety. Stress-induced abnormalities in neuronal activity lead to long-lasting changes in anxiety, hence targeting the mechanisms underlying these effects in stress-sensitive brain regions may provide new insight for treatment. In rodent and human studies, the bed nucleus of the stria terminalis (BNST) has emerged as a key brain region translating stress into sustained changes in anxiety. Repeated stress increases the activity of BNST output neurons, which produce the stress hormone corticotropin-releasing factor (CRF). This parallels a state of anxiety, fear and hypervigilance, hallmarks of stress-induced mental disorders. Accordingly, activity of the BNST is elevated in patients suffering from GAD. Therefore, the factors that regulate BNST output have untapped potential as novel therapeutic targets. We propose that one such target may be engaging local inhibitory interneurons to strengthen inhibition of BNST output neurons. Oxytocin (OT) has been shown to inhibit the stress response and reduce anxiety, but the sites or cellular targets of these OT actions remain unclear. BNST neurons express OT receptors and receive OT input from the paraventricular nucleus of the hypothalamus (PVN). Our preliminary data indicate that OT activates a subset of inhibitory interneurons in the BNST. Based on this premise, the main objective of this proposal is to establish whether by activating the inhibitory interneurons, OT inhibits BNST output and thus reduces anxiety. The central hypothesis of this proposal is that OT ameliorates stress-induced anxiety-like behavior by engaging interneurons that provide inhibitory control over BNST output (CRF) neurons. This innovative concept will be investigated using cutting- edge experimental approaches, including OT promoter-driven expression of inhibitory DREADDs (Designer Receptors Exclusively Activated by Designer Drugs) to selectively suppress the release of OT in the projection from the PVN to the BNST (OT PVN BNST pathway). Rigorously designed experiments combining behavioral and electrophysiological measures will test the central hypothesis with the following specific aims: 1) Determine the role of OT in the BNST in the regulation of anxiety and fear in male and female rats and 2) Determine the role of OT in regulating the activity of BNST neurons in male and female rats. Accomplishing these Aims will fill a significant knowledge gap by establishing the role of OT in the BNST in ameliorating the effects of stress. We expect that by refining our understanding of central OT effects this proposal will have a positive impact on uncovering the mechanisms underlying the neurobiology of stress-induced psychiatric disorders. Project Summary/Abstract Page 6