CRF-Oxytocin Interaction in the Regulation of Stress and Affect

根据世界卫生组织的数据，全球有1.21亿人患有抑郁症。慢性应激和因此增加的促肾上腺皮质激素释放因子（CRF）信号被认为是抑郁症的潜在危险因素。相反，抑郁症与中枢催产素（OT）系统功能下降有关，该系统也被证明可以减轻压力反应。因此，本研究旨在探索CRF和OT系统在两个关键大脑区域中对压力和情感行为的反应之间的复杂相互作用。我们最近的研究表明，在下丘脑室旁核（PVN）和终纹床核（BNST）中表达这些神经递质的神经元之间存在密切的神经解剖学回路，PVN是应激轴的中心枢纽，而终纹床核是负责应激反应的情感成分的大脑区域。因此，PVN中的OT神经元选择性地表达CRF 2型受体（CRFR2），并向BNST发送催产素输入，其中OT轴突和终端也表达CRFR2。值得注意的是，这些OT阳性轴突与BNST的CRF神经元进行细胞周围接触，后者反过来表达高水平的OT受体（OTR） （Dabrowska et al., 2011）。在此，我们提出了一个新的概念，即在CRF和OT系统之间存在一个反馈回路，它可以调节应激反应和情感行为。在本模型中，应激后局部释放CRF会激活PVN OT神经元上的CRFR2，诱导PVN体树突性OT释放和BNST终端OT释放，从而影响情绪行为。因此，CRF和OT系统之间的相互作用可能在调节情感行为中发挥关键作用，我假设CRFR2是这种关系的关键组成部分。因此，根据本提案，我将利用综合神经解剖学、神经生理学、体内微透析和行为方法确定CRFR2在CRF-OT关系中的作用。针对性我将回答以下关键问题:1)什么是CRF的起源终端,使在PVN与magnocellular OT神经元突触联系,SA2)是否和如何这些OT神经元的兴奋性PVN可调制CRFR2激活,SA3)是否以及如何激活BNST CRFR2影响无法释放的,PVN和SA4)是否和如何激活CRFR2和OTR BNST影响情感行为控制和长期强调老鼠?四种分析方法将用于直接解决这些问题：SA1)基于腺病毒的神经元示迹研究结合共聚焦显微镜，SA2) PVN中OT神经元的体外全细胞膜片钳电生理记录，SA3) BNST和PVN中OT的体内微透析，SA4)行为实验，以确定CRFR2在CRF-OT相互作用中调节慢性应激后情感行为的作用。在功能水平上理解PVN和BNST之间反馈回路的本质将是我们理解应激性情感障碍病因学的一个里程碑，并可能最终为治疗策略提供新的方向。我正在追求成为美国国立卫生研究院资助的独立研究者的职业道路，我已经拥有独立执行具体目标1和4的动机和专业知识。然而，为了成功地开展具体目标2和3中提出的工作，我将需要额外的指导。我已经准备了详细的指导计划，并将在导师（K99）阶段接受世界知名科学家的具体培训。我的主要导师唐纳德·雷尼博士和我的共同导师拉里·杨博士都是埃默里大学精神病学和行为科学系由美国国立卫生研究院资助的资深教员。他们的实验室位于耶基斯国家灵长类动物研究中心。Rainnie博士是体外膜片钳记录技术的公认专家，他在扩展杏仁核神经元的电生理记录方面拥有超过20年的经验。Dr. Rainnie曾发表过关于CRF等神经递质调控这些神经元的重要论文，他将亲自培训我全细胞膜片钳技术，并教授我神经生理学的基础知识。Young博士是世界知名的催产素和社会行为神经生物学专家，他将提供催产素和催产素受体药理学方面的培训，他将指导我在该项目中与催产素相关的研究。此外，杨医生将帮助我在体内微透析催产素与他目前的合作者来自德国。马克斯·普朗克精神病学研究所的Rainer Landgraf博士将训练我使用他实验室开发的放射免疫测定技术，该技术具有测量PVN和BNST细胞外催产素水平所需的必要灵敏度。雷根斯堡大学的Oliver Bosch博士将指导我定制适合神经肽孔径的微透析探针，以及探针的验证和最佳植入PVN和BNST。我的指导团队将以协作和有组织的方式工作，以促进我在奖项指导阶段结束时向独立研究事业的过渡。K99/R00职业发展奖将使我能够在情感行为的神经肽调节研究方面进行专业的多学科培训，并有望将我的发现转化为新的更好的精神疾病治疗方法。指导阶段将加速我的发展，成为一个独立的研究者在学术设置。在导师（K99）奖项阶段结束后，我将成为一名极具竞争力的学术初级教师职位申请人。我的职业目标是进行独立研究，了解压力和影响是如何相互作用的，并阐明它们各自在抑郁症和焦虑症等精神障碍病因学中的作用。

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.