Long-term alcohol drinking alters stress engagement of BNST circuit elements

项目摘要 酒精使用障碍是一种慢性复发性脑部疾病，通常是由压力引起的。的来源 压力可以是外部的，例如作用于受影响个人的环境应激源，和/或 内部，内源性信号挑战系统，要求更多的酒精。此K99/R00应用程序旨在 研究长期间歇性饮酒和压力的神经回路。强啡肽 (Dyn)/kappa阿片受体神经肽系统已被证明参与了焦虑期 临床前研究中的酒精依赖。这项建议的K99部分将涉及更广泛的培训 使用细胞特有的光遗传学和化学遗传学来影响酒精影响的行为。具体地说，这 Proposal测试了在长时间戒断过程中对捕食者气味做出反应的压力应对行为的控制 来自终纹床核(BNST)的酒精，这是大脑中具有动态适应的区域 长期酗酒。在第二个K99阶段期间，将使用基于发现的 一种识别酒精饮酒和压力增加后全脑回路图的方法。去调查一下 BNST输入是以无偏见的方式招募的，一种可诱导的cre依赖逆行病毒将用于 FOS-cRE(活性群体靶向重组，TRAP2)小鼠识别BNST的激活输入 对急性捕食者气味应激是时间锁定的；这些输入可能受到以下历史的差异调制 我在喝酒。接下来，将通过iDISCO+全脑组织清除来识别被捕获的BNST电路 用光片显微镜和计算分析结合Allen的免疫标记方法 脑图集。此外，将进行Dyn细胞的全细胞膜片钳记录以描述突触 应激、酒精暴露的BNST投射通路的改变。这些K99实验将产生 新发现的在酒精和压力后招募的全脑神经系统作为未来的途径 R00阶段的独立研究。R00研究将评估不同电路的贡献，使用 包括体内光遗传学和抑制特定BNST投射的多路DREADD方法。这 因此，研究将增强我们对大脑功能回路的理解，并向 开发针对压力和酒精障碍的靶向治疗方法。博士后候选人，劳拉博士 Hwa将利用K99/R00职业发展奖在 北卡罗来纳大学及其合格的指导团队将成为酒精领域的神经科学家 和压力相互作用。她希望在一家R1学院领导一个强有力的研究和教学项目 机构，准备探索酒精和压力在病理行为中的复杂关系 通过先进的神经科学技术。

Project Summary Alcohol use disorder is a chronically relapsing brain disease that is often precipitated by stress. The source of the stress can either be external, such as an environmental stressor acting upon the affected individual, and/or internal, with endogenous signals challenging the system for more alcohol. This K99/R00 application aims to study the neural circuits modulating long-term, intermittent alcohol (IA) drinking and stress. The dynorphin (DYN)/kappa opioid receptor neuropeptide system has been shown to be involved in the dysphoric phases of alcohol dependence in preclinical studies. The K99 portion of this proposal will involve more extensive training using cell-specific optogenetics and chemogenetics to influence alcohol-affected behavior. Specifically, this proposal tests the control of stress coping behavior in response to a predator odor during protracted withdrawal from alcohol in the bed nucleus of the stria terminalis (BNST), a brain area that has DYN adaptations following chronic IA drinking. Additional training will be gained during the second K99 phase using a discovery-based approach to identify whole brain circuit mapping after escalated alcohol drinking and stress. To probe which BNST inputs are recruited in an unbiased manner, an inducible cre-dependent retrograde virus will be used in fos-cre (Targeted Recombination of Active Populations, TRAP2) mice to identify activated inputs to the BNST that are time-locked to acute predator odor stress; these inputs may be differentially modulated by a history of IA drinking. Next, the TRAPed BNST circuitry will be identified through the iDISCO+ whole brain tissue clearing and immunolabeling method using light sheet microscopy and computational analysis aligned with the Allen Brain Atlas. Further, whole cell patch clamp recordings of DYN cells will be performed to describe the synaptic alterations in the stressed, alcohol-exposed BNST-projecting pathways. These K99 experiments will yield newly identified whole brain neural systems recruited after alcohol and stress as future avenues for independent studies for the R00 phase. The R00 studies will assess the contributions of distinct circuits using both in vivo optogenetics and a multiplexed DREADD approach to inhibit specific BNST projections. This research would thereby enhance our understanding of the functional circuitry of the brain and inform the development of targeted treatments for stress and alcohol disorders. The postdoctoral candidate, Dr. Lara Hwa, will use the K99/R00 career development award to master these modern neuroscience techniques at the University of North Carolina with her qualified mentoring team to become an expert neuroscientist in alcohol and stress interactions. She hopes to lead a strong research and teaching program at an R1 academic institution, poised to probe the complex relationship of alcohol and stress underlying pathological behavior through advanced neuroscience techniques.