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，通常由压力引起。的来源 压力可以是外部的，例如作用于受影响个体的环境压力源，和/或 内部，内源性信号挑战系统更多的酒精。此K99/R 00应用程序旨在 研究调节长期间歇性饮酒和压力的神经回路。强啡肽 (DYN)已经证明，/κ阿片受体神经肽系统参与了抑郁症的烦躁阶段。 酒精依赖的临床前研究本提案的K99部分将涉及更广泛的培训 使用细胞特异性光遗传学和化学遗传学来影响酒精影响的行为。具体来说， 一项提案测试了在长期戒断过程中，对捕食者气味的压力应对行为的控制 终纹床核（BNST）中的酒精，这是一个大脑区域， 长期酗酒在第二个K99阶段，将使用基于发现的 方法来确定全脑电路映射后，逐步增加饮酒和压力。为了探索 BNST输入以无偏的方式招募，将使用诱导型cre依赖性逆行病毒， fos-cre（活动群体的靶向扩增，TRAP 2）小鼠，以识别BNST的激活输入 这些输入可能被不同的历史所调制， 喝酒。接下来，将通过iDISCO+全脑组织清除来识别TRAPed BNST电路 和免疫标记方法，使用光片显微镜和与艾伦 大脑图谱此外，DYN细胞的全细胞膜片钳记录将被执行以描述突触的突触结构。 在压力下，酒精暴露的BNST投射途径的改变。这些K99实验将产生 新发现的全脑神经系统在酒精和压力后被招募为未来的 R 00阶段的独立研究。R 00研究将评估不同回路的贡献， 在体内光遗传学和多重DREADD方法两者中抑制特异性BNST投射。这 因此，研究将增强我们对大脑功能回路的理解，并为我们提供信息。 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，将使用K99/R 00职业发展奖，掌握这些现代神经科学技术， 北卡罗来纳州大学与她合格的指导团队，成为一个专家神经学家在酒精 和压力的相互作用。她希望在R1学术机构领导一个强大的研究和教学计划。 该机构准备探索酒精和压力之间的复杂关系，这些关系是病理行为的基础 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.