Molecular genetic dissection of central amygdala microcircuitry underlying fear a

描述(申请人提供)：精神障碍，如创伤后应激障碍、抑郁症和广泛性焦虑症，越来越多地被认为是特定大脑回路的功能障碍，而不是全球“大脑化学”的变化。为了在了解潜在疾病机制的基础上开发新的治疗方法，有必要了解受影响回路的正常功能。在这项应用中，我们建议应用新的、基于基因的技术来操纵神经元功能和绘制神经元连接图，以剖析条件性恐惧及其消亡背后的微电路。我们的重点是了解位于杏仁中央核(CEA)的中间神经元亚群的功能，CEA是大脑中参与情绪的区域。这些神经元中的一个亚群以蛋白激酶C-4(PKC-4)的表达为标志。我们的初步数据表明，这些神经元的遗传失活增强了条件性冻结，这表明这些神经元通常可能起到选通CEA输出的作用。利用最近开发的基于伊维菌素(IVM)门控氯离子通道的神经元沉默遗传系统，以及针对CEA中PKC-4细胞特异表达这一异构体通道的“交叉”策略，我们将检验这一假设，并研究这些神经元在恐惧学习和恐惧消退以及在无条件恐惧和焦虑(特定目标I)中的功能作用。在特定的目标II中，我们将使用基于光(通道视紫红质-2)或化学激活的神经元激活策略来进一步研究这些神经元的作用。这些实验将分别测试PKC-4神经元在杏仁核介导的情绪行为中的必要性和充分性。在特定的目标III中，我们将使用基于遗传的神经元跟踪和电生理技术来映射这些神经元的输入和输出。最后，在特定的目标IV中，我们将检验这一假设，即抗焦虑药物，如苯二氮卓类药物的行为效应需要激活PKC-4神经元。这些研究应该开始在特定神经元亚型的粒度水平上提供杏仁核的功能解剖，并可能为精神疾病的治疗干预确定新的细胞靶点。公共卫生相关性：抑郁症、精神分裂症和创伤后应激障碍(PTSD)等精神疾病对公共健康造成重大影响，但目前诊断和治疗这些疾病的方法并不充分。为了开发新一代更有效、副作用更少的治疗这些疾病的方法，有必要确定潜在的大脑回路受损，了解这些回路在情绪行为中的正常功能，并描述这一功能在给定的障碍中是如何改变的。本提案应用了一系列新的、基于基因的工具，以以前从未实现的特异性水平剖析神经回路功能，以了解控制信息通过杏仁核流动的“门控”机制，杏仁核是学习(和“忘记”)恐惧的重要大脑结构。

DESCRIPTION (provided by applicant): Psychiatric disorders, such as PTSD, depression and generalized anxiety disorder, are increasingly being recognized as dysfunctions of specific brain circuits, rather than alterations in global "brain chemistry." In order to develop new therapeutic approaches based on an understanding of underlying disease mechanisms, it is necessary to understand the normal function of the affected circuits. In this application, we propose to apply new, genetically based, techniques for manipulating neuronal function and mapping neuronal connectivity, to dissect the microcircuitry that underlies conditioned fear and its extinction. Our focus is on understanding the function of subpopulations of interneurons located in the central nucleus of the amygdala (CeA), a brain region involved in emotion. One subset of these neurons is marked by expression of protein kinase C-4 (PKC-4). Our preliminary data indicate that genetically based inactivation of these neurons enhances conditioned freezing, suggesting that these neurons may normally act to gate output from CeA. Using a recently developed genetic system for neuronal silencing, based on an ivermectin (IVM)-gated chloride channel, and an "intersectional" strategy to target expression of this heteromeric channel exclusively to PKC-4 cells in CeA, we will test this hypothesis and investigate the functional role of these neurons in fear learning and fear extinction, as well as in unconditional fear and anxiety (Specific Aim I). In Specific Aim II, we will further investigate the role of these neurons using neuronal activation strategies based on light (channelrhodopsin-2) or chemical activation. These experiments will test the necessity and sufficiency, respectively, of PKC-4 neurons in emotional behaviors mediated by the amygdala. In Specific Aim III, we will map the inputs and outputs to and from these neurons, using genetically based neuronal tracing and electrophysiological techniques. Finally, in Specific Aim IV we will test the hypothesis that activation of PKC-4 neurons is required for the behavioral effects of anxiolytic drugs, such as benzodiazepines. These studies should begin to provide a functional dissection of the amygdala at the level of granularity of specific neuronal subtypes, and may identify new cellular targets for therapeutic intervention in psychiatric disorders. PUBLIC HEALTH RELEVANCE: Psychiatric disorders, such as depression, schizophrenia and post-traumatic stress disorder (PTSD), exact a significant toll on public health, yet current methods to diagnose and treat them are inadequate. In order to develop a new generation of more effective treatments for these illnesses, with fewer side-effects, it is necessary to identify the underlying brain circuits that are impaired, understand the normal function of these circuits in emotional behavior, and describe how this function is altered in a given disorder. The present proposal applies an arsenal of new, genetically based, tools for dissecting neural circuit function at a level of specificity that has not previously been achieved, to understand the 'gating' mechanisms that control the flow of information through the amygdala, a brain structure important in learning (and "unlearning") fear.