Molecular genetic dissection of central amygdala microcircuitry underlying fear a

描述（由申请人提供）：精神疾病，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脑回路的功能障碍，而不是全球“脑化学”的改变。“为了在了解潜在疾病机制的基础上开发新的治疗方法，有必要了解受影响电路的正常功能。在这个应用程序中，我们建议应用新的，基于遗传的，操纵神经元功能和映射神经元连接的技术，解剖微电路的基础条件性恐惧及其灭绝。我们的重点是了解位于杏仁核中央核（CeA）的中间神经元亚群的功能，这是一个涉及情绪的大脑区域。这些神经元的一个子集以蛋白激酶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冻结，这表明这些神经元可能正常发挥作用，从CeA门输出。使用最近开发的遗传系统的神经元沉默，伊维菌素（IVM）门控氯离子通道的基础上，和一个“交叉”的策略，专门针对表达这种异源通道的PKC-4细胞在CeA，我们将测试这一假设，并调查这些神经元的功能作用，在恐惧学习和恐惧灭绝，以及在无条件的恐惧和焦虑（具体目标I）。在特定目标II中，我们将进一步研究这些神经元的作用，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。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。在具体目标III中，我们将使用基于遗传的神经元追踪和电生理技术来映射这些神经元的输入和输出。最后，在具体目标IV中，我们将检验以下假设：PKC-4神经元的激活是抗焦虑药物（如苯二氮卓类）的行为效应所必需的。这些研究应该开始提供一个杏仁核的功能解剖在特定的神经元亚型的粒度水平，并可能确定新的细胞靶点的治疗干预精神疾病。公共卫生相关性：抑郁症、精神分裂症和创伤后应激障碍等精神疾病对公共卫生造成重大损害，但目前诊断和治疗这些疾病的方法不足。为了开发新一代更有效的治疗方法，减少副作用，有必要识别受损的潜在脑回路，了解这些回路在情绪行为中的正常功能，并描述这种功能在特定疾病中是如何改变的。目前的建议应用了一个新的，基于遗传的工具库，用于解剖神经回路功能的特异性水平，以前没有实现，以了解控制信息通过杏仁核，学习（和“忘记”）恐惧的重要大脑结构的流动的“门控”机制。

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.