Intact Circuit Assessment of Aging Dopamine Neurons vis Optogenetics and CLARITY

描述（由申请人提供）：随着年龄的增长，运动功能下降，精细、快速运动和协调能力下降。实验研究将年龄依赖性运动障碍与多巴胺能（DA）通路功能障碍联系起来，多巴胺能通路起源于黑质致密部（SNc）。然而，我们不了解DA神经元的活动如何在体内不同层次的神经神经元中随着年龄的增长而变化，什么类型的活动变化发生在神经退行性变之前，这些活动变化如何影响行为，以及恢复受干扰的活动是否可以延缓神经退行性变和/或行为缺陷。为了首次在完整的神经回路中表征老化的神经多巴胺能回路的功能和解剖结构，我们建议利用神经科学领域的两项强大技术进步：一是在体内以高时间精度（光遗传学）对神经元活动进行细胞类型特异性双向控制；另一种是完整脑回路制图和表型分析，无需切片（CLARITY）。光遗传学使用微生物视蛋白，这是一种光敏蛋白，可以通过靶向启动子在特定细胞中表达，并以毫秒级的速度打开/关闭，从而提供具有高空间、时间和遗传特异性的细胞功能控制。它们能够控制神经回路的电活动，并赋予特定神经元表型功能的可逆增益和丧失，这使我们能够以前所未有的方式研究神经系统和疾病。为了靶向SNc DA神经元亚群，我们将利用TH- Cre转基因系以及局部立体定向视蛋白传递和靶向光照。我们假设，在整个衰老过程中，不同SNc层的DA神经元具有不同的行为贡献（Aim 1），这是由于其内在兴奋性（Aim 2）的差异和突触输入的变化（Aim 3）。该建议结合了强大的互补技术（光遗传学、电生理学和CLARITY的神经解剖学），通过对完整回路的研究，促进我们对多巴胺能功能和对整个衰老过程行为的贡献的理解。PI参与了这两项技术的开发，我们的实验室处于理想的位置，可以将这些技术应用于衰老的大脑，重点是DA系统。更好地了解衰老SNc中DA神经元的特性有助于确定回路靶点和/或行为/营养方法，以延缓/逆转这些神经元和运动功能的年龄相关改变。

DESCRIPTION (provided by applicant): During aging, motor function declines, with deficits in fine and fast movement and coordination. Experimental studies associate age-dependent motor deficits with the malfunction of dopaminergic (DA) pathway, which originates in the substantia nigra pars compacta (SNc). However we do not understand how the activity of DA neurons varies throughout aging in the different tiers of nigral neurons in vivo, what type of activity changes precede neurodegeneration, how these activity changes affect behavior, and whether restoring perturbed activity can delay neurodegeneration and/or behavioral deficits. To characterize, for the first time in the intact circuit, the function and anatomy of aging nigral dopaminergic circuits, we propose to use two powerful technological advances in neuroscience: one for cell-type specific bidirectional control of neuronal activity in vivo with high temporal precision (optogenetics); and one for intact brain circuit mapping and phenotyping, slicing-free (CLARITY). Optogenetics uses microbial opsins, light-sensitive proteins that can be expressed in specified cells via targeting promoters and turned on/off with millisecond speed, thus providing control of cell function with high spatial, temporal, and genetic specificity. Their abilty to control the electrical activity of neural circuits and confer reversible gain and loss of functin of specific neuronal phenotypes allows us to study neural systems and diseases in unprecedented manner. To target subsets of SNc DA neurons we will take advantage of the TH- Cre transgenic lines as well as localized stereotaxic opsin delivery and targeted light application We hypothesize that throughout aging, DA neurons in different SNc tiers have distinct behavioral contributions (Aim 1), which is due to differences in their intrinsic excitability (Aim 2) and changes in synaptic inputs (Aim 3). This proposal combines powerful complementary techniques (optogenetics, electrophysiology, and neuroanatomy by CLARITY) to advance our understanding of dopaminergic function and contribution to behavior throughout aging by performing studies in the intact circuit. The PI has been involved in the development of both techniques and our laboratory is ideally positioned to apply these techniques to the aging brain with a focus on the DA system. A better understanding of the properties of DA neurons in the aging SNc can aid in identifying circuit targets and/or behavioral/nutritional methods to delay/reverse age-related alterations in these neurons and in motor functions.