Intact Circuit Assessment of Aging Dopamine Neurons vis Optogenetics and CLARITY

描述（由申请人提供）：在衰老过程中，运动功能下降，精细和快速运动和协调能力不足。实验研究表明，年龄依赖性运动功能障碍与多巴胺（DA）通路功能障碍有关，DA通路起源于黑质黑质（SNc）。然而，我们不知道DA神经元的活动如何在体内黑质神经元的不同层次中在整个衰老过程中变化，什么类型的活动变化先于神经退行性变，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迟神经退行性变和/或行为缺陷。为了表征，第一次在完整的电路，功能和解剖学的老化黑质多巴胺能电路，我们建议使用两个强大的技术进步，在神经科学：一个细胞类型特异性双向控制体内神经元活动的时间精度高（光遗传学）;和一个完整的脑电路映射和表型，切片免费（biologity）。光遗传学使用微生物视蛋白，可以通过靶向启动子在特定细胞中表达并以毫秒级速度打开/关闭的光敏蛋白，从而提供具有高度空间，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可逆的功能获得和丧失，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神经元的子集，我们将利用TH-Cre转基因系以及局部立体定位视蛋白递送和靶向光应用。我们假设，在整个衰老过程中，不同SNc层中的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的差异（Aim 2）和突触输入的变化（Aim 3）。 该提案结合了强大的互补技术（光遗传学，电生理学和神经解剖学），通过在完整电路中进行研究，促进我们对多巴胺能功能和对整个衰老过程中行为的贡献的理解。PI参与了这两种技术的开发，我们的实验室非常适合将这些技术应用于衰老的大脑，重点是DA系统。更好地了解老化SNc中DA神经元的特性可以帮助识别电路目标和/或行为/营养方法，以延迟/逆转这些神经元和运动功能中与年龄相关的改变。

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.