Intact Circuit Assessment of Aging Dopamine Neurons vis Optogenetics and CLARITY

描述(申请人提供)：随着年龄的增长，运动功能下降，精细和快速的运动和协调能力受损。实验研究表明，起源于黑质致密部(SNC)的多巴胺(DA)通路功能障碍与年龄相关的运动障碍有关。然而，我们还不清楚在体内黑质神经元不同层次中DA神经元的活动是如何随着年龄的增长而变化的，在神经退行性变之前是什么类型的活动变化，这些活动变化是如何影响行为的，以及恢复扰动的活动是否可以延缓神经退化和/或行为缺陷。为了首次在完整的回路中表征老化的黑质多巴胺能回路的功能和解剖结构，我们建议使用神经科学中的两项强大的技术进步：一项用于高时间精度的活体神经元活动的细胞类型特异性双向控制(光遗传学)；另一项用于完整的脑回路映射和表型分析，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，并以毫秒的速度打开/关闭，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，并提供特定神经元表型功能的可逆获得和丧失，使我们能够以前所未有的方式研究神经系统和疾病。为了针对SNC DA神经元的亚群，我们将利用TH-CRE转基因系以及局部立体定位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神经元有不同的行为贡献(目标1)，这是由于它们内在兴奋性的差异(目标2)和突触输入的变化(目标3)。这一建议结合了强大的互补技术(光遗传学、电生理学和神经解剖学)，通过在完整的电路中进行研究，促进了我们对多巴胺能功能和在整个衰老过程中对行为的贡献的理解。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老化的大脑，重点放在DA系统上。更好地了解衰老黑质中DA神经元的特性有助于识别电路靶点和/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.