Optogenetic approaches to study complex neuronal circuits during cognitive aging

描述(由申请人提供)：衰老过程中的认知障碍是导致老年人发病率和死亡率的主要因素，也是当今医学面临的主要挑战之一。兴奋性/抑制性突触比率的紊乱可能是导致衰老过程中认知功能下降的最终共同途径，需要开发新药来恢复改变的突触功能。本项目的目标是建立新的衰老研究实验模型，利用视紫红质-2-黄色荧光蛋白ChR2(H134R)-EYFP BAC转基因小鼠研究囊泡性伽马氨基丁酸(GABA)转运体(VGAT)的选择性激活，并利用囊泡性谷氨酸转运体2(VGlu)特异性转基因小鼠研究选择性谷氨酸(GLU)突触终末激活。我们将建立一个在4-6，10-12和20-22个月龄使用的群体。我们将使用光遗传刺激、膜片钳记录、钙离子敏感的荧光成像、共聚焦显微镜、水迷宫行为特征和单细胞RT-PCR(SCRT-PCR)来鉴定细胞。热量限制将被用来逆转或预防与年龄相关的突触和认知缺陷，以支持突触变化与认知的相关性，并建立治疗干预的实用性。我们有了一种新的“减少突触准备”，可用于光遗传操作，这将使我们能够识别导致衰老的关键突触参数。控制神经元兴奋性的光遗传学方法是基于光激活通道Rhodospin-2(ChR2)的细胞类型特异性表达，ChR2是一种阳离子渗透通道，使细胞能够对蓝光做出反应去极化。我们假设，在衰老过程中，基底前脑胆碱能神经元的GABA能和GLU能突触传递发生改变，从而导致突触兴奋与抑制的比率(E/I)增加，从而导致认知功能障碍。如果没有这些转基因小鼠品系中存在的光遗传资源，支持这一假设将是极其困难的。我们的最终目标是通过使用更好的研究工具和合理的药物设计来恢复“年轻的突触”，从而改善认知障碍老年人的生活质量。这些研究很重要，因为光遗传学技术正在成为选择性研究大脑中神经递质系统的强大工具；然而，人们对ChR2-EYFP在神经元衰老过程中的功能表达知之甚少。这个项目是创新的，因为我们将在一个前所未有的水平上选择性地刺激和定量分离的GABA能和GLU突触，以在我们减少的突触准备中进行衰老研究。我们将首次在细胞水平上量化ChR2在啮齿动物衰老晚期的功能表达，并将为未来的衰老研究建立一个廉价的动物模型。

DESCRIPTION (provided by applicant): Cognitive impairment during aging is a primary factor contributing to morbidity and mortality in the elderly and is one of the major challenges facing medicine today. It is thought that disruption of the excitatory/inhibitory synaptic ratio may represent the final common pathway contributing to cognitive decline during aging, and that new drug development is needed to resurrect altered synaptic function. The goal of this project is to develop new experimental models for aging research utilizing channelrhodopsin-2-yellow fluorescent protein ChR2(H134R)-EYFP BAC transgenic mice specific for the vesicular gamma-aminobutyric acid (GABA) transporter (VGAT) to study selective GABAergic synaptic terminal activation, and transgenic mice specific for the vesicular glutamate transporter 2 (Vglut2) to study selective glutamatergic (GLU) synaptic terminal activation. We will establish a colony for use at 4-6, 10-12 and 20-22 mo of age. We will employ optogenetic stimulation, patch-clamp recording, Ca2+ sensitive fluorescent imaging, confocal microscopy, water maze behavioral characterization and single cell RT-PCR (scRT-PCR) for cell identification. Caloric restriction wil be used to reverse or prevent age-related synaptic and cognitive deficits in order to support the relevance of the synaptic change to cognition and to establish the practicality for therapeutic intervention. We have a new "reduced synaptic preparation" amenable to optogenetic manipulation that will allow us to identify critical synaptic parameters contributing to detrimenta aging. The optogenetic method to control neuronal excitability is based on the cell-type specific expression of the light-activated channelrhodospin-2 (ChR2) which is a cation-permeable channel that enables cell depolarization in response to blue light. We hypothesize that GABAergic and GLU synaptic transmission is altered in basal forebrain cholinergic neurons during aging such that an increased ratio of synaptic excitation to inhibition (E/I ratio) contribues to cognitive impairment. It would be exceedingly difficult to support this hypothesis without the optogenetic resources present in these transgenic mouse lines. Our ultimate objective is to improve the quality of life in cognitively-impaired aged individuals by restoring "youthful synapses" through the use of better research tools and rational drug design. These studies are important because optogenetic techniques are emerging as a powerful tool to selectively study neurotransmitter systems in the brain; however, little is known about the functional expression of ChR2- EYFP in neurons across aging. This project is innovative because we will selectively stimulate and quantitate isolated GABAergic and GLU synapses at an unprecedented level for aging research in our reduced synaptic preparation. We will be the first to quantitate ChR2 functional expression at the cellular level across late aging in rodents, and we will establish an inexpensive animal model for future aging research.