Optogenetic approaches to study complex neuronal circuits during cognitive aging

描述（由申请人提供）：衰老期间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。人们认为兴奋性/抑制性突触比例的破坏可能代表了衰老期间导致认知能力下降的最终共同途径，并且需要开发新药来恢复改变的突触功能。本项目的目的是利用通道视紫红质-2-黄色荧光蛋白ChR 2（H134 R）-EYFP BAC转基因小鼠研究选择性GABA能突触末端激活，和对囊泡谷氨酸转运体2（VEGF 2）特异的转基因小鼠，以研究选择性谷氨酸能（GLU）突触末端激活。我们将在4-6、10-12和20-22月龄建立一个使用的群体。我们将采用光遗传学刺激，膜片钳记录，钙敏感的荧光成像，共聚焦显微镜，水迷宫行为表征和单细胞RT-PCR（scRT-PCR）的细胞鉴定。热量限制将用于逆转或预防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。我们有一种新的“减少突触准备”，可以进行光遗传学操作，这将使我们能够识别导致损伤老化的关键突触参数。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（ChR 2）的细胞类型特异性表达，所述光激活通道rhodospin-2（ChR 2）是阳离子可渗透通道，其能够响应蓝光使细胞去极化。我们假设，GABA能和GLU突触传递在基底前脑胆碱能神经元在老化过程中发生改变，这样，突触兴奋抑制比（E/I比）的增加有助于认知障碍。如果没有这些转基因小鼠品系中存在的光遗传学资源，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，通过恢复“年轻的突触”来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ChR 2- EYFP在衰老过程中神经元中的功能表达知之甚少。这个项目是创新的，因为我们将选择性地刺激和定量分离的GABA能和GLU突触在一个前所未有的水平老化研究在我们减少突触准备。我们将是第一个在啮齿动物衰老后期在细胞水平上定量ChR 2功能表达的人，我们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.