Genetic approaches to image neural activity in the brain

描述（由申请人提供）：目标是探索基于遗传学的方法来绘制小鼠嗅觉系统中特定神经元群体的活动。该方法利用了完整小鼠神经系统中神经元活动的遗传编码指示器（synapto-pHluorin），以及可以从该指示器中光谱分离出的新型荧光蛋白。该方法将用于解决嗅觉回路的感觉处理的基本问题。具体目的1是通过建立体内检测肾小球活性的方法来测试气味受体配体的生理相关性。基因靶向将用于标记由表达特定气味受体的嗅觉感觉神经元形成的肾小球。靶肾小球的反应将与背球其他肾小球的反应进行比较，以确定确定配体的相对亲和力。将使用先前确定的气味剂，并筛选新的化学物质，以在体内识别高亲和力配体。这些实验将为从各种分析中放置功能性or配体数据提供必要的背景，并将为未来旨在操纵小鼠嗅觉受体表达和嗅觉功能的实验铺平道路。特异性目的2是将synaptophorin靶向嗅球二尖瓣细胞，在体内研究其在肾小球层的侧向抑制作用。这将首次允许在体内对二尖瓣细胞原代树突的突触释放进行选择性成像。该方法解决了一个关于嗅球回路嗅觉处理的根本重要问题，这是目前可用方法无法解决的。这些研究探索了一种新的技术来建立研究神经元功能的实验系统。这些技术的进一步发展有望为研究大脑功能提供一个新的窗口，通过对基因定义的神经元群体的活动进行光学成像，而这些神经元群体以前很难在完整的神经组织中进行功能分离。

DESCRIPTION (provided by applicant): The goal is to explore genetically-based methods for mapping activity in defined populations of neurons in the mouse olfactory system. The approach makes use of a genetically-encoded indicator for neuronal activity (synapto-pHluorin) in the intact mouse nervous system, and novel fluorescent proteins that can be spectrally separated from the indicator. The approach will be used to address fundamental questions of sensory processing by olfactory circuits. Specific Aim 1 is to test the physiological relevance of odorant receptor ligands by establishing an in vivo assay to image glomerular activity. Gene targeting will be used to label the glomeruli formed by olfactory sensory neurons that express defined odorant receptors. Responses from targeted glomeruli will be compared with responses from other glomeruli in the dorsal bulb to determine relative affinities for defined ligands. Previously identified odorants will be used and new chemical will be screened to identify high-affinity ligands in vivo. The experiments will provide a necessary context in which to place functional OR-ligand data from a variety of assays, and will pave the way for future experiments that aim to manipulate odorant receptor expression and olfactory function in mice. Specific Aim 2 is to target synaptopHluorin to mitral cells of the olfactory bulb to study lateral inhibition in the glomerular layer in vivo. This will permit, for the first time, selective imaging of synaptic release from mitral cell primary dendrites in vivo. The approach addresses a fundamentally important question regarding olfactory processing by olfactory bulb circuits that can not be addressed using currently available methods. These studies explore a new technique to set up experimental systems for studying neuronal function in vivo. Further development of these technologies promise to provide a novel window into brain function by allowing optical imaging of activity in genetically-defined neuronal populations that were previously difficult to isolate functionally in intact nervous tissue.