Two-photon Calcium Imaging of Specific Neuronal Phenotypes

描述（由申请人提供）：双光子激光扫描显微镜（TPLSM）的最新发展与荧光钙指示剂的改进相结合，为神经元活动的体内成像开辟了前所未有的可能性。今天，钙信号的TPLSM是用于研究健康和疾病中的神经元回路和神经元-胶质细胞-血管相互作用的广泛使用的工具。目前将钙指示剂引入脑组织的技术导致神经元和神经胶质的非选择性标记。虽然可以通过添加选择性标记物将神经胶质细胞与神经元区分开，但不能区分不同类型的神经元（兴奋性和多种类型的抑制性细胞）。然而，神经元行为是细胞类型特异性的，并且对所鉴定的神经元细胞类型的生理学和病理生理学的理解对于开发治疗和预防人类疾病的新策略是至关重要的。因此，特定细胞类型的体内识别和功能成像是生物医学成像中未解决的中心问题。原则上，这可以通过使用遗传方法来实现。然而，细胞类型特异性转基因表达仅部分成功。本提案的目标是开发一种替代策略，用于利用特定神经元细胞类型中钙信号的差异行为来识别神经元细胞类型。TPLSM将用于详细评估野生型动物体内可观察到的细胞类型特异性神经生理学特性的独特钙“特征”。这些发现将通过转基因小鼠和体外脑切片制备中鉴定的神经元的测量来验证。该项目的主要可交付成果是一种用于“在线”鉴定兴奋性和多种类型抑制性神经元的工具，它将能够从多种鉴定的细胞类型同时进行钙成像，而无需额外的启动子特异性遗传标记。这种方法将大大推进用于基础神经科学研究和人类疾病动物模型研究的体内双光子钙成像。公共卫生相关性组成哺乳动物大脑的神经元细胞类型的生理鉴定是神经科学中尚未解决的中心问题。拟议的项目将通过提供识别特定细胞类型的工具，推进双光子激光扫描显微镜在神经元组装成像中的应用。同时在体内成像的识别神经元表型是至关重要的，以确定在神经元网络的正常生理状态和脑疾病的偏差程度。

DESCRIPTION (provided by applicant): Recent developments in two-photon laser scanning microscopy (TPLSM) in combination with improvement of fluorescent calcium indicators have opened an unprecedented possibility for in vivo imaging of neuronal activity. Today, TPLSM of calcium signals is a widely used tool for studying of neuronal circuits and neuron-glial-vascular interactions in health and disease. Current techniques of introduction of calcium indicators into the brain tissue result in non-selective labeling of neurons and glia. While glial cells can be distinguished from neurons by adding selective markers, different types of neurons (excitatory and multiple types of inhibitory cells) cannot be discriminated. Yet, neuronal behavior is cell-type specific and understanding of the physiology and pathophysiology of identified neuronal cell types is of prime importance for development of new strategies of treatment and prevention of human disease. Thus, in vivo identification and functional imaging of specific cell types is a central unresolved problem in biomedical imaging. In principle, this can be achieved through use of genetic methods. However, cell-type specific transgenic expression has been only partially successful. The goal of this proposal is to develop an alternative strategy for identification of neuronal cell types taking an advantage of differential behavior of calcium signals in specific neuronal cell types. TPLSM will be used for detailed assessment of unique calcium "signatures" of cell-type specific neurophysiological properties observable in vivo in wild type animals. The findings will be validated by measurements from identified neurons in transgenic mice and in vitro brain slice preparations. The main deliverable of this project, a tool for "on-line" identification of excitatory and multiple types of inhibitory neurons, will enable simultaneous calcium imaging from multiple identified cell types with no need in additional promoter- specific genetic labeling. This approach will significantly advance in vivo two-photon calcium imaging used in basic neuroscience research and for investigation of animal models of human disease. PUBLIC HEALTH RELEVANCE Physiological identification of the neuronal cell types that comprise the mammalian brain is a central unresolved problem in neuroscience. The proposed project will advance application of two-photon laser scanning microscopy to imaging of neuronal assemblies by providing a tool for identification of specific cell types. Simultaneous in vivo imaging of identified neuronal phenotypes is crucial to define the normal state of physiology in neuronal networks and the degree of deviation in brain disease.