Development and use of the Two-Photon Optogenetic toolbox

描述（由申请人提供）：神经回路是人类各种行为的基础。神经系统的异常状态，无论是急性还是慢性，都是导致心理健康疾病的原因，影响着超过四分之一的18岁以上的美国人。“执行”区域，如人类的背外侧前额叶皮层（dlPFC）（大约相当于啮齿动物的内侧前额叶皮层（mPFC））是特别感兴趣的主题，因为对人类的功能成像、电生理和死后研究表明，这些区域的细胞和神经生理异常与精神疾病（如精神分裂症、情绪障碍和成瘾）有关。dlPFC的巨大复杂性以及它与许多不同大脑区域的相互作用使得区分这些疾病的特定病理神经回路极其困难。我们如何探测神经系统，以了解神经回路中的活动模式如何导致行为？我们能更好地理解病理状态吗？为了解决这些问题，我们的实验室设计了基于特定神经元亚型遗传特征的光学激活和沉默神经元的方法，（例如CaMKII-alpha作为兴奋性锥体神经元的标记物或抑制性神经元子集中的小白蛋白），使用光门控离子通道和泵，一种称为“光遗传学”的方法。在本提案的第一部分中，我们的目标是通过使用双光子（2P）显微镜利用这些蛋白质的光激活的独特特征来扩展这些技术，以获得超越遗传特征的特异性；更具体地说，该项目将在单个细胞的分辨率下实现神经系统的空间受限激活和沉默（2P光遗传学）。此外，我们将使用单个神经元的2P激活对不同神经元亚型的mPFC第V层的功能性神经元连接进行高通量映射，以便更好地了解它们如何作为一个单元发挥作用。该提案的这一部分还将展示该工具在使用离体制备的大脑所有区域的通用性，这将为研究正常和病理神经系统状态的研究人员提供丰富的信息。在本项目的第二部分，我们将使用2P光遗传学来了解mPFC的动态皮质功能。局部皮质过程，如同步性，其功能障碍与精神疾病（如精神分裂症）有关，将被研究以了解导致这些现象的潜在细胞和网络机制。综上所述，这些工具有望帮助我们深入了解mPFC中不同神经元之间的功能连接矩阵，以及它们如何协同工作，提供有效的信息处理，从而产生行为。

DESCRIPTION (provided by applicant): Neural circuitry underlies human behavior in all of its forms. Aberrant states in the nervous system, both acute and chronic are the cause of mental health diseases that affect more than a quarter of Americans over the age of 18. 'Executive' regions such as the dorsolateral prefrontal cortex (dlPFC) in humans (approximately equivalent to the medial prefrontal cortex (mPFC) in rodents) are the subject of particular interest as functional imaging, electrophysiological, and post-mortem studies of humans have shown cellular and neurophysiological abnormalities in those regions are associated with psychiatric disorders (e.g. schizophrenia, mood disorders, and addiction). The great complexity of the dlPFC in addition to its interplay with many different brain regions has made distinguishing particular pathological neural circuitry to these diseases extremely difficult. How can we probe the nervous system to understand how activity patterns in neural circuits lead to behavior? Can we then better understand pathological states? In order to tackle such problems, our laboratory has devised ways of optically activating and silencing neurons in specific neuronal subtypes based on their genetic signatures, (e.g. CaMKII-alpha as a marker for excitatory pyramidal neurons or parvalbumin in a subset of inhibitory neurons), using light gated ion channels and pumps, a method termed 'Optogenetics'. In the first part of this proposal, we aim to expand on these technologies by utilizing the unique features of light activation of such proteins by using two-photon (2P) microscopy in order to attain specificity beyond genetic signatures; more specifically, this project will achieve spatially restricted activation and silencing of the nervous system at the resolution of a single cell (2P optogenetics). Further, we will use 2P activation of individual neurons for high-throughput mapping of functional neuronal connectivity in layer V of mPFC of different neuronal subtypes in order to better understand how they function as a unit. This part of the proposal will also show this tool's generalizability to all areas of the brain using ex vivo preparations, which will provide a wealth of information for researchers investigating both normal and pathological nervous system states. In the second part of this project, we will use 2P optogenetics to understand dynamic cortical function in the mPFC. Local cortical processes such as synchrony whose dysfunction has been implicated in psychiatric disorders (e.g. schizophrenia) will be investigated to understand the underlying cellular and network mechanisms that lead to these phenomena. Taken together, these tools will hopefully lend insight into the functional connectivity matrix between different neurons in the mPFC and how they work together to provide efficient processing of information that gives rise to behavior. PUBLIC HEALTH RELEVANCE: Schizophrenia affects 1% of the population and is thought to arise from the dysfunction of different regions of the brain, most notably the dorso-lateral prefrontal cortex (dlPFC). We propose to study an approximately analogous brain region to the dlPFC in rodents known as the medial prefrontal cortex (mPFC) in order to understand how specific neurons are connected to each other and effect dynamic cortical processes known to be disrupted in patients with schizophrenia. Understanding the underlying neural circuit function of the mPFC will likely help facilitate more targeted therapies to patients suffering from schizophrenia and other psychosis.