Channel Structure-Based Tools for Precise Interrogation of Circuitry and Behavior

项目摘要 光遗传学技术，设计用于控制神经回路的定义元素， 精确性，已得到广泛使用，包括与精神疾病有关的问题（例如与焦虑有关的 自由移动的哺乳动物）。这种方法的关键特征包括使用光激活调节剂， 跨膜离子电导，例如通道视紫红质（ChR），其在时间上精确， 直接递送（例如通过病毒载体）到靶向脑区域。我们将继续扩大 并通过父母奖改善光遗传工具箱，以了解 抑制性通道视紫红质传导氯离子，使用这个和其他结构数据来设计红色激活的 激发和抑制的通道，并利用这些和其他强大的光遗传学工具， 在行为哺乳动物中开发和应用全光快速闭环电路箝位。能够精确地 在对精神病学显着的目标神经元群体中表达分子工具，包括光遗传学工具 疾病是至关重要的，以扩大这些多重工程，下一代光遗传学工具的效用， 尖端的全光学生理学该项目能够精确靶向光遗传学和其他 分子工具，高度定义的神经元亚群，并扩大了广泛适用的关键方法 在神经科学中。 Lief Fenno医学博士，哲学博士、之前发明了一种技术，称为INTRSECT，允许表达 在由多个参数定义的神经元亚群中的分子神经科学工具（例如，仅 同时表达SST和PV的神经元，但不表达单独的SST或PV的神经元）。使用受保护 由该补充计划资助的研究时间，他将进行重点研究，1）工程师和 验证INTRSECT构建体的扩展工具箱以包括兴奋性和抑制性光遗传学工具， 荧光团和遗传编码的钙指示剂; 2）通过 Flp重组酶识别盒的迭代工程化和筛选; 3）应用一种新的，廉价的， 光学设备，以获得长期寻求和高度新颖的数据描述的表达动力学的腺， 体内相关病毒;以及4）通过以下方式扩展和完善INTRSECT方法的靶向分辨率： 本发明涉及一种新正向重组酶，VCre的适应，并应用这些新病毒来表达光遗传学 神经元中的试剂基于三个独立的标准。该项目将产生一个改进的，扩大的， 经过验证和扩展的试剂套件，可实现无限数量的当前无法实现的项目 在神经科学研究中。

PROJECT SUMMARY Optogenetic technologies, designed for control of defined elements of neural circuitry with high temporal precision, have come into broad use, including for questions relating to psychiatric disease (e.g. anxiety-related behaviors in freely-moving mammals). Key features of this approach include use of light-activated regulators of transmembrane ion conductance such as channelrhodopsins (ChRs) which are temporally precise as well as straightforward to deliver (for example by viral vectors) to targeted brain regions. We are continuing to expand and improve the optogenetic toolbox through the parent award in order to understand the structure of inhibitory channelrhodopsins that conduct chloride, to use this and other structural data to design red-activated channels for excitation and inhibition, and to leverage these and other powerful optogenetic tools in order to develop and apply all-optical fast closed-loop circuit clamping in behaving mammals. Being able to precisely express molecular tools, including optogenetic ones, in targeted populations of neurons salient to psychiatric disease is critical to extending the utility of these multiply-engineered, next-generation optogenetic tools and cutting-edge, all-optical physiology. This project enables the precise targeting of optogenetic and other molecular tools to highly defined neuron sub-populations and expands a critical approach widely applicable in neuroscience. Lief Fenno, M.D., Ph.D., previously invented a technique, called INTRSECT, that allows for the expression of molecular neuroscience tools in neuron sub-populations defined by multiple parameters (for instance, only neurons expressing both SST AND PV, but not neurons expressing either SST or PV alone). Using protected research time funded by this supplemental program, he will conduct focused research to 1) engineer and validate an expanded toolbox of INTRSECT constructs to include excitatory and inhibitory optogenetic tools, fluorophores, and genetically-encoded calcium indicators; 2) Improve the efficacy of Flp recombinase through iterative engineering and screening of the Flp recombinase recognition cassette; 3) Apply a novel, inexpensive, optical device to acquire long-sought and highly novel data describing the expression kinetics of Adeno- Associated Viruses in vivo; and 4) Extend and refine the targeting resolution of the INTRSECT approach through the novel adaptation of an orthologous recombinase, VCre and apply these new viruses to express optogenetic reagents in neurons based on three separate criteria. This project will produce an improved, expanded, validated, and extended suite of reagents to enable an unlimited number of currently unapproachable projects in neuroscience research.