New approaches for chemical-genetic targeting of specific circuits and cell types in the mammalian brain

本研究的目标是建立新的鲁棒方法来操纵特定的电路和遗传 用小分子模型生物的大脑中定义了神经元类型。虽然一些化学遗传学 虽然已经有一些技术可用，但是这些技术具有限制其实用性的缺点。我们最近的工作 表明这些障碍可以通过使用蛋白质功能的急性控制策略来克服 含有去稳定化结构域（DD）的感兴趣的（POI）与廉价的市售药物TMP。 该化合物有效地穿过血脑屏障，以快速的时间过程稳定DD-POI，并且 本身不会产生不良副作用。由于DD标签几乎可以连接到任何蛋白质上，因此TMP诱导的 稳定化适用于广泛的实验范例。我们建议产生小鼠等位基因 和编码DD-POI融合体的病毒载体，其适用于从超微结构成像到 评估复杂行为。我们将开发依赖TMP的DNA和基因组重组工具 编辑（Aim 1），用与光学标记相容的报告基因急性标记行为相关的神经元群体。 成像和电子显微镜（Aim 2），和神经递质分泌的细胞类型特异性控制（Aim 3）。我们 一个合作小组将通过结合基因组学、全脑和神经科学来展示这些技术的优势。 成像、连续电子显微镜、电生理学、光遗传学和行为学。新工具将被比较 与现有技术并排，然后分发给神经科学界。我们预计 这些努力将大大有利于未来对正常大脑及其机制的研究。 神经系统疾病

The goal of this research is to establish new robust methods for manipulation of specific circuits and genetically defined neuron types in brains of model organisms with small molecules. While several chemical-genetic techniques are already available, these techniques have drawbacks that limit their utility. Our recent work demonstrates that these obstacles can be overcome by using a strategy for acute control of function of proteins of interest (POI) containing destabilizing domains (DD) with the inexpensive commercially available drug, TMP. This compound efficiently crosses the blood-brain barrier, stabilizes DD-POIs with a rapid time course, and does not produce undesired side effects by itself. Since DD tags can be attached to virtually any protein, TMP-inducible stabilization is applicable to a broad spectrum of experimental paradigms. We propose to generate mouse alleles and viral vectors encoding DD-POI fusions suitable for applications ranging from ultra-structural imaging to assessment of complex behaviors. We will develop tools for TMP-dependent recombination of DNA and genome editing (Aim1), acute labeling of behaviorally relevant neuron populations with a reporter compatible with optical imaging and electron microscopy (Aim2), and cell-type-specific control of neurotransmitter secretion (Aim3). Our collaborative team will demonstrate the advantages of these techniques by combining genomics, whole brain imaging, serial electron microscopy, electrophysiology, optogenetics and behavior. New tools will be compared side-by-side against existing technologies, and then distributed to the neuroscience community. We anticipate that these efforts will significantly benefit future studies of the normal brain and mechanisms underlying neurological diseases.