Innovation of methods for in vivo monitoring and manipulation of neurotensin circuits

项目总结/摘要 神经肽是控制多种行为的神经回路的重要调节剂， 饮食压力学习和奖励虽然以前的遗传和药理学研究 提供了关键的洞察肽功能的基本原则，技术限制， 限制了我们有效探测肽能电路的能力，许多问题仍然没有答案。 值得注意的是，肽能神经元通常释放一种以上的神经肽，并且通常也释放一种 快速神经递质，如谷氨酸或GABA。目前的遗传小鼠模型和病毒工具无法 有效且高效地分离这些不同神经递质和神经肽组分。这里我 拟利用下丘脑外侧部（LH）到腹侧被盖的神经降压素（NTS）投射 区域（VTA）作为开发新型病毒工具的模型肽能电路， 监测肽释放和肽相关基因的快速细胞类型特异性敲除。众所周知，NTS 与腹侧被盖区的多巴胺神经元相互作用以促进食欲行为，我们发现刺激 从LH到VTA的NTS投射启动消耗性和食欲行为表型。这些 神经元除了NTS外还释放GABA，目前还不清楚这些递质成分各自的作用 在指导观察到的行为中发挥作用。我们已经开发了一种单载体条件病毒CRISPR/Cas9 能够以细胞类型特异性方式高效地快速敲除任何给定基因的系统。我 我建议使用该系统敲除编码NTS的基因（Nts）或编码囊泡的基因， GABA转运蛋白Vgat（Slc 32a 1）在LH NTS神经元中的作用，并将测试去除任何一种成分如何影响 由该回路的刺激引起的行为。我也会用这种病毒CRISPR方法来靶向NTS 腹侧被盖区的受体此外，我建议验证NTS的荧光传感器，并在体内使用该工具， 监测经历食欲行为的小鼠中真实的时间的肽释放。完成本提案将 通过调节多巴胺来回答有关NTS行为调节的关键生物学问题 系统，并将建立可行性，以产生新的病毒为基础的工具包，可用于调查 整个大脑的肽能回路。

Project Summary/Abstract Neuropeptides are essential modulators of neural circuits governing a wide variety of behaviors including eating, drinking, stress, learning, and reward. While previous genetic and pharmacological studies have provided critical insight into the underlying principles governing peptide function, technical limitations have constrained our ability to effectively probe peptidergic circuits, and many questions remain unanswered. Notably, peptidergic neurons commonly release more than one neuropeptide, and also typically release one fast neurotransmitter, such as glutamate or GABA. Current genetic mouse models and viral tools are unable to effectively and efficiently separate these different neurotransmitter and neuropeptide components. Here I propose to use the neurotensin (NTS) projections from the lateral hypothalamus (LH) to the ventral tegmental area (VTA) as a model peptidergic circuit for the development of novel viral tools that will enable real-time monitoring of peptide release and rapid cell-type specific knockout of peptide-related genes. NTS is known to interact with dopamine neurons in the VTA to promote appetitive behaviors, and we have found that stimulation of NTS projections from the LH to the VTA initiates consumptive and appetitive behavioral phenotypes. These neurons release GABA in addition to NTS, and it is unclear what role each of these transmitter components plays in directing the observed behaviors. We have developed a single-vector conditional viral CRISPR/Cas9 system that enables rapid knockout of any given gene in a cell-type specific manner with high efficiency. I propose to use this system to knock out the gene encoding NTS (Nts) or the gene encoding the vesicular GABA transporter Vgat (Slc32a1) in LH NTS neurons, and will test how removing either component affects the behaviors induced by stimulation of this circuit. I will also use this viral CRISPR method to target NTS receptors in the VTA. In addition, I propose to validate a fluorescent sensor for NTS and use this tool in vivo to monitor peptide release in real time in mice undergoing appetitive behaviors. Completion of this proposal will answer critical biological questions about NTS regulation of behavior via its modulation of the dopamine system and will establish feasibility for generating new viral-based tool kits that can be applied to investigate peptidergic circuits throughout the brain.