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

项目摘要/摘要 神经肽是控制多种行为的神经回路的基本调节器，包括 吃、喝、压力、学习和奖励。虽然之前的遗传和药理学研究已经 提供了对支配多肽功能的基本原理的批判性见解，技术限制 限制了我们有效探索肽能回路的能力，许多问题仍然没有答案。 值得注意的是，肽能神经元通常会释放不止一种神经肽，而且通常也会释放一种。 快速神经递质，如谷氨酸或GABA。目前的小鼠遗传模型和病毒工具无法 有效和高效地分离这些不同的神经递质和神经肽成分。在这里，我 建议使用神经降压素(NTS)从下丘脑外侧到腹侧被盖的投射 区域(VTA)作为模型肽能电路，用于开发新的病毒工具，使实时 监测多肽的释放和多肽相关基因的快速细胞型特异性敲除。NTS是已知的 与VTA中的多巴胺神经元相互作用，促进食欲行为，我们发现刺激 黄体生成素向下丘脑室旁核的投射启动了食欲和食欲行为表型。这些 除了NTS外，神经元还会释放GABA，目前还不清楚这些递质成分中的每一个都扮演着什么角色 在指导观察到的行为方面发挥了作用。我们开发了单载体条件病毒CRISPR/Cas9 一种能够以细胞类型特定的方式高效地快速敲除任何给定基因的系统。我 建议使用该系统敲除编码NTS(NTS)的基因或编码水泡的基因 GABA转运体Vgat(Slc32a1)，并将测试去除这两种成分中的任何一种对 刺激这一回路所引起的行为。我也将使用这种病毒CRISPR方法来针对NTS VTA中的受体。此外，我建议验证NTS的荧光传感器，并在体内使用该工具来 实时监测发生食欲行为的小鼠的多肽释放。完成这项提案将 回答有关NTS通过其对多巴胺的调节来调节行为的关键生物学问题 系统，并将确定生成新的基于病毒的工具包的可行性，这些工具包可用于调查 整个大脑中都有肽能回路。

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.