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

神经降压素回路体内监测和操作方法的创新

基本信息

批准号：
10063052
负责人：
Marta E Soden
金额：
$ 19.44万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-12-01 至 2022-10-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10063052
关键词：
Address Adult Affect Animals Anxiety Appetite Regulation Appetitive Behavior Bathing Behavior Behavioral Biological Biological Models Brain CRISPR/Cas technology Cell Culture Techniques Cell Line Cell physiology Clustered Regularly Interspaced Short Palindromic Repeats Consummatory Behavior Cre driver Cytomegalovirus Desire for food Detection Development Disease Eating Electrophysiology (science)Enzymes Fright Functional disorder Genes Genetic Genetic Models Genetic study Glutamates Goals Guide RNA Hypothalamic structure Infusion procedures Knock-out Lateral Learning Left Link Measures Mental disorders Methods Modality Modeling Motivation Motor Activity Mus Mutation Nature Neurons Neuropeptides Neurosecretory Systems Neurotensin Neurotensin Receptors Neurotransmitters Nonsense Codon Optics Peptide Signal Sequences Peptides Pharmacology Pharmacology Study Physiological Play Post-Traumatic Stress Disorders Preparation Regulation Resolution Rewards Rodent Role Schizophrenia Signal Transduction Signaling Molecule Slice Stress Substance abuse problem System Systems Development Techniques Testing Time Validation Ventral Tegmental Area Viral adeno-associated viral vector base behavioral phenotyping cell type design dopamine system dopaminergic neuron drinking drug of abuse experimental study gamma-Aminobutyric Acid hormone regulation in vivo in vivo imaging in vivo monitoring innovation insight interest loss of function mutation mesolimbic system mouse model neural circuit novel optical sensor optogenetics pancreatic secretory trypsin inhibitor I real time monitoring sensor small molecule tool vector vesicular GABA transporter virtual virus genetics

项目摘要

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.

项目摘要/摘要神经肽是神经回路的重要调节剂饮食，饮酒，压力，学习和奖励。虽然以前的遗传学和药理研究已经提供了有关管理肽功能的基本原则的重要洞察力，技术限制有限制了我们有效探测肽能电路的能力，许多问题仍然没有得到答复。值得注意的是，肽能神经元通常释放多个神经肽，并且通常释放一个快速神经递质，例如谷氨酸或GABA。当前的遗传鼠标模型和病毒工具无法有效，有效地将这些不同的神经递质和神经肽成分分开。我在这里建议使用从下丘脑（LH）到腹侧对段的神经素（NTS）投影区域（VTA）作为用于开发新型病毒工具的模型肽能电路监测肽释放和与肽相关基因的快速细胞类型特异性敲除。 NTS已知与VTA中的多巴胺神经元相互作用，以促进食欲，我们发现刺激 NTS从LH到VTA的预测启动了消费性和食欲的行为表型。这些神经元除NTS外释放GABA，目前尚不清楚这些发射器组件中的每一个作用发挥指导观察到的行为。我们已经开发了单媒体有条件的病毒crispr/cas9 可以以高效率以细胞类型的方式快速敲除任何给定基因的系统。我建议使用该系统淘汰编码NTS（NTS）的基因或编码囊泡的基因 LH NTS神经元中的GABA Transporter VGAT（SLC32A1），并将测试去除任何一个成分如何影响该电路刺激引起的行为。我还将使用这种病毒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，并在体内使用此工具在接受食欲行为的小鼠中实时监测肽释放。该提议的完成将回答有关NTS行为调节多巴胺的关键生物学问题系统并将建立可行性，以生成新的基于病毒的工具套件，可用于调查整个大脑的肽能电路。