vGLUT2-Tomato mice: a novel tool to study Basal Forebrain Glutamate Neurons

vGLUT2-番茄小鼠：研究基底前脑谷氨酸神经元的新工具

基本信息

批准号：
8950810
负责人：
Ritchie Edward Brown
金额：
$ 19.31万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-07-15 至 2017-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8950810
关键词：
Adenosine Agonist Alzheimer&apos s Disease Attention Behavior Brain region Calcium Calcium-Binding Proteins Calculi Carbachol Cells Choline O-Acetyltransferase Cholinergic Agonists Cholinergic Receptors Coma Data Disease Electrophysiology (science)Enzymes Excitatory Amino Acid Antagonists Exhibits Fiber Genes Glutamate Transporter Glutamates Goals Green Fluorescent Proteins In Vitro Injection of therapeutic agent Interneurons Knock-in Mouse Label Lead Membrane Membrane Potentials Methods Modeling Mouse Strains Mus Neurons Neurosciences Research Neurotransmitters Optics Parvalbumins Pattern Pharmacogenetics Physiologic pulse Prefrontal Cortex Promoter Regions Property Proteins REM Sleep Rattus Reporter Rest Role Sleep Sleep Disorders Staining method Stains T-Type Calcium Channels Techniques Testing Time Tomatoes Tracer Transgenic Mice Validation Viral Vector Wakefulness Whole-Cell Recordings adeno-associated viral vector basal forebrain basal forebrain cholinergic neurons calbindin cholinergic cholinergic neuron combinatorial electrical property enhanced green fluorescent protein in vivo inhibitor/antagonist insight mouse Cre recombinase mouse model nervous system disorder neuron component neuronal circuitry neuropsychiatry novel optogenetics patch clamp postsynaptic presynaptic public health relevance recombinase red fluorescent protein selective expression time use tool

项目摘要

DESCRIPTION (provided by applicant): Methods to identify different classes of neurons and decipher their properties represent the Rosetta stone of neuroscience research. These tools are essential in order to understand how to selectively manipulate the activity of defined neuronal circuits in nervous system disorders. Thus, our goal here is to validate a novel mouse model to investigate glutamatergic neurons in the basal forebrain (BF), an important brain region controlling activation of the cortex, attention and sleep. The BF is impaired in coma, sleep disorders and Alzheimer's disease. Thus, a better understanding of its component neurons and their properties may lead to novel treatments for these disorders. We will study BF neurons which use the vesicular glutamate transporter, type 2 (vGluT2), the major subtype of BF glutamate neurons, including those which are project to the cortex. Mice expressing the bacterial enzyme, Cre Recombinase (Cre), under the control of the vGluT2 gene promoter region will be crossed with a reporter mouse strain which expressing a red fluorescent protein tdTomato in the presence of Cre, to yield vGluT2-Tomato mice. These novel mice will allow online identification of BF vGluT2 neurons, and is an essential first step prior to in vivo studiesof the role of BF glutamate neurons in sleep-wake behavior. Use of tdTomato as a reporter will allow combinatorial approaches using pre-existing transgenic mice and viral vectors expressing green/yellow fluorescent proteins. Aim1 will validate the selective expression of tdTomato in BF glutamatergic neurons. We will confirm that tdTomato is not expressed in the two other major BF neurotransmitter classes, cholinergic and GABAergic neurons, and test if a subset of putative cortically-projecting BF vGluT2 neurons expresses the calcium-binding protein, calbindin. Optogenetic techniques will validate the release of glutamate and reveal the targets of BF glutamate neurons. Aim 2 will reveal the intrinsic electrical properties of BF glutamate neurons for the first time using whole-cell patch-clamp recordings from vGluT2-Tomato neurons. Excitingly, our preliminary data indicate that a subset of medium-large sized, putative cortically projecting neurons exhibits a unique pattern of burst firing, reminiscent of the firing patterns of putative glutamate neurons recorded in vivo. Injections of retrograde tracer in the prefrontal cortex will be used to definitively identify cortically-projecting BF glutamate neurons. Aim3 will investigate the effects of a cholinergic receptor agonist and adenosine on BF glutamate neurons in vitro. BF cholinergic neurons are involved in cortical activation during wakefulness and REM sleep, and degenerate early in the course of Alzheimer's disease. Our preliminary data suggests that cortically-projecting BF glutamate neurons are hyperpolarized by cholinergic inputs, facilitating rhythmic firing during waking and REM sleep by de-inactivating a low-threshold calcium current. Adenosine is an important homeostatic sleep factor. We predict adenosine promotes sleep via postsynaptic inhibition of local BF glutamate interneurons and presynaptic inhibition of excitatory inputs to cortically-projecting BF glutamate neurons.

描述（由申请人提供）：识别不同类别的神经元并破译其特性的方法代表了神经科学研究的罗塞塔石碑，这些工具对于理解如何选择性地操纵神经系统疾病中定义的神经回路的活动至关重要。，我们的目标是验证一种新型小鼠模型，以研究基底前脑 (BF) 中的谷氨酸能神经元，基底前脑是控制皮质、注意力和睡眠的重要大脑区域。BF 在昏迷、睡眠障碍中受损。因此，更好地了解其组成神经元及其特性可能会导致这些疾病的新治疗方法，我们将研究使用 2 型囊泡谷氨酸转运蛋白（vGluT2）（BF 谷氨酸神经元的主要亚型）的 BF 神经元。，包括那些投射到皮质的小鼠，其在 vGluT2 基因启动子区域的控制下表达细菌酶 Cre 重组酶（Cre），将与报告小鼠品系杂交。在 Cre 存在的情况下表达红色荧光蛋白 tdTomato，以产生 vGluT2-Tomato 小鼠。这些新型小鼠将允许在线识别 BF vGluT2 神经元，并且是体内研究 BF 谷氨酸神经元的作用之前的重要第一步。使用 tdTomato 作为报告基因将允许使用预先存在的转基因小鼠和表达绿色/黄色荧光蛋白的病毒载体的组合方法来验证 Aim1。 tdTomato 在 BF 谷氨酸能神经元中的选择性表达我们将确认 tdTomato 在另外两种主要 BF 神经递质类别（胆碱能和 GABA 能神经元）中不表达，并测试假定的皮质投射 BF vGluT2 神经元的子集是否表达钙结合蛋白。、钙结合蛋白。光遗传学技术将验证谷氨酸的释放并揭示 BF 谷氨酸神经元的目标。图 2 将首次使用 vGluT2-Tomato 神经元的全细胞膜片钳记录揭示 BF 谷氨酸神经元的内在电特性。令人兴奋的是，我们的初步数据表明，中大型、假定的皮质投射神经元的子集表现出独特的连发射击模式，让人想起体内记录的假定谷氨酸神经元将用于明确识别皮质投射的 BF 谷氨酸神经元，Aim3 将在体外研究胆碱能受体激动剂和腺苷对 BF 谷氨酸神经元的影响。参与清醒和快速眼动睡眠期间的皮质激活，并在阿尔茨海默病的早期退化。初步数据表明，皮质投射的 BF 谷氨酸神经元被胆碱能输入超极化，通过使低阈值钙电流失活来促进清醒和快速眼动睡眠期间的节律放电，我们预测腺苷通过突触后促进睡眠。抑制局部 BF 谷氨酸中间神经元和突触前抑制皮质投射 BF 的兴奋性输入谷氨酸神经元。