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

 描述（由申请人提供）：识别不同类别的神经元并破译其属性的方法代表了神经科学研究的罗塞塔石碑。这些工具是必不可少的，以了解如何选择性地操纵神经系统疾病中定义的神经元回路的活动。因此，我们的目标是验证一种新的小鼠模型，以研究基底前脑（BF）中的神经元，这是一个重要的大脑区域，控制皮层，注意力和睡眠的激活。BF在昏迷、睡眠障碍和阿尔茨海默病中受损。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疗法。我们将研究使用囊泡谷氨酸转运体2型（vGluT 2）的BF神经元，这是BF谷氨酸神经元的主要亚型，包括那些投射到皮层的神经元。将在vGluT 2基因启动子区的控制下表达细菌酶Cre转移酶（Cre）的小鼠与在Cre存在下表达红色荧光蛋白tdTomato的报告小鼠品系杂交，以产生vGluT 2-Tomato小鼠。这些新的小鼠将允许在线识别BF vGluT 2神经元，并且是在体内研究BF谷氨酸神经元在睡眠-觉醒行为中的作用之前的重要的第一步。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。 Aim 1将验证tdTomato在BF神经元中的选择性表达。我们将确认tdTomato在另外两个主要的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 vGluT 2神经元的子集是否表达钙结合蛋白。光遗传学技术将验证谷氨酸的释放并揭示BF谷氨酸神经元的靶点。目的2首次采用全细胞膜片钳技术对vGluT 2-Tomato神经元进行记录，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，一个中大型的，假定的皮质投射神经元的子集表现出独特的突发放电模式，让人想起的放电模式， 体内记录的推定谷氨酸神经元。在前额叶皮质中注射逆行示踪剂将用于明确识别皮质投射的BF谷氨酸神经元。Aim 3将研究胆碱能受体激动剂和腺苷对体外BF谷氨酸神经元的影响。BF胆碱能神经元参与觉醒和REM睡眠期间的皮质激活，并在阿尔茨海默病的早期过程中退化。我们的初步数据表明，皮质投射BF谷氨酸神经元超极化胆碱能输入，促进清醒和REM睡眠期间的节律性放电去失活的低阈值钙电流。腺苷是一种重要的稳态睡眠因子。我们预测腺苷通过突触后抑制局部BF谷氨酸中间神经元和突触前抑制皮质投射BF谷氨酸神经元的兴奋性输入来促进睡眠。

 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.