Hypothalamic arousal systems

下丘脑唤醒系统

基本信息

批准号：
9244861
负责人：
CLIFFORD B SAPER
金额：
$ 55.63万
依托单位：
BETH ISRAEL DEACONESS MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-07-01 至 2019-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9244861
关键词：
Acute Alpha Cell Animals Antibiotics Area Arousal Axon Behavior Brain Brain Injuries Cell Nucleus Cells Cerebral cortex Chloride Channels Chlorides Chronic Clozapine Coma Diffuse Diphtheria Toxin Disease Drowsiness Enterobacteria phage P1 Cre recombinase Exons Felis catus Financial compensation Genes Glutamates Green Fluorescent Proteins Hippocampus (Brain)Histamine Histidine Decarboxylase Hypersomnias Hypothalamic structure Individual Injectable Invertebrates Ivermectin Label Lateral Lateral Hypothalamic Area Learning Lesion Long-Term Effects Measures Mediating Methods Monkeys Mus Muscarinic M3 Receptor Mutate Narcolepsy Neurons Neurotransmitters Output Oxides Play Population Proteins Rattus Recovery Reporting Role Signal Transduction Site Sleep Stretching Stroke System Testing Venus Wakefulness Work adeno-associated viral vector alpha Toxin awake experimental study gamma-Aminobutyric Acid glutamatergic signaling hypocretin killings melanin-concentrating hormone mutant nerve supply promoter public health relevance receptor red fluorescent protein synthetic enzyme transmission process vector vesicular GABA transporter vesicular glutamate transporter 2

项目摘要

DESCRIPTION (provided by applicant): This project seeks to study a population of glutamatergic neurons in the supramammillary region of the hypothalamus (SUM), which may be a previously unrecognized but important part of the ascending arousal system. Earlier studies have shown that SUM neurons diffusely innervate the cerebral cortex, and that lesions in this area cause profound somnolence not explained by involving nearby orexin or histaminergic neurons. Our preliminary observations indicate that the SUM neurons have massive projections to many components of the arousal system, and that they may be necessary to maintain a normal waking state. We will test this hypothesis by first examining the projections of the glutamatergic SUM neurons, using a conditional adeno- associated viral vector (AAV) containing the gene for GFP which is only produced in Cre-expressing neurons, in mice expressing Cre recombinase under the vesicular glutamate 2 (Vglut2) promoter. We next will selectively lesion the SUM glutamatergic neurons, and study the projections of their remaining non- glutamatergic neighbors, by using a conditional AAV that expresses mCherry in Cre- neurons, and the diphtheria toxin A (lethal) subunit in Cre+ neurons, and recording subsequent wake-sleep behavior. To determine whether the Vglut2+ neurons in the SUM may use other neurotransmitters than glutamate to cause arousal, we will then use AAV-Cre-2A-Venus to delete the Vglut2 gene in the SUM in Vlgut2-flox/flox mice, and express the fluorescent protein Venus in the same cells. We will measure the effect on wake-sleep behavior and correlate that with which target areas contain Venus+ axons, in which Vglut2 has been deleted. We will similarly study the role of GABA in some neurons in the SUM using Vgat-flox/flox mice. Finally, we will examine the acute effects on wake-sleep of either inhibiting or stimulating the Vglut2 neurons in the SUM, to determine the role of compensation in chronic deletion studies. We will inject Vglut2-Cre mice with either a conditional hM3-mCherry vector, which expresses a mutant M3 muscarinic receptor in Cre+ neurons that is activated by clozapine-N-oxide and stimulates neurons; or a conditional ivermectin vector, which expresses YFP fused with the ivermectin receptor, an invertebrate chloride channel that is activated by the antibiotic drug ivermectin, which inhibits neurons. We will then study the acute effects on wake-sleep of either activating or inhibiting the SUM Vglut2+ neurons, and correlate this with the innervation of SUM targets by axons labeled with the fluorescent proteins. This work will characterize the targets of the SUM glutamatergic neurons, and the role of those projections in mediating acute and chronic effects on sleep and wakefulness. These findings will test the hypothesis that the SUM neurons may plan an even more important role than the nearby orexin or histaminergic cell groups in maintaining a normal waking state.

描述（由申请人提供）：该项目旨在研究下丘脑乳头上区（SUM）中的谷氨酸能神经元群体，这可能是上行唤醒系统中以前未被识别但重要的部分。早期的研究表明，SUM 神经元广泛地支配大脑皮层，该区域的损伤会导致严重的嗜睡，而这不能用附近的食欲素或组胺能神经元来解释。我们的初步观察表明，SUM 神经元对唤醒系统的许多组成部分都有大量的投射，它们可能是维持正常清醒状态所必需的。我们将通过首先检查谷氨酸能 SUM 神经元的投射来测试这一假设，使用条件腺相关病毒载体（AAV），该载体包含仅在表达 Cre 的神经元中产生的 GFP 基因，在囊泡下表达 Cre 重组酶的小鼠中谷氨酸 2 (Vglut2) 启动子。接下来，我们将选择性地损害 SUM 谷氨酸能神经元，并通过使用在 Cre- 神经元中表达 mCherry 的条件 AAV 和 Cre+ 神经元中表达白喉毒素 A（致命）亚基来研究其剩余非谷氨酸神经元的投射，并记录随后的唤醒-睡眠行为。为了确定 SUM 中的 Vglut2+ 神经元是否可能使用谷氨酸以外的其他神经递质引起唤醒，我们将使用 AAV-Cre-2A-Venus 删除 Vlgut2-flox/flox 小鼠中 SUM 中的 Vglut2 基因，并表达荧光同一细胞中的蛋白质金星。我们将测量对唤醒-睡眠行为的影响，并将其与包含 Vglut2 已被删除的金星+轴突的目标区域相关联。我们将使用 Vgat-flox/flox 小鼠类似地研究 GABA 在 SUM 中某些神经元中的作用。最后，我们将检查抑制或刺激 SUM 中的 Vglut2 神经元对清醒睡眠的急性影响，以确定补偿在慢性缺失研究中的作用。我们将向 Vglut2-Cre 小鼠注射条件 hM3-mCherry 载体，该载体在 Cre+ 神经元中表达突变型 M3 毒蕈碱受体，该受体被氯氮平-N-氧化物激活并刺激神经元；或条件性伊维菌素载体，表达与伊维菌素受体融合的YFP，伊维菌素受体是一种无脊椎动物氯离子通道，被抗生素药物伊维菌素激活，抑制神经元。然后，我们将研究激活或抑制 SUM Vglut2+ 神经元对清醒睡眠的急性影响，并将其与荧光蛋白标记的轴突对 SUM 目标的神经支配联系起来。这项工作将描述 SUM 谷氨酸能神经元的目标，以及这些投射在调节睡眠和觉醒的急性和慢性影响中的作用。这些发现将检验这样的假设：SUM 神经元在维持正常清醒状态方面可能比附近的食欲素或组胺能细胞群发挥更重要的作用。