Hypothalamic arousal systems

下丘脑唤醒系统

• 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
• 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

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超氨甲状腺超乳腺癌（SUM）的谷氨酸能神经元的人群，这可能是先前未知但重要的唤起系统的重要部分。较早的研究表明，总和神经元扩散地支配了大脑皮质，并且该区域的病变不会引起深深的逗休息，这不会通过附近的Orexin或Hentainer蛋白或组胺能神经元涉及。我们的初步观察表明，总和神经元对唤醒系统的许多组成部分具有大量的预测，并且对于维持正常的唤醒状态可能是必要的。我们将首先使用条件腺能和相关的病毒载体（AAV）首先检查谷氨酸能和神经元的预测，该病毒载体（AAV）含有GFP基因，该基因仅在表达CRE的神经元中产生，在表达CRE重组酶的小鼠中，表达CRE重组酶在Vesicular Glutamate 2（Vglut2）下（Vglut2）。接下来，我们将通过使用条件AAV和在Cre-themeron中表达MCHERRY的条件AAV以及在CRE+ Neurons中表达MCHERRY的条件AAV，并在CRE+ Neurons中使用二脑毒素A（杀伤力），并记录了后续的Wake-Spelepepercy。为了确定总和中的vglut2+神经元是否可以使用其他神经递质引起唤醒，然后我们将使用aav-cre-2a-venus在vlgut2-flox/flox小鼠中删除vglut2基因，并在同一细胞中表达荧光蛋白venus。我们将衡量对唤醒性行为的影响，并将目标区域含有金星+轴突的影响，其中vglut2已被删除。我们将使用VGAT-FLOX/FLOX小鼠在总和中类似地研究GABA在某些神经元中的作用。最后，我们将检查对总和中抑制或刺激vglut2神经元的唤醒性的急性影响，以确定补偿在慢性缺失研究中的作用。我们将向有条件的HM3-MCHERRY载体注入VGLUT2-CRE小鼠，该载体在CRE+神经元中表达突变的M3毒蕈碱受体，该神经元被氯氮平-N-氧化物激活并刺激神经元激活。或有条件的伊维菌素载体，其表达与伊维菌素受体融合的YFP，这是一种无脊椎动物氯化物通道，该通道被抑制神经元的抗生素药物伊维菌素激活。然后，我们将研究对激活或抑制SUM VGLUT2+神经元的唤醒尾巴的急性影响，并将其与与荧光蛋白标记的轴突的神经靶神经联系起来。这项工作将表征总和谷氨酸能神经元的靶标，以及这些预测在介导急性和慢性对睡眠和清醒作用中的作用。这些发现将检验以下假设：总和神经元可能比附近的Orexin或Extamin能细胞基团在维持正常的唤醒状态中更重要的作用。