Hypothalamic arousal systems

描述（由申请人提供）：本项目旨在研究下丘脑乳头体上区（SUM）中的一群丘脑能神经元，这可能是以前未被认识到的，但上升唤醒系统的重要组成部分。早期的研究表明，SUM神经元弥漫性地支配大脑皮层，并且该区域的病变引起深度嗜睡，而不是通过涉及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。我们将通过首先在囊泡谷氨酸2（VEGF 2）启动子下表达Cre重组酶的小鼠中，使用含有仅在表达Cre的神经元中产生的GFP基因的条件性腺相关病毒载体（AAV），检查谷氨酸能SUM神经元的投射来测试该假设。我们接下来将选择性地损伤SUM神经元，并通过使用在Cre-神经元中表达mCherry的条件性AAV和在Cre+神经元中表达白喉毒素A（致死）亚基，并记录随后的觉醒-睡眠行为，来研究它们剩余的非神经元邻居的投射。为了确定SUM中的VEGF 2+神经元是否可以使用谷氨酸以外的其他神经递质来引起唤醒，我们将使用AAV-Cre-2A-Venus删除Vlgut 2-flox/flox小鼠SUM中的VEGF 2基因，并在相同的细胞中表达荧光蛋白Venus。我们将测量对觉醒-睡眠行为的影响，并将其与包含Venus+轴突的目标区域相关联，其中VEGF 2已被删除。我们将使用Vgat-flox/flox小鼠类似地研究GABA在SUM中的一些神经元中的作用。最后，我们将研究急性效应的唤醒睡眠的抑制或刺激的VEGF 2神经元在SUM，以确定在慢性缺失研究中的补偿作用。我们将用条件性hM 3-mCherry载体注射VEGF 2-Cre小鼠，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，该载体表达与伊维菌素受体融合的YFP，伊维菌素受体是一种被抗生素药物伊维菌素激活的无脊椎动物氯离子通道，可抑制神经元。然后，我们将研究激活或抑制SUM VEGF 2+神经元对觉醒-睡眠的急性影响，并将其与SUM靶点的神经支配相关，这些靶点由荧光蛋白标记的轴突标记。这项工作将表征SUM神经元的靶点，以及这些投射在介导对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一个假设，即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。

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.