Genetic dissection of sleep regulation by ventrolateral preoptic area

腹外侧视前区睡眠调节的基因剖析

• 批准号: 8425041
• 负责人: Vetrivelan Ramalingam
• 金额: $ 20.99万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8425041
• 关键词: Address; Animals; Apoptotic; Architecture; Area; Arousal; BID protein; Brain; Brain Stem; Cell Death; Cells; Chemicals; Dependovirus; Disease; Dissection; Dorsal; Economics; Exons; Galanin; General Population; Generations; Genes; Genetic; High Prevalence; Homeostasis; Hour; Hypothalamic structure; Ibotenic Acid; Immunohistochemistry; Injection of therapeutic agent; Knock-out; Knockout Mice; Laboratories; Lesion; Light; Maintenance; Mediating; Methods; Modeling; Mus; Neurons; Neurotoxins; Neurotransmitters; Outcome; Pharmacological Treatment; Play; Preoptic Areas; Protein Biosynthesis; Proteins; Rattus; Regulation; Research; Role; Secondary to; Site; Sleep; Sleep Deprivation; Sleep Fragmentations; Sleeplessness; Structure; Synaptic Vesicles; System; Transgenes; Transgenic Mice; Wakefulness; adeno-associated viral vector; base; cell type; design; gamma-Aminobutyric Acid; insight; killings; knockout gene; locus ceruleus structure; mammilloinfundibular nucleus structure; midbrain central gray substance; neurochemistry; neuromechanism; neuron loss; neurotransmission; preoptic nucleus; presynaptic; recombinase; sleep regulation; social; vesicular GABA transporter

DESCRIPTION (provided by applicant): Previous studies have shown that the ventrolateral preoptic nucleus (VLPO) is a critical structure for promoting sleep. The VLPO is reciprocally connected with major wake-promoting centers in the caudal hypothalamus and the brainstem, and the loss of VLPO neurons (chemical lesions) produces profound insomnia and sleep fragmentation. Neurochemical studies identified galanin as a specific marker for the sleep-active VLPO neurons. However, past VLPO lesion studies also knocked out many other cell types (other than galaninergic neurons) in and around the VLPO region, and thus the effect of selectively lesioning the galaninergic VLPO neurons on sleep-wakefulness is not known. In this project, we will selectively destroy the galaninergic VLPO neurons by stereotaxically injecting adeno-associated viral vectors (AAV) containing transcriptionally blocked pro-apoptotic transgene, truncated BH3-interacting domain death agonist (tBID), into the preoptic hypothalamus of transgenic mice that express Cre-recombinase (Cre) specifically in the galaninergic neurons (Gal-Cre knockin mice). Injection of AAV-tBID into the preoptic hypothalamus acts selectively on the Cre- containing galaninergic VLPO neurons and triggers apoptotic cell death. We will study the changes in sleep- wake amounts, architecture and state transitions in these mice that lack galaninergic VLPO neurons (Specific Aim 1). We anticipate that the loss of these neurons will result in severe insomnia and sleep fragmentation. Interestingly, all the galaninergic neurons in the VLPO contain GABA. The VLPO-GABA has been hypothesized to mediate the inhibition of wake-promoting cell groups during sleep, although it has never been definitively determined. Hence, we will focally and selectively eliminate GABA release from the VLPO neurons and evaluate sleep-wakefulness changes that are secondary to the loss of VLPO GABA neurotransmission (Specific Aim 2). Specific elimination of GABA release will be achieved -1) by delivering AAV-Cre into the VLPO of conditional vesicular GABA transporter knockout (VGAT) mice that contain loxP sites flanking exon 2 of the VGAT gene and 2) by crossing the conditional VGAT mice with Gal-cre mice. Addition of Cre excises VGAT exon 2 and makes the entire gene non-functional, which results in the absence of functional VGAT protein in the VLPO neurons. As VGAT is critical for transporting GABA into the synaptic vesicles, the loss of VGAT will result in selective loss of GABA neurotransmission from the VLPO neurons. Studying sleep- wakefulness in these animals will provide critical insight into the specific contribution of VLPO-GABA in sleep regulation and in sleep-wake transitions. Collectively, the outcomes of this proposal will help us better understand the hypothalamic control mechanisms controlling sleep generation, maintenance and state transitions. This information will ultimately be important in designing pharmacological treatment for insomnia and arousal disorders.

描述（由申请人提供）：先前的研究表明，腹外侧视前核（VLPO）是促进睡眠的关键结构。VLPO与尾侧下丘脑和脑干中的主要唤醒促进中心紧密相连，VLPO神经元的丢失（化学损伤）会导致严重的失眠和睡眠碎片。神经化学研究确定甘丙肽作为睡眠活跃VLPO神经元的特异性标记物。然而，过去的VLPO损伤研究也敲除了VLPO区域内和周围的许多其他细胞类型（除了甘丙肽能神经元），因此选择性损伤甘丙肽能VLPO神经元对睡眠-觉醒的影响尚不清楚。在这个项目中，我们将选择性地破坏Galaninergic VLPO神经元立体定向注射腺相关病毒载体（AAV）含有转录阻断促凋亡转基因，截短BH 3-相互作用域死亡激动剂（tBID），到视前下丘脑的转基因小鼠，表达Cre重组酶（Cre），特别是在galaninergic神经元（Gal-Cre敲入小鼠）。将AAV-tBID注射到视前下丘脑中选择性地作用于含Cre的甘丙肽能VLPO神经元并触发凋亡性细胞死亡。我们将研究这些缺乏甘丙肽能VLPO神经元的小鼠的睡眠-觉醒量、结构和状态转换的变化（具体目标1）。我们预计，这些神经元的丢失将导致严重的失眠和睡眠碎片。有趣的是，VLPO中的所有Galaninergic神经元都含有GABA。VLPO-GABA被假设为在睡眠期间介导促醒细胞群的抑制，尽管它从未被明确确定。因此，我们将集中并选择性地消除VLPO神经元的GABA释放，并评估继发于VLPO GABA神经传递丧失的睡眠-觉醒变化（具体目标2）。GABA释放的特异性消除将通过以下方式实现：1）将AAV-Cre递送到含有VGAT基因外显子2侧翼的loxP位点的条件性囊泡GABA转运蛋白敲除（VGAT）小鼠的VLPO中，和2）将条件性VGAT小鼠与Gal-cre小鼠杂交。添加Cre切除VGAT外显子2并使整个基因无功能，这导致VLPO神经元中不存在功能性VGAT蛋白。由于VGAT对于将GABA转运到突触囊泡中至关重要，因此VGAT的丧失将导致来自VLPO神经元的GABA神经传递的选择性丧失。在这些动物中研究睡眠-觉醒将提供对VLPO-GABA在睡眠调节和睡眠-觉醒转换中的具体贡献的关键见解。总的来说，这一提议的结果将帮助我们更好地了解控制睡眠产生、维持和状态转换的下丘脑控制机制。这些信息最终将在设计失眠和觉醒障碍的药物治疗中发挥重要作用。