Genetic dissection of sleep regulation by ventrolateral preoptic area

描述(申请人提供)：以前的研究表明，视前外侧核(VLPO)是促进睡眠的关键结构。VLPO与下丘脑尾侧和脑干的主要觉醒促进中心相互联系，VLPO神经元的丢失(化学损害)会导致严重的失眠和睡眠碎片。神经化学研究证实甘丙素是睡眠活跃的VLPO神经元的特殊标记。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类型的细胞(除甘氨酸能神经元外)，因此选择性地损毁VLPO神经元对睡眠觉醒的影响尚不清楚。在这个项目中，我们将通过立体定向向在甘丙胺能神经元中特异性表达Cre重组酶(Cre)的转基因小鼠(Gal-Cre敲门小鼠)的视前下丘脑中注射含有转录阻断的促凋亡转基因基因截短型BH3相互作用域死亡激动剂(TBID)的腺相关病毒载体(AAV)来选择性地破坏Gal-Cre能VLPO神经元。视前下丘脑注射AAV-TBID选择性地作用于含CRE的VLPO神经元，并引发细胞死亡。我们将研究这些缺乏Galanine能VLPO神经元的小鼠在睡眠-觉醒数量、结构和状态转换方面的变化(特定目标1)。我们预计，这些神经元的丧失将导致严重的失眠和睡眠碎片。有趣的是，VLPO内的所有Galanine能神经元都含有GABA。VLPO-GABA被假设为在睡眠中介导对促进觉醒的细胞群的抑制，尽管它从未被确定。因此，我们将有针对性和选择性地消除VLPO神经元中GABA的释放，并评估由于VLPO GABA神经传递丧失而导致的睡眠觉醒变化(特定目标2)。通过将AAV-Cre导入条件性囊泡GABA转运体敲除(VGAT)小鼠的VLPO中(其包含VGAT基因外显子2两侧的loxP位点)和2)将条件VGAT小鼠与Gal-cre小鼠杂交，将实现GABA释放的特异性消除。Cre的加入使VGAT外显子2缺失，使整个基因失去功能，导致VLPO神经元中缺乏功能性VGAT蛋白。由于VGAT是将GABA运输到突触小泡的关键，VGAT的缺失将导致VLPO神经元GABA神经传递的选择性丧失。研究这些动物的睡眠-觉醒状态将为深入了解VLPO-GABA在睡眠调节和睡眠-觉醒转换中的具体作用提供重要的见解。总而言之，这一提议的结果将帮助我们更好地理解控制睡眠产生、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治疗失眠和觉醒障碍的药物治疗中发挥重要作用。

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.