Genetic dissection of sleep regulation by ventrolateral preoptic area

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 与下丘脑尾部和脑干的主要唤醒中心相互连接，VLPO 神经元的丧失（化学损伤）会导致严重的失眠和睡眠碎片。神经化学研究发现甘丙肽是睡眠活跃 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（除甘丙肽能神经元外），因此选择性损伤甘丙肽能神经元对睡眠-觉醒的影响尚不清楚。在这个项目中，我们将通过立体定向注射含有转录阻断的促凋亡转基因、截短的BH3相互作用域死亡激动剂（tBID）的腺相关病毒载体（AAV）到在甘氨酸能神经元中特异性表达Cre重组酶（Cre）的转基因小鼠（Gal-Cre敲入小鼠）的视前下丘脑，选择性地破坏甘氨酸能VLPO神经元。将 AAV-tBID 注射到视前下丘脑选择性地作用于含有 Cre 的甘氨酸能 VLPO 神经元并引发细胞凋亡。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损失将导致严重的失眠和睡眠碎片。有趣的是，VLPO 中的所有甘丙肽能神经元都含有 GABA。据推测，VLPO-GABA 可以介导睡眠期间促醒细胞群的抑制，尽管这一点尚未得到明确确定。因此，我们将集中选择性地消除 VLPO 神经元的 GABA 释放，并评估 VLPO GABA 神经传递丧失继发的睡眠-觉醒变化（具体目标 2）。 GABA 释放的特异性消除将实现 -1) 通过将 AAV-Cre 递送至条件性囊泡 GABA 转运蛋白敲除 (VGAT) 小鼠的 VLPO 中，该小鼠含有 VGAT 基因外显子 2 侧翼的 loxP 位点，以及 2) 通过将条件性 VGAT 小鼠与 Gal-cre 小鼠杂交。添加 Cre 会切除 VGAT 外显子 2，并使整个基因失去功能，从而导致 VLPO 神经元中缺乏功能性 VGAT 蛋白。由于 VGAT 对于将 GABA 转运到突触小泡至关重要，因此 VGAT 的丧失将导致 VLPO 神经元选择性丧失 GABA 神经传递。研究这些动物的睡眠-觉醒将为了解 VLPO-GABA 在睡眠调节和睡眠-觉醒转变中的具体贡献提供重要的见解。总的来说，该提案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。这些信息最终对于设计失眠和觉醒障碍的药物治疗非常重要。

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.