Optogenetic Control of Vigilance State Transition

描述(申请人提供)：下克汀类，也被称为食欲素，是两种神经肽递质，专门由下丘脑中的几千个神经元产生。对小鼠、狗和人类的研究表明，下丘脑泌素系统功能的丧失会导致发作性睡病/猝倒。我们最近已经证明，使用光激活的阳离子通道Channel视紫红质2(ChR2)对遗传定义的Hcrt神经元进行光刺激会增加睡眠到觉醒转变的概率。然而，Hcrt神经元在清醒动物中的作用仍然知之甚少。在这里，我们将使用Hcrt神经元的光基因操作来测试这些神经元在觉醒过程中的相活动是否足以稳定觉醒。首先，我们将使用慢病毒将ChR2引入Hcrt神经元，并使用不同的光刺激模式来操纵Hcrt神经元在清醒时的活动，以测试持续活动是否延长了清醒时间。我们将通过微透析监测光刺激引起的Hcrt释放的变化。我们还将监测运动活动、焦虑、进食、行为和睡眠/觉醒参数。为了确定Hcrt诱导觉醒的机制，我们将光基因刺激Hcrt系统的三个主要突触后靶点：蓝斑的去甲肾上腺素能神经元、腹侧被盖区的多巴胺能神经元和基底前脑的胆碱能神经元。在第三个特定目标中，我们将寻求证据证明，在适当的条件下，在几分钟内阻断时相活动足以诱导野生型小鼠的猝倒。与药理学研究不同，光遗传学提供了前所未有的毫秒级时间分辨率，这使得对啮齿动物等睡眠不稳定物种的神经递质功能进行警觉状态特定分析。我们的数据将扩大最先进的光遗传学方法在睡眠研究中的使用，并将提供下肌醇系统如何稳定唤醒的机械模型。这些实验还可能带来治疗发作性睡病、失眠和其他睡眠障碍的新的和增强的治疗方法。

DESCRIPTION (provided by applicant): The hypocretins, also known as orexins, are two neuropeptide transmitters produced exclusively by a few thousand neurons in the hypothalamus. Studies in mice, dogs and humans have shown that loss-of- function of the hypocretin system results in narcolepsy/cataplexy. We have recently shown that optical stimulation of genetically defined Hcrt neurons using the light activated cation channel, Channelrhodopsin 2 (ChR2) increases the probability of sleep-to-wake transitions. However, the role of Hcrt neurons in awake animals is still poorly understood. Here, we will use optogenetic manipulation of Hcrt neurons to test whether phasic activity of these neurons during wakefulness is sufficient to stabilize arousal. First, we will introduce ChR2 in Hcrt neurons using a lentivirus and we will use different photostimulation patterns to manipulate Hcrt neuronal activity during wakefulness to test whether sustained activity extends waking. We will monitor photostimulation-induced changes in Hcrt release by microdialysis. We will also monitor locomotor activity, anxiety, feeding, behavior and sleep/wake parameters. To determine the mechanism by which Hcrt induces wakefulness, we will optogenetically stimulate three of the main postsynaptic targets of the Hcrt system: noradrenergic neurons in the locus coeruleus, dopaminergic neurons in the ventral tegmental area, and cholinergic neurons in the basal forebrain. In a third specific aim, we will seek evidence demonstrating that blocking phasic activity during a few minutes under the appropriate conditions is sufficient to induce cataplexy in wild-type mice. Unlike pharmacological studies, optogenetics offers unprecedented millisecond scale temporal resolution, which allows vigilance state-specific analysis of neurotransmitter function in species with unconsolidated sleep such as rodents. Our data will expand the use of state-of-the-art optogenetic methods in sleep studies and will provide mechanistic models on how the hypocretinergic system stabilizes arousal. These experiments may also lead to new and enhanced treatments for narcolepsy, insomnia and other sleep disorders.