The Tuberal Hypothalamus and Arousal State Control

黑色素浓集激素（MCH）和下丘脑泌素/食欲素（HCRT）表达神经元混合在一起 下丘脑结节中的细胞群，它们广泛地投射到整个大脑中的许多相同的终端， 领域的然而，HCRT系统与清醒的控制有关，因为睡眠 当这些细胞退化时，会导致嗜睡症，这个系统也参与能量代谢。 相反，MCH系统主要与食物摄入和能量代谢有关， 最近的研究已经确定MCH神经元也参与睡眠和觉醒的调节。 这一假设的基础是HCRT系统是唤醒稳定和REM抑制 而MCH系统是睡眠促进和REM稳定的。我们将通过确定 通过去除HCRT或MCH神经元部分消融的小鼠的表型 两种条件性小鼠模型的饮食中的强力霉素。然后，我们将评估是否部分消融 HCRT神经元导致发作性睡病的表型而不伴有cataesthetic以及cataesthetic是否加重 通过同时消除两个神经元群体。我们还将评估是否直接连接 存在于这些细胞群之间，使用光遗传学辅助神经解剖追踪和全细胞贴片， 在存在和不存在选择性HCRT和MCH受体拮抗剂的情况下的钳位电生理学。到 为了评估当HCRT神经元退化时大脑中发生了什么，就像在人类嗜睡症中一样，我们将使用 条件性HCRT神经元消融模型，以确定MCH群体的兴奋性如何受到以下因素的影响： HCRT输入的慢性丧失。我们还将使用条件性MCH神经元消融来评估 MCH对HCRT神经元兴奋性的损失。基于头部固定的有限数量细胞的记录， 在动物中，HCRT和MCH神经元已被报道在睡眠-觉醒期间具有相互活动 周期，其中HCRT神经元在主动觉醒期间具有最高的放电率，MCH神经元在主动觉醒期间具有最高的放电率。 主要活跃于REM睡眠期。为了确定这一结论的准确性，我们将使用基因- 编码的Ca 2+指标和显微内窥镜成像，以测量数百个HCRT和MCH的活性， 神经元在睡眠/清醒周期中的变化，在不受限制的自由移动的动物中。评估HCRT是否 和MCH神经元在功能上是相互关联的，我们将通过药物遗传学激活一个群体， 在存在选择性HCRT和MCH受体的情况下对另一群体中的Ca 2+荧光成像 对手。最后，由于这两个群体投射到许多相同的大脑区域，我们将评估 他们对已知参与唤醒状态控制的大脑区域的相对输入，特别是蓝斑， 结节乳头核、内侧隔和杏仁核。总之，这些实验应该提供一个 更完整地了解这两个人群的解剖和功能连接， 选择性丧失一个种群或另一个种群的后果。

Melanin-concentrating hormone (MCH) and hypocretin/orexin (HCRT)-expressing neurons are intermingled populations in the tuberal hypothalamus that project widely throughout the brain to many of the same terminal fields. Whereas the HCRT system has been implicated in the control of wakefulness because the sleep disorder narcolepsy results when these cells degenerate, this system is also involved in energy metabolism. Conversely, the MCH system has primarily been associated with food intake and energy metabolism, but recent studies have established that MCH neurons also participate in the regulation of sleep and wakefulness. The hypothesis underlying this proposal is that the HCRT system is wake-stabilizing and REM-inhibiting whereas the MCH system is sleep-facilitating and REM-stabilizing. We will test this hypothesis by determining the phenotype of mice in which either the HCRT or MCH neurons have been partially ablated by removal of doxycycline in the diet of two conditional mouse models. We will then evaluate whether partial ablation of the HCRT neurons results in a phenotype of narcolepsy without cataplexy and whether cataplexy is exacerbated by simultaneously eliminating both neuronal populations. We will also assess whether direct connectivity exists between these cell groups using optogenetically-assisted neuroanatomical tracing and whole-cell patch- clamp electrophysiology in the presence and absence of selective HCRT and MCH receptor antagonists. To assess what occurs in the brain when the HCRT neurons degenerate as in human narcolepsy, we will use the conditional HCRT neuron ablation model to determine how the excitability of the MCH population is affected by chronic loss of HCRT input. We will also use conditional MCH neuron ablation to assess the converse effect of MCH loss on HCRT neuron excitability. Based on recordings from a limited number of cells in head-fixed animals, the HCRT and MCH neurons have been reported to have reciprocal activity across the sleep-wake cycle with HCRT neurons having their highest firing rates during active wakefulness and MCH neurons being primarily active during REM sleep. To determine the accuracy of this conclusion, we will use genetically- encoded Ca2+ indicators and microendoscopic imaging to measure the activity of hundreds of HCRT and MCH neurons across the sleep/wake cycle in unrestrained, freely-moving animals. To evaluate whether the HCRT and MCH neurons are functionally interconnected, we will pharmacogenetically activate one population while imaging Ca2+ fluorescence in the other population in the presence of selective HCRT and MCH receptor antagonists. Lastly, since these two populations project to many of the same brain regions, we will assess their relative input to brain areas known to be involved in arousal state control, specifically, the locus coeruleus, tuberomammillary nucleus, medial septum, and the amygdala. Together, these experiments should provide a more complete picture of the anatomical and functional connectivity of these two populations and the consequences of selective loss of one population or the other.