The Tuberal Hypothalamus and Arousal State Control

黑色素浓缩激素(MCH)和下丘脑促性腺激素/食欲素(HCRT)表达的神经元混合在一起 结节下丘脑中广泛投射到许多相同终末的群体 菲尔兹。鉴于HCRT系统被认为与控制觉醒有关，因为睡眠 当这些细胞退化时，这个系统也参与了能量代谢，从而导致发作性睡病。 相反，妇幼保健系统主要与食物摄入量和能量代谢有关，但 最近的研究证实，MCH神经元也参与了睡眠和觉醒的调节。 支持这一建议的假设是，HCRT系统是稳定尾迹和抑制REM的 而妇幼保健系统是促进睡眠和稳定快速眼动的系统。我们将通过确定以下内容来检验这一假设 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饮食中的多西环素。然后我们将评估部分消融是否会导致 HCRT神经元导致无猝发的发作性睡病的表型以及猝发是否加重 通过同时消灭这两个神经元群体。我们还将评估直接连接是否 利用光基因辅助神经解剖示踪和全细胞贴片在这些细胞组之间存在- 存在和不存在选择性HCRT和MCH受体拮抗剂时的钳夹电生理学。至 评估当HCRT神经元退化时大脑中发生了什么，就像人类发作性睡病一样，我们将使用 条件HCRT神经元消融模型确定MCH群体的兴奋性如何受 HCRT输入的慢性丢失。我们还将使用有条件的MCH神经元消融术来评估 MCH缺失对HCRT神经元兴奋性的影响。基于来自有限数量的磁头固定单元的记录 据报道，动物的HCRT和MCH神经元在睡眠觉醒过程中具有相互作用的活动。 在主动觉醒期间，HCRT神经元的放电频率最高，而MCH神经元 主要在快速眼动睡眠期间活跃。为了确定这一结论的准确性，我们将使用基因- 编码的钙指示剂和显微内窥镜成像测量数百个HCRT和MCH的活性 自由活动的动物的睡眠/清醒周期中的神经元。评估HCRT是否 和MCH神经元在功能上是相互联系的，我们将通过药物遗传学激活一个种群，同时 在选择性HCRT和MCH受体存在的情况下成像其他种群中的钙荧光 对抗者。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 它们对已知参与觉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，这些实验应该会提供一种 这两个群体的解剖和功能连接的更完整的图景 有选择地失去一个人口或另一个人口的后果。

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.