Astrocyte modulation of sleep

内源性节律行为在进化上是保守的，对生命是必不可少的。在哺乳动物和 在无脊椎动物模型中，除了神经元回路外，还有对神经胶质细胞的已知要求 昼夜行为和睡眠的调节1-4。利用果蝇模型，我们的实验室首先展示了 在活体内，星形胶质细胞是正常的昼夜节律行为所必需的5，6，我们在这个建议中表明 星形胶质细胞也调节睡眠。在这项应用的初步研究中，我们已经证明 苍蝇星形胶质细胞通过增加细胞内钙离子激活促进睡眠，而抑制星形胶质细胞 囊泡的运输和释放会导致睡眠减少。在相关研究(现已发表)中，我们已经表明 一种被称为NKT的富含星形胶质细胞的分泌蛋白可以促进睡眠。 假设神经元睡眠调节回路受制于星形胶质细胞的调节。Aim 1a将测试这一点 通过同时激活或抑制星形胶质细胞并记录已知睡眠的兴奋性- 使用遗传编码的电压传感器调节神经元群体。这将识别神经元组 这表明通过激活或抑制星形胶质细胞来改变兴奋性。在对目标1b的补充研究中，我们 将在所有已知的睡眠调节神经元组中表达膜拴系版本的NKT以识别 那些受神经胶质蛋白调节的细胞。NKT是一种分泌的星形细胞蛋白，但这个亚目标将利用 膜系NKT的神经元表达以确定其细胞靶点。加在一起，目标1a和1b将 识别受星形胶质细胞调节的睡眠调节神经元。AIM 2将使用翻译核糖体 亲和纯化(TRAP)方法与全基因组RNA-SEQ相结合来验证基因 睡眠调节神经元的表达谱因星形胶质细胞的调节而改变。对这一目标的研究将 重点识别脊椎动物的神经因子(受体、细胞内信号成分) 可能参与星形胶质细胞调节睡眠的同源基因。记录可行性的初步结果 这些目标都包括在提案中。这两个目标结合起来将提供有关神经元的有价值的信息。 星形胶质细胞调控的靶点，并确定对星形胶质细胞信号做出反应的神经元分子通路。 这类通路可能对神经胶质细胞与神经元之间的通讯或更直接地调节睡眠很重要。

Endogenous rhythmic behaviors are evolutionarily conserved and essential for life. Both in mammalian and invertebrate models, there are known requirements for glial cells, in addition to neuronal circuits, in the regulation of circadian behavior and sleep 1-4. Using the Drosophila model, our lab was the first to demonstrate that astrocytes were required, in vivo, for normal circadian behavior 5, 6, and we show in this proposal that astrocytes also modulate sleep. In preliminary studies for this application, we have demonstrated that activation of fly astrocytes, by increasing intracellular Ca2+, promotes sleep whereas inhibition of astrocyte vesicle trafficking and release leads to decreased sleep. In related studies (now published), we have shown that an astrocyte-enriched, secreted protein known as NKT promotes sleep 7. Based on these findings, we hypothesize that neuronal sleep-regulating circuits are subject to astrocyte modulation. Aim 1a will test this hypothesis by simultaneously activating or inhibiting astrocytes and recording the excitability of known sleep- regulating neuronal populations using genetically encoded voltage sensors. This will identify neuronal groups that show altered excitability with activation or inhibition of astrocytes. In complementary studies of Aim 1b, we will express membrane-tethered versions of NKT in all known sleep-regulating neuronal groups to identify those subject to modulation by the glial protein. NKT is a secreted astrocyte protein, but this subaim will utilize neuronal expression of membrane-tethered NKTs to identify its cellular targets. Together, Aims 1a and 1b will identify sleep-regulating neurons that are subject to astrocyte modulation. Aim 2 will use Translating Ribosome Affinity Purification (TRAP) methods combined with genome-wide RNA-seq to test the hypothesis that the gene expression profile of sleep-regulating neurons is altered by astrocyte modulation. The studies of this aim will focus on identification of neuronal factors (receptors, intracellular signaling components) with vertebrate orthologs that may mediate the astrocyte modulation of sleep. Preliminary results documenting feasibility for the aims are included in the proposal. Together, the two aims will provide valuable information about neuronal targets of astrocyte modulation and identify neuronal molecular pathways that respond to astrocyte signals. Such pathways may be important for glia-to-neuron communication or more directly the regulation of sleep.