Behavioral state-dependent microglia Ca2+ dynamics

我们建议研究我们在清醒行为中发现的一种新形式的小胶质细胞钙信号 老鼠。这是到目前为止发现的第一个小胶质细胞钙信号，它代表了对积极、警惕的直接反应 短潜伏期(&lt；10秒)内的行为和神经元活动。由于实验的复杂性 程序和可用数据的匮乏对清醒状态下的小胶质细胞钙动力学知之甚少 行为不端的老鼠。这与钙离子信号对小胶质细胞功能的预期重要性形成了鲜明的对比。 从对培养的小胶质细胞的研究中，我们知道基本的细胞过程，如细胞骨架重排， 它们是细胞运动和吞噬的先决条件，也是大多数分泌的机制 细胞因子等信号分子的变化依赖于细胞内钙离子的波动。小胶质细胞表达 许多膜蛋白潜在地将细胞外信号转化为细胞内钙离子升高。 其中包括嘌呤能受体、免疫系统相关受体以及离子通道。具体来说， 小胶质细胞表达L类钙通道(LTCC)。LTCC引起了特别的关注，因为敌手 这类离子通道中，传统上用于控制血压的，已经显示出治疗作用 有可能限制伴随神经退行性疾病而来的神经炎症和认知损害。 小胶质细胞LTCC在这种治疗潜力中的作用尚未得到很好的证实，也不清楚。 当这些离子通道正常激活时。对于这项提议，我们将使用双光子显微镜 在小胶质细胞特异表达遗传编码的钙指示剂GCaMP6f的转基因小鼠上。这个 老鼠将被头固定在电动直线跑步机上，以精确控制强制训练的速度和持续时间 除监测自愿运动事件外，还将监测运动事件。运动代表着一种可靠的 诱导适度唤醒的手段，一种高度警惕的行为状态。为了避免由 实验条件研究将包括通过慢性颅窗对小胶质细胞进行成像 手术后几周以及急性手术实验的局部药理学。我们的预赛 药理学实验表明，运动诱导的小胶质细胞钙升高依赖于LTCCs。在……里面 目的1我们将对新发现的运动诱导的小胶质细胞钙离子进行系统的表征 立面。我们将研究反应的动力学限制，并测试V1小胶质细胞钙离子 反应受视觉刺激的影响。在目标2中，我们将结合小胶质细胞选择性基因缺失和 药理学实验，以确定哪个LTCC亚型对这些反应起主要作用。 在成功完成拟议的实验后，我们将为 旨在揭示警觉依赖的小胶质细胞钙离子后果的未来研究的合理设计 对电路活动和行为的响应。这些发现可能为以小胶质细胞为中心的研究开辟新的途径 神经退行性疾病和神经精神疾病的治疗干预。

We propose to investigate a novel form of microglia Ca2+ signal that we have discovered in awake behaving mice. It is the first microglia Ca2+ signal identified so far that represents a direct response to active, vigilant behavior and neuronal activity within short latency (<10 seconds). Due to the complexity of experimental procedures and the paucity of available data very little is known about microglia Ca2+ dynamics in awake behaving mice. This is in striking contrast to the expected importance of Ca2+ signaling for microglia function. From work on cultured microglia it is known that basic cellular processes such as cytoskeletal rearrangements, which are a prerequisite for cell motility and phagocytosis, as well as most mechanisms underlying the secretion of signaling molecules such as cytokines depend on intracellular fluctuations of Ca2+. Microglia express numerous membrane proteins to potentially translate extracellular signals into intracellular Ca2+ elevations. Among these are purinergic receptors, immune system-related receptors as well as ion channels. Specifically, microglia express L-type Ca2+ channels (LTCCs). LTCCs have attracted special attention because antagonists of this class of ion channels, traditionally applied to control blood pressure, have demonstrated therapeutic potential for limiting neuroinflammation and cognitive impairments accompanying neurodegenerative diseases. The contribution of microglia LTCCs to this therapeutic potential is not yet well established and it is not clear when these ion channels become normally activated. For this proposal we will employ two-photon microscopy on transgenic mice with microglia-specific expression of the genetically-encoded Ca2+ indicator GCaMP6f. The mice will be head-fixed on a motorized linear treadmill for precise control over speed and duration of enforced locomotion events in addition to the monitoring of voluntary locomotion events. Locomotion represents a reliable means to induce moderate arousal, a behavioral state of heightened vigilance. To avoid a bias by the experimental conditions the studies will include imaging of microglia through chronic cranial windows several weeks following surgery as well as acute surgery experiments for topical pharmacology. Our preliminary pharmacological experiments suggest that locomotion-induced microglia Ca2+ elevations depend on LTCCs. In Aim 1 we will conduct a systematic characterization of the newly discovered locomotion-induced microglia Ca2+ elevations. We will investigate the kinetic constraints of the responses and test whether V1 microglia Ca2+ responses are affected by visual stimulation. In Aim 2 we will combine microglia-selective gene deletion and pharmacological experiments to determine which LTCC subtype predominantly contributes to the responses. Upon successful completion of the proposed experiments we will have established a rigorous foundation for rational design of future studies aimed at unraveling the consequences of vigilance-dependent microglia Ca2+ responses for circuit activity and behavior. These findings may then open new avenues for microglia-centered therapeutic interventions in neurodegenerative and neuropsychiatric diseases.