Behavioral state-dependent microglia Ca2+ dynamics

行为状态依赖性小胶质细胞 Ca2 动力学

• 批准号: 10593572
• 负责人: Martin Paukert
• 金额: $ 23.25万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-01-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10593572
• 关键词: Acute; Affect; Anesthesia procedures; Animals; Area; Arousal; Astrocytes; Attention; Behavior; Behavioral; Brain; Cell physiology; Cells; Cephalic; Chronic; Cytoskeleton; Data; Deterioration; Development; Endocytosis; Event; Foundations; Future; Gene Deletion; Head; Image; Immune; Immune system; Impaired cognition; Impairment; Individual; Inflammatory; Investigation; Ion Channel; Ion Channel Gating; Ischemia; Kinetics; Length; Link; Locomotion; Macrophage; Mechanics; Membrane; Membrane Proteins; Microglia; Molecular; Monitor; Motor; Mus; Neurodegenerative Disorders; Neurons; Operative Surgical Procedures; Pathologic; Phagocytes; Phagocytosis; Pharmacology; Photic Stimulation; Population; Potassium; Procedures; Process; Purinoceptor; Regulation; Reporting; Residual state; Signal Transduction; Signaling Molecule; Slice; Source; Speed; Synapses; Testing; Therapeutic; Therapeutic Intervention; Transgenic Mice; Translating; Trauma; Trees; Visual; Work; antagonist; attenuation; awake; blood pressure control; brain cell; cell motility; cognitive performance; conditional knockout; cytokine; density; experience; experimental study; extracellular; genetic manipulation; interest; nervous system disorder; neuroinflammation; neuropsychiatric disorder; novel; pharmacologic; rational design; receptor; response; selective expression; sensory input; targeted treatment; treadmill; two photon microscopy; vigilance; visual processing; voltage

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.

我们建议研究一种新的形式的小胶质细胞钙信号，我们已经发现在清醒的行为 小鼠这是迄今为止发现的第一个小胶质细胞Ca 2+信号，它代表了对活跃、警惕的 短潜伏期（<10秒）内的行为和神经元活动。由于实验的复杂性， 程序和缺乏可用的数据很少知道小胶质细胞Ca 2+动态清醒 行为老鼠这与Ca 2+信号对小胶质细胞功能的预期重要性形成鲜明对比。 从对培养的小胶质细胞的研究可知，基本的细胞过程如细胞骨架重排， 这是细胞运动和吞噬作用的先决条件，以及分泌的大多数机制 细胞因子等信号分子的活性依赖于细胞内Ca ~（2+）的波动。小胶质细胞表达 许多膜蛋白可能将细胞外信号翻译成细胞内Ca 2+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。具体地说， 小胶质细胞表达L型钙通道（LTCCs）。LTCC引起了特别的关注，因为拮抗剂 传统上用于控制血压的这类离子通道中， 限制伴随神经退行性疾病的神经炎症和认知障碍的潜力。 小胶质细胞LTCC对这种治疗潜力的贡献尚未得到很好的确定， 当这些离子通道被正常激活时。对于这个建议，我们将采用双光子显微镜 在转基因小鼠与小胶质细胞特异性表达的遗传编码的Ca 2+指标GCaMP 6 f。的 将小鼠头部固定在电动线性跑步机上，以精确控制强制运动的速度和持续时间。 运动事件，除了监测自愿运动事件。运动是一种可靠的 意味着诱导中度觉醒，一种高度警惕的行为状态。为了避免偏见， 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， 手术后数周以及局部药理学的急性手术实验。我们的初步 药理学实验表明运动诱导的小胶质细胞Ca 2+升高依赖于LTCC。在 目的1我们将对新发现的运动诱导的小胶质细胞Ca 2+进行系统的表征 海拔我们将研究反应的动力学约束，并测试V1小胶质细胞Ca 2 + 反应受到视觉刺激的影响。在目标2中，我们将结合联合收割机小胶质细胞选择性基因缺失和 药理学实验以确定哪种LTCC亚型主要有助于响应。 在成功完成拟议的实验后，我们将为以下方面奠定坚实的基础： 未来研究的合理设计旨在揭示警惕依赖性小胶质细胞Ca 2+的后果 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 神经退行性疾病和神经精神疾病的治疗干预。