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型的小胶质细胞Ca2+信号 老鼠。这是到目前为止确定的第一个小胶质细胞Ca2+信号，代表了对主动，警惕性的直接响应 短潜伏期内的行为和神经元活性（<10秒）。由于实验的复杂性 关于清醒中的小胶质细胞CA2+动力学的过程和可用数据的匮乏知之甚少 行为小鼠。这与Ca2+信号对小胶质细胞功能的预期重要性形成鲜明对比。 从培养的小胶质细胞的工作中，众所周知，基本的细胞过程，例如细胞骨架重排， 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 信号分子（例如细胞因子）的依赖于Ca2+的细胞内波动。小胶质细胞表达 许多膜蛋白可能会将细胞外信号转化为细胞内Ca2+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型Ca2+通道（LTCCS）。 LTCC引起了特别的注意，因为对手 传统上用于控制血压的这类离子通道中已显示出治疗性 限制神经退行性疾病伴随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不确定，尚不清楚 当这些离子通道通常被激活时。对于此提案，我们将采用两光子显微镜 在具有遗传编码的Ca2+指标GCAMP6F的小胶质细胞特异性表达的转基因小鼠上。这 小鼠将在电动线性跑步机上固定，以精确控制速度和执行持续时间 运动事件除了监测自愿运动事件。运动代表一个可靠的 诱发中等唤醒的手段，一种行为的警惕性。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几个 手术后的几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Ca2+升高取决于LTCC。在 AIM 1我们将对新发现的运动诱导的小胶质细胞CA2+进行系统表征 海拔。我们将研究响应的动力学约束，并测试V1小胶质细胞CA2+是否 反应受视觉刺激的影响。在AIM 2中，我们将结合小胶质细胞选择性基因缺失和 确定哪种LTCC亚型的药理实验主要有助于反应。 成功完成拟议的实验后，我们将为 未来研究的理性设计旨在揭示依赖警惕性小胶质细胞CA2+的后果 电路活动和行为的响应。这些发现可能会以小胶质细胞为中心开放新途径 神经退行性和神经精神疾病的治疗干预措施。