Behavioral state-dependent microglia Ca2+ dynamics

行为状态依赖性小胶质细胞 Ca2 动力学

基本信息

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
项目类别：
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

项目摘要

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形式的小胶质细胞 Ca2+ 信号，我们在清醒行为中发现了这种信号老鼠。这是迄今为止发现的第一个小胶质细胞 Ca2+ 信号，代表对活跃、警惕的直接反应短潜伏期（<10 秒）内的行为和神经元活动。由于实验的复杂性程序和可用数据的缺乏对清醒状态下小胶质细胞 Ca2+ 动力学知之甚少行为老鼠。这与 Ca2+ 信号传导对小胶质细胞功能的预期重要性形成鲜明对比。通过对培养的小胶质细胞的研究，我们知道基本的细胞过程，例如细胞骨架重排，这是细胞运动和吞噬作用以及大多数分泌机制的先决条件细胞因子等信号分子的表达依赖于细胞内 Ca2+ 的波动。小胶质细胞表达许多膜蛋白可能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 (LTCC)。 LTCC 引起了特别关注，因为拮抗剂传统上用于控制血压的此类离子通道已被证明具有治疗作用限制神经退行性疾病伴随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确定且尚不清楚当这些离子通道正常激活时。对于这个建议，我们将采用双光子显微镜在具有小胶质细胞特异性表达基因编码 Ca2+ 指示剂 GCaMP6f 的转基因小鼠上。这小鼠将被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除了监测自愿运动事件外，还监测运动事件。运动代表着可靠意味着引起适度的觉醒，一种高度警惕的行为状态。为了避免偏见实验条件这些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，运动引起的小胶质细胞 Ca2+ 升高取决于 LTCC。在目标 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。我们将研究反应的动力学约束并测试 V1 小胶质细胞 Ca2+ 是否反应受到视觉刺激的影响。在目标 2 中，我们将结合小胶质细胞选择性基因删除和药理学实验以确定哪种 LTCC 亚型对反应有主要贡献。成功完成拟议的实验后，我们将为未来研究的合理设计旨在揭示警惕依赖性小胶质细胞 Ca2+ 的后果对电路活动和行为的反应。这些发现可能为以小胶质细胞为中心的研究开辟新途径神经退行性疾病和神经精神疾病的治疗干预。