The role of P2Y6 receptors in microglial calcium signaling: Investigations in the awake animal during the basal state and epilepsy development

小胶质细胞是中枢神经系统（CNS）的常驻免疫细胞。在中枢神经系统中，钙信号传导是星形胶质细胞和神经元中的重要次级信使系统。然而，在小胶质细胞中，钙信号传导并不完全清楚。在这项提案中，我们研究了健康和疾病中的小胶质细胞钙信号。在我们的第一个目标中，我们使用体内双光子显微镜探索小胶质细胞钙信号是否发生在清醒的小鼠中，以及该信号是否依赖于神经元活动。小胶质细胞中的活动依赖性钙信号表明这种细胞类型可以严格地感知神经元变化并做出反应。感知神经元活动的能力在癫痫发展过程中可能特别重要。在我们的第二个目标中，我们探索在癫痫发展模型中小胶质细胞钙活性是否以及如何变化。在目标2中，我们使用体内纵向双光子成像来研究癫痫发生过程中钙信号的变化。我们假设嘌呤能受体P2 Y 6对健康和疾病中的小胶质细胞钙信号传导是必不可少的。我们的训练计划利用选择性药理学和基因敲除策略来了解小胶质细胞钙信号传导的机制。我们提出的研究是理解关键的次级信使系统及其在小胶质细胞-神经元相互作用中的作用的重要的第一步。这些目标的成功完成将促进我们对癫痫发生过程的理解（NINDS癫痫研究基准II）。研究将在著名的转化研究和临床护理中心马约诊所进行。项目导师是小胶质细胞生理学/病理生理学（Dr. Long-Jun Wu）和癫痫发生机制（Dr. Greg Worrell）方面的专家。

Microglia are the resident immune cells of the central nervous system (CNS). In the CNS, calcium signaling is an essential secondary messenger system in astrocytes and neurons. However, in microglia, calcium signaling is not completely understood. In this proposal, we investigate microglial calcium signaling in health and disease. In our first aim, we explore whether microglial calcium signaling occurs in the awake mouse and whether that signaling is neuronal activity dependent using in vivo two-photon microscopy. Activity-dependent calcium signaling in microglia would suggest that this cell type could critically sense and respond to neuronal changes. The ability to sense neuronal activity may be particularly important during epilepsy development. In our second aim, we explore whether and how microglial calcium activity changes in a model of epilepsy development. In aim 2, we use in vivo, longitudinal two-photon imaging to study changes in calcium signaling during epileptogenesis. We hypothesize that the purinergic receptor, P2Y6, is essential for microglial calcium signaling in both health and disease. Our training plan utilizes selective pharmacology and gene knockout strategies to understand the mechanisms of microglial calcium signaling. Our proposed research is an important first step in understanding a critical secondary messenger system and its role in microglial-neuron interactions. Successful completion of these aims will advance our understanding of the epileptogenic process (NINDS Benchmark II for Epilepsy Research). Research will be performed at the Mayo Clinic, a renowned center for translational research and clinical care. Project mentors are experts in microglial physiology/pathophysiology (Dr. Long-Jun Wu), and mechanisms of epileptogenesis (Dr. Greg Worrell).