Investigating Astrocytic Glutamate and Potassium Dynamics in the Healthy and Injured Brain

项目摘要 在过去的二十年里，星形胶质细胞在脑生理学中的作用变得更加清晰。星形胶质细胞对照 通过调节突触传递、离子梯度等来调节神经元功能。它们迅速感知到 对突触活动作出反应。谷氨酸是中枢神经系统中的主要兴奋性神经递质， 对正常的大脑功能至关重要在健康的大脑中，谷氨酸一旦从神经元释放出来， 通过兴奋性氨基酸转运体（EAAT）GLT-1和GLAST进入星形胶质细胞。此前，使用 我们的实验室已经表明，健康成年小鼠皮层中基于iGluSnFR的谷氨酸成像和电生理学 在神经元活动爆发后，谷氨酸的摄取会减慢三倍。我们怀疑， 发生是因为神经元活动产生动作电位，导致细胞外钾离子局灶性增加 ([K+]e）。我们发现，[K+]e的增加驱动局部星形胶质细胞去极化，引起电压依赖性 抑制EAAT和延长细胞外谷氨酸瞬变。我们的模型表明， 由K+通道Kir4.1介导的EAAT和星形胶质细胞的K+摄取都可以导致破坏 谷氨酸动力学，但这些通道和转运蛋白发挥的确切作用是未知的。我们假设 EAAT，如GLT-1，驱动摄取，而Kir4.1形状活动依赖性减缓谷氨酸。 本F99/K 00 D-SPAN建议书包含2个目标，详细说明了我的目标和目的。在目标1中，我描述我的 到目前为止的进展以及我提出的完成博士学位的研究。关键的初步数据， 小鼠创伤性脑损伤（TBI）的受控皮质撞击（CCI）模型显示损伤后3天（1）谷氨酸 衰减时间常数减慢，（2）峰值谷氨酸反应增加，（3）GLT-1表达降低， （4）Kir4.1表达无改变。为了更好地理解Kir4.1对谷氨酸清除的重要性，我们 将利用Kir4.1fl/fl小鼠系和AAV来条件性地和局部地敲除星形胶质细胞中的Kir4.1。当 完成后，这项研究将提供新的见解谷氨酸动力学是如何改变TBI，Kir4.1的作用 以及星形胶质细胞中这些分子变化如何驱动突触活动。 在目标2中，我概述了我的博士后愿望，即研究胶质细胞的功能以及胶质细胞如何在神经系统中发挥作用。 疾病我将确定一个强大的博士后实验室和机构，促进创新的科学， 研究，合作，多样性，专业成长，并推动生物医学领域。与支撑 在我的赞助人克里斯·杜拉博士的帮助下，我通过参加多个会议和研讨会， 在高影响力的期刊上发表，并在我的信心中成长。我已经确定了我的优势和领域， 成长为科学家，导师和科学界的成员。最终，我相信我是一个完美的 感谢并受益于这样一个绝佳的机会，成为D-SPAN社区的一部分， 这个奖项将推动我在学术和专业上追求成为一名独立的研究科学家。

PROJECT SUMMARY Over the past two decades, the role of astrocytes in brain physiology has become clearer. Astrocytes control neuronal function by modulating synaptic transmission, ion gradients, and more. They sense and rapidly respond to synaptic activity. Glutamate, the primary excitatory neurotransmitter in the central nervous system, is essential for normal brain function. In the healthy brain, once released from neurons, glutamate is transported into astrocytes by the excitatory amino acid transporters (EAATs) GLT-1 and GLAST. Previously, using iGluSnFR-based glutamate imaging and electrophysiology in the healthy adult mouse cortex, our lab has shown that glutamate uptake is slowed up to threefold following bursts of neuronal activity. We suspected that this occurs because neuronal activity generates action potentials, causing focal increases in extracellular potassium ([K+]e). We showed that increases in [K+]e drives local astrocyte depolarization, causing voltage-dependent inhibition of EAATs and prolonging extracellular glutamate transients. Our model suggests that dysregulation of both EAATs and astrocytic K+ uptake, mediated by the K+ channel Kir4.1, can both contribute to disrupted glutamate dynamics, but the exact role these channels and transporters play is unknown. We hypothesize that EAATs, like GLT-1, drive uptake while Kir4.1 shapes activity-dependent slowing of glutamate. This F99/K00 D-SPAN proposal encompasses 2 Aims detailing my goals and objectives. In Aim 1, I describe my progress thus far as well as my proposed research to complete my Ph.D. Key preliminary data using the controlled cortical impact (CCI) model of traumatic brain injury (TBI) in mice show 3 days after injury (1) glutamate decay time constants are slowed, (2) peak glutamate response is increased, (3) GLT-1 expression is decreased, and (4) Kir4.1 expression is not altered. To better understand Kir4.1’s importance to glutamate clearance, we will utilize a Kir4.1fl/fl mouse line and AAVs to conditionally and focally knockout Kir4.1 in astrocytes. When completed, this study will provide novel insight into how glutamate dynamics are altered by TBI, the role of Kir4.1 in shaping glutamate uptake, and how these molecular changes in astrocytes drive synaptic activity. In Aim 2, I outline my post-doctoral aspirations of studying glial function and how glia contribute to neurological disease. I will identify a strong post-doctoral laboratory and institution that promotes innovative scientific research, collaboration, diversity, professional growth, and pushes forward the biomedical field. With the support of my sponsor, Dr. Chris Dulla, I have grown as a scientist by attending multiple conferences and workshops, published in high impact journals, and grown in my confidence. I have identified my strengths and areas for growth as a scientist, mentor, and member of the scientific community. Ultimately, I believe I am at a perfect juncture to appreciate and benefit from such a fantastic opportunity to be a part of the D-SPAN community and this award will propel me in my academic and professional pursuits of being an independent research scientist.