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

研究健康和受伤大脑中星形胶质细胞谷氨酸和钾的动态

• 批准号: 10754425
• 负责人: Jacqueline P Garcia
• 金额: $ 4.44万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10754425
• 关键词: Action Potentials; Address; Adult; Affect; Amino Acid Transporter; Animals; Area; Astrocytes; Award; Binding; Biology; Brain; Brain Injuries; Buffers; Cell Communication; Cells; Central Nervous System; Collaborations; Communities; Complex; Data; Data Set; Distal; Doctor of Philosophy; Educational workshop; Electrophysiology (science); Ensure; Event; Excitatory Amino Acids; Extracellular Space; Flow Cytometry; Functional Imaging; Functional disorder; GLAST Protein; Genetic; Glutamate Receptor; Glutamates; Goals; Growth; Image; Immunohistochemistry; Injury; Institution; Ions; Journals; Knock-out; Laboratories; Mediating; Membrane Potentials; Mentors; Modeling; Molecular; Motion; Mus; Neuroglia; Neurologic Dysfunctions; Neurological Models; Neurons; Neurotransmitters; Pathologic; Pathway interactions; Patient-Focused Outcomes; Patients; Peripheral; Persons; Phase; Physiology; Play; Positioning Attribute; Postdoctoral Fellow; Potassium; Potassium Channel; Process; Publishing; Regulation; Reporting; Research; Research Personnel; Research Project Grants; Role; Scientist; Series; Shapes; Signal Transduction; Synapses; Synaptic Transmission; Technical Expertise; Techniques; Testing; Therapeutic Intervention; Time; Training; Traumatic Brain Injury; Western Blotting; Work; aspirate; career; cell type; controlled cortical impact; experience; experimental study; extracellular; genetic manipulation; glutamatergic signaling; improved; innovation; insight; inward rectifier potassium channel; member; mortality; nervous system disorder; neural repair; neurotransmission; novel; response; single-cell RNA sequencing; success; symposium; transcriptome sequencing; uptake; voltage

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.

项目摘要 在过去的二十年中，星形胶质细胞在脑生理学中的作用变得更加清晰。星形胶质细胞控制 神经元功能通过调节突触传递，离子梯度等。他们感觉到很快 响应突触活动。谷氨酸是中枢神经系统中的主要兴奋性神经递质，是 对正常的大脑功能必不可少。在健康的大脑中，一旦从神经元释放，谷氨酸就会运输 通过兴奋性氨基酸转运蛋白（EAATS）GLT-1和GLAST进入星形胶质细胞。以前使用 基于IGLUSNFR的基于健康的成年小鼠皮层中的谷氨酸成像和电生理学，我们的实验室已显示 神经元活性爆发后，这种谷氨酸摄取量减慢了三倍。我们怀疑这 发生由于神经元活性会产生动作电位，从而导致细胞外钾的局灶性增加 （[K+] E）。我们表明，[K+] E的增加驱动了局部星形胶质细胞沉积，从而导致电压依赖性 抑制EAATS和延长细胞外谷氨酸瞬变。我们的模型表明失调 由K+ Channel Kir4.1介导的EAATS和星形胶质细胞K+摄取都可能导致破坏 谷氨酸动力学，但是这些通道和转运蛋白播放的确切作用尚不清楚。我们假设这一点 像GLT-1一样，EAATS推动吸收，而Kir4.1塑造了谷氨酸的活动依赖性减慢。 该F99/K00 D-SPAN提案涵盖2旨在详细介绍我的目标和目标。在AIM 1中，我描述了我的 到目前为止，进步以及我提出的研究以完成我的博士学位。密钥初步数据使用 损伤后3天显示的小鼠创伤性脑损伤（TBI）的受控皮质冲击（CCI）模型（1）谷氨酸 衰减时间常数放慢，（2）增加谷氨酸峰响应，（3）GLT-1表达降低， （4）KIR4.1表达不会改变。为了更好地了解Kir4.1对谷氨酸清除的重要性，我们 将利用KIR4.1FL/FL鼠标线和AAVS在星形胶质细胞中进行条件和局部敲除Kir4.1。什么时候 完成后，这项研究将提供新的洞察力，以了解TBI如何改变谷氨酸动力学，Kir4.1的作用。 在塑造谷氨酸摄取以及这些分子变化的星形胶质细胞中的合成活性时。 在AIM 2中，我概述了研究神经胶质功能以及神经胶质对神经系统的贡献的愿望 疾病。我将确定强大的博士后实验室和机构，以促进创新的科学 研究，协作，多样性，专业增长，并推动了生物医学领域。在支持下 我的赞助商克里斯·杜拉（Chris Dulla）博士，通过参加多个会议和讲习班，成长为科学家 发表在高影响期刊上，并充满信心。我已经确定了我的优势和领域 成长为科学家，心理和科学界成员。最终，我相信我是一个完美的 从如此奇妙的机会中欣赏和受益的关头，成为D-Span社区的一部分， 该奖项将推动我成为一名独立研究科学家的学术和专业追求。