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.

项目总结 在过去的二十年里，星形胶质细胞在大脑生理学中的作用变得更加清晰。星形胶质细胞控制 神经元通过调节突触传递、离子梯度等来发挥作用。他们能感觉到并迅速 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，是 对正常的大脑功能来说是必不可少的。在健康的大脑中，谷氨酸一旦从神经元释放出来，就会被运输 通过兴奋性氨基酸转运体(EAATs)GLT-1和GLAST进入星形胶质细胞。以前，使用 我们的实验室已经在健康的成年小鼠皮质中显示了基于iGluSnFR的谷氨酸成像和电生理学 谷氨酸的摄取在神经元活动爆发后减慢到原来的三倍。我们怀疑这是 发生是因为神经元活动产生动作电位，导致细胞外钾局灶性增加 ([K+]e)。我们发现，[K+]e的增加驱动星形胶质细胞局部去极化，导致电压依赖 抑制EAATs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K+摄取均可导致细胞功能紊乱 谷氨酸动力学，但这些通道和转运体发挥的确切作用尚不清楚。我们假设 EAAT，像GLT-1一样，驱动摄取，而Kir4.1形成依赖活性的谷氨酸减速。 这份F99/K00 D-SPAN提案包含2个目标，详细说明了我的目标和目标。在目标1中，我描述了我的 到目前为止的进展以及我为完成博士学位而提议的研究。主要初步数据使用 小鼠创伤性脑损伤后3天控制性皮质撞击(CCI)模型(1)谷氨酸 衰变时间常数减慢，(2)谷氨酸峰值反应增加，(3)GLT-1表达降低， (4)Kir4.1的表达没有改变。为了更好地理解Kir4.1‘S对谷氨酸清除的重要性，我们 将利用Kir4.1fl/fl小鼠系和AAVs在星形胶质细胞中有条件地和局部地敲除Kir4.1。什么时候 完成后，这项研究将为脑损伤如何改变谷氨酸动态提供新的见解，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，我概述了我对研究神经胶质功能和神经胶质细胞如何对神经学有贡献的博士后抱负。 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 研究、协作、多样化、专业成长，推动生物医学领域向前发展。在支持下 在我的赞助人克里斯·杜拉博士中，我通过参加多次会议和研讨会，成长为一名科学家， 在影响很大的期刊上发表，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、导师和科学界的一员。最终，我相信我是在一个完美的 成为D-SPAN社区的一部分并从中受益的绝佳机会 这一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。