Targeting Kir4.1 To Control Brain Excitability And Seizures

神经元活动可以迅速提高大脑中的细胞外钾。钾的升高与神经细胞兴奋性的增加有机械上的联系，可能会导致异常的网络行为，如癫痫。星形胶质细胞是维持钾平衡的关键，通过与钠钾泵相连的缓冲机制。然而，新的证据将细胞外钾的动态调节主要归因于Kir4.1类型的星形胶质细胞通道，而这些通道的下调与癫痫敏感性的增强以及从长期来看，脑组织的硬化性异常有关。尽管Kir4.1对脑兴奋性控制很重要，但Kir4.1的表达、钾动力学、局部神经元兴奋性和突触功能之间的因果关系仍然知之甚少。缺乏进展的原因是对细胞外钾升高的影响知之甚少，而且往往是抵消的后果，缺乏监测脑组织中钾动态的工具，以及很难获得海绵状的星形胶质细胞的纳米形态。因此，本研究的主要目的是了解星形胶质细胞依赖于Kir4.1的钾缓冲是如何调节神经兴奋性和突触回路功能的，以及是否从药物或基因上针对这些机制可以改变对失控兴奋和癫痫的易感性。因此，中心假设是星形胶质细胞Kir4.1的不同表达以一种机械可预测的方式调节细胞的兴奋性和突触信号传递。该提案的关键翻译方面是，对Kir4.1表达的受控操作应该可以改善神经兴奋性的病理变化，如癫痫或皮质扩散性抑制期间的病理变化。为了实现我们的目标，我们将利用我们新颖和尖端的实验和理论方法。我们已经建立了基因打靶方案，以便在Kir4.1-高表达星形胶质细胞中进行单细胞研究，同时监测局部突触连接的神经递质释放。我们已经开始了一项新的纳米工程技术来监测钾，这涉及到将比率光学传感器封装到离子渗透的、生物兼容的微胶囊中。我们已经建立了一个新的生物物理建模平台，可以从理论上探索现实星形胶质细胞模型中细胞内和细胞外钾的动态变化。我们开发了一种新的基于柔性石墨烯晶体管阵列的多电极皮层脑电成像技术，可以在清醒动物身上进行全频段电流记录。这些和相关的方法突破，在大量试点和原则验证数据的支持下，帮助我们制定了实现主要目标的可行研究战略。该计划包括五个工作方案中涉及的五个具体目标。这一结果将为星形胶质细胞通过依赖Kir4.1调控细胞外钾动力学来调节神经元兴奋性的机制提供新的知识。基于这些知识，可以开发一种治疗策略，帮助降低大脑对失控兴奋的敏感性，例如癫痫和相关疾病。

Neuronal activity can rapidly elevate extracellular potassium in the brain. Potassium elevations are mechanistically related to increases in nerve cell excitability, potentially leading to abnormal network behaviours such as in epilepsy. Astroglia are essential for maintaining potassium balance, through a buffering mechanism engaging their sodium-potassium pumps. However, new evidence attributes the dynamic regulation of extracellular potassium mainly to astroglial channels of the Kir4.1 type whereas downregulation of these channels has been associated with enhanced seizure susceptibility and, in the long term, sclerotic abnormalities of brain tissue. Despite the importance of Kir4.1 for brain excitability control, the causal relationships between Kir4.1 expression, potassium dynamics, local neuronal excitability and synaptic function remain poorly understood. The lack of progress stems from the poorly understood and often counteracting consequences of extracellular potassium rises, from the lack of tools to monitor potassium dynamics in brain tissue, and from the poor access to the sponge-like, nanoscopic morphology of astroglia. The main goal of the present proposal is therefore to understand how the Kir4.1-dependent potassium buffering by astrocytes regulates neural excitability and synaptic circuit function, and whether targeting these mechanisms, pharmacologically or genetically, can alter susceptibility to runaway excitation and seizures. Thus, the central hypothesis is that the varied expression of astroglial Kir4.1 regulates, in a mechanistically predictable manner, cell excitability and synaptic signal transfer. The key translational aspect of the proposal is that the controlled manipulation of Kir4.1 expression should ameliorate pathological changes in neural excitability, such as those during epilepsy or cortical spreading depression. To achieve our goal, we will take advantage of our novel and cutting-edge experimental and theoretical approaches. We have established gene-targeting protocols to enable single-cell studies in Kir4.1-overexpressing astrocytes while simultaneously monitoring neurotransmitter release at local synaptic connections. We have embarked on a novel nanoengineering technology to monitor potassium, which involves encapsulation of the ratiometric optical sensor into ion-permeable, biologically compatible microcapsules. We have established a novel biophysical modelling platform that enables theoretical probing of the intra- and extracellular potassium dynamics in realistic astrocyte models. We have developed a novel multi-electrode electrocorticography technique based on flexible graphene transistor arrays enabling full-band current recordings in awake animals. These and related methodological breakthroughs, backed by a large body of pilot and proof-of-principle data, helped us to formulate a feasible research strategy for achieving our main goal. The plan includes five specific objectives addressed in five work packages. The results will provide new knowledge about the mechanisms by which astroglia regulate neuronal excitability through the Kir4.1-dependent control of extracellular potassium dynamics. Based on such knowledge, a therapeutic strategy could be developed that helps reduce brain susceptibility to runaway excitation, such as seen in epilepsy and related disorders.