Targeting Kir4.1 To Control Brain Excitability And Seizures

神经元活动可迅速升高脑中的细胞外钾。钾升高与神经细胞兴奋性增加有关，可能导致异常网络行为，如癫痫。星形胶质细胞是维持钾平衡所必需的，通过缓冲机制参与其钠钾泵。然而，新的证据将细胞外钾的动态调节主要归因于Kir4.1型星形胶质细胞通道，而这些通道的下调与癫痫发作易感性增强以及长期脑组织的神经功能异常相关。尽管Kir4.1对大脑兴奋性控制的重要性，Kir4.1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。缺乏进展源于对细胞外钾升高的认识不足和经常抵消的后果，缺乏监测脑组织中钾动力学的工具，以及难以获得星形胶质细胞的海绵状纳米形态。因此，本提案的主要目标是了解星形胶质细胞的Kir4.1依赖性钾缓冲如何调节神经兴奋性和突触回路功能，以及靶向这些机制，无论是神经系统还是遗传学，都可以改变对失控兴奋和癫痫发作的易感性。因此，中心假设是星形胶质细胞Kir4.1的不同表达以机械可预测的方式调节细胞兴奋性和突触信号传递。该提案的关键翻译方面是Kir4.1表达的受控操纵应改善神经兴奋性的病理变化，例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靶向方案，使单细胞研究Kir4.1过表达的星形胶质细胞，同时监测神经递质释放在当地的突触连接。我们已经着手研究一种新的纳米工程技术来监测钾，该技术涉及将比率光学传感器封装到离子可渗透的生物相容性微胶囊中。我们已经建立了一个新的生物物理建模平台，使理论探测内和细胞外钾动力学在现实的星形胶质细胞模型。我们已经开发了一种新的多电极皮层电图技术的基础上灵活的石墨烯晶体管阵列，使清醒的动物全波段电流记录。这些以及相关的方法学突破，在大量试点和原理验证数据的支持下，帮助我们制定了一个可行的研究策略，以实现我们的主要目标。该计划包括五个具体目标，分为五个工作包。这些结果将为星形胶质细胞通过Kir4.1依赖性控制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，例如在癫痫和相关疾病中看到的。

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.