ASTROCYTIC REGULATION OF NEURONAL EXCITABILITY IN VIVO

神经元的过度兴奋性和兴奋性毒性被认为是许多临床疾病的基础。不幸的是，人们对导致神经元过度活动的机制知之甚少。有力的间接证据表明，神经元的兴奋性受到[K]o微小变化的影响，星形胶质细胞在[K]o的调节中起着重要作用。星形胶质细胞被认为将[K]o维持在正常神经元活动所需的狭窄限制内，将K从神经元活动区中移除(这一过程被称为空间缓冲)。空间缓冲被认为是通过内向整流钾通道(KIR)摄取K，并将K分散到通过缝隙连接连接的星形胶质细胞的合胞体中来完成的。我们建议验证空间缓冲依赖于星形胶质细胞KIR和缝隙连接表达的假设，以及消除星形胶质细胞中的这些会导致[K]O、神经元兴奋性和癫痫发作活动的增加。Cre/loxP技术和可诱导的星形胶质细胞特异性基因表达系统将用于进行连接蛋白43(Cn43)和Kir4.1的条件性敲除(CKO)；这些蛋白质分别是星形胶质细胞之间缝隙连接通讯(GJC)和K摄取所必需的。消除星形细胞的cn43和kir的效果将使用海马脑片进行原位检测，并使用高兴奋性模型进行体内检测。这些研究将解决以下问题。首先，在体内，cn43或kir4.1的CKO是否会导致自发性癫痫发作或增加对促癫痫刺激的敏感性？第二，cn43或kir4.1的CKO是否会导致神经元在原位活动时[K]o异常升高？第三，cn43或kir4.1的CKO是否导致海马片自发的癫痫样活动或对癫痫刺激的敏感性增加？第四，cn43或kir4.1的CKO是否影响Schaffer侧支-CA1锥体神经元突触的突触传递的有效性或可塑性？我们的前提是，虽然星形胶质细胞表现出使它们能够在体内调节神经元活动的特性，但开发模型系统以检验星形胶质细胞在大脑功能和功能障碍中的作用是必不可少的。这项建议的目标是开发这样一个模型系统，并测试星形胶质细胞通过调节[K]O的能力来调节神经元活动的假设。

Neuronal hyperexcitability and excitotoxicity are thought to underlie a number of clinical disorders. Unfortunately, very little is known about the mechanisms that lead to excessive neuronal activity. Strong indirect evidence indicates that neuronal excitability is influenced by small changes in [K+]o and that astrocytes are important in the regulation of [K+]o. Astrocytes are thought to maintain [K+]o within the narrow limits required for normal neuronal activity be removing K+ from areas of neuronal activity (a process referred to as spatial buffering). Spatial buffering is thought to be accomplished by the uptake of K+ through an inwardly rectifying K+ channel (Kir) and the dissipation of K+ into a syncytium of astrocytes connected by gap junctions. We propose to test the hypothesis that spatial buffering is dependent on the expression of astrocytic KirS and gap junctions and that elimination of these in astrocytes leads to increases in [K+]o, neuronal excitability and seizure activity. Cre/loxP technology and an inducible, astrocyte-specific gene expression system will be used to carry out conditional knockouts (cKO) of connexin43 (cn43) and Kir4.1; these proteins are necessary for gap junction communication (gjc) between and K+ uptake into astrocytes, respectively. The effects of eliminating astrocytic cn43 and KirS will be examined in situ using hippocampal brain slices and in vivo using models of hyperexcitability. The following questions will be addressed in these studies. First, in vivo, doe a cKO of cn43 or Kir4.1 lead to spontaneous seizures or an increased sensitivity to seizure- promoting stimuli? Second, does a cKO of cn43 or Kir4.1 lead to abnormal increases in [K+]o during neuronal activity in situ? Third, does a cKO of cn43 or Kir4.1 lead to spontaneous seizure- like activity or an increased sensitivity to seizure-promoting stimuli in hippocampal slices? And fourth, does a cKO of cn43 or Kir4.1 affect the efficacy or plasticity of synaptic transmission at the Schaffer collateral-CA1 pyramidal neuron synapse? It is our premise that while it is clear that astrocytes exhibit properties that would enable them to modulate neuronal activity in vivo, it is essential to develop model systems whereby the role of astrocytes in brain function and dysfunction can be examined. The goal of this proposal is to develop such a model system and to test the hypothesis that astrocytes regulate neuronal activity through their ability to modulate [K+]o.