Ionic Homeostasis and Seizure Regulation

描述（由申请人提供）：这项研究的长期目标是了解神经元如何同步进入癫痫发作放电，以及大脑如何终止同步活动。在最后的资助期，实验探索了细胞外钾的调节机制，以及这种调节如何影响神经元的兴奋性，并开始研究pH的调节。初步证据表明，细胞外pH的变化有助于神经元活动的调节，并可能有助于大脑区域的相对癫痫敏感性。初步数据还表明，星形胶质细胞在细胞外钙的调节中发挥重要作用，从而影响神经元的兴奋性，并可能影响神经元的易感性。在此资助期间，我们将继续关注离子环境在神经元同步中的作用，重点是pH和钙。假设1 -神经元活动期间pH值的区域差异是由于该区域神经元中活性酸转运体的差异。这将通过确定在体内和体外齿状回中与CA1相比哪些pH调节过程不同来进行测试。将测量CA1细胞内pH值，并将其与癫痫发作持续时间和细胞外pH值进行比较，以确定细胞内pH值是否反映细胞外pH值，以及它是否有助于CA1的癫痫发作终止。解剖学和药理学研究将确定哪些离子转运体参与了齿状回和CA1的pH调节，并检查神经元活动期间和之后的变化。假设2 -神经元活动期间细胞外钙的恢复是由于星形胶质细胞钙的外排。这一假设将通过测定体内和体外刺激过程中CA1和齿状回细胞外钙的变化来验证。药物阻滞剂将决定不同电压门控钙通道的作用和钙从内部储存释放的作用。在持续的神经元活动期间，星形胶质细胞在细胞外钙恢复中的作用将使用胶质特异性毒素进行测试。这一假设将通过测量海马切片中神经胶质细胞和神经元在刺激训练过程中细胞内钙的变化，同时测量细胞外钙来直接验证。

DESCRIPTION (provided by applicant): The long-term goal of this research is to understand how neurons become synchronized into seizure discharges and how the brain terminates the synchronized activity. In the last grant period experiments explored the mechanisms underlying regulation of extracellular potassium and how this regulation might influence neuronal excitability and started to examine regulation of pH. Preliminary evidence suggests that the changes in extracellular pH contribute to regulation of neuronal activity and may contribute to the relative seizure sensitivity of brain regions. Preliminary data also suggest that astrocytes play a significant role in regulation of extracellular calcium, thus influencing neuronal excitability and possibly neuronal vulnerability. In this grant period, we will continue to focus on the role of the ionic environment in neuronal synchronization, with an emphasis on pH and calcium. Hypothesis 1 -The regional differences in pH during neuronal activity are due to differences in active acid transporters in the neurons in the region. This will be tested by determining which pH regulatory processes are different in the dentate gyrus compared to CA1, both in vivo and in vitro. Intracellular pH in CA1 will be measured and compared to seizure duration and extracellular pH to determine whether intracellular pH mirrors extracellular pH and whether it contributes to seizure termination in CA1. Anatomical and pharmacological studies will determine which ion transporters are involved in the regulation of pH in the dentate gyrus and CA1, with examination of changes during and after neuronal activity. Hypothesis 2 - Recovery of the extracellular calcium during neuronal activity is due to efflux of calcium from astrocytes. This hypothesis will be tested by determining extracellular calcium changes in CA1 and the dentate gyrus in vivo and in vitro during trains of stimulation. Pharmacological blockers will determine the role of the different voltage-gated calcium channels and the role of calcium released from internal stores. The role of astrocytes in the recovery of extracellular calcium during continued neuronal activity will be tested using glial specific toxins. This hypothesis will be directly tested by measuring changes in intracellular calcium in glial cells and neurons during stimulus trains in hippocampal slices while measuring extracellular calcium.