Ion channels in suprachiasmatic nucleus neurons

描述（由申请人提供）：下丘脑腹侧的视交叉上核（SCN）包含大脑的主时钟，控制生理功能的24小时昼夜节律。最近的工作已经确定在SCN神经元的分子钟，由转录因子和激酶的相互作用，在调节反馈途径的许多组件。一个尚未回答的主要问题是分子钟如何与神经元的电活动联系起来。 这项研究的目的是了解控制SCN神经元自发放电的离子电导及其放电率的昼夜变化。我们将在脑切片中完整的SCN神经元和维持起搏的急性分离神经元的制备上进行补充实验。脑切片制备允许在最不受干扰的条件下进行记录，而解离的神经元使电压钳记录具有高的时间和电压分辨率，并允许快速的溶液交换和容易可逆的药物和通道阻断剂的应用。电流的各种组件的基础上的通道的电生理学和药理学鉴定将通过在神经元中表达的通道的免疫细胞化学鉴定来补充。 除了表征直接驱动SCN神经元起搏的离子电流外，我们还将表征对调节其频率很重要的其他电流。然后，我们将确定这些电流的幅度，电压依赖性或动力学的变化，以产生放电频率的昼夜变化。最后，我们将试图发现分子钟是如何与电起搏联系在一起的，并确定电时钟是否受激酶或其他第二信使途径活性改变的调节。了解参与调节SCN神经元兴奋性的机制将有助于了解神经系统的正常功能以及睡眠，注意力和激素释放的失调。

DESCRIPTION (provided by applicant): The suprachiasmatic nucleus (SCN) in the ventral hypothalamus contains the master clock of the brain, controlling the 24-hour circadian rhythm of physiological functions. Recent work has identified in SCN neurons many components of a molecular clock, composed of transcription factors and kinases that interact in regulatory feedback pathways. A major unanswered question is how the molecular clock is connected to the electrical activity of the neurons. The goal of the proposed research is to understand the ionic conductances that control spontaneous firing of SCN neurons and the circadian variation in their firing rate. We will do complementary experiments on intact SCN neurons in brain slice and on a preparation of acutely-dissociated neurons in which pacemaking is maintained. The brain slice preparation allows recordings under the most undisturbed conditions, while dissociated neurons enable voltage-clamp recordings with high time- and voltage-resolution and permit fast solution exchanges and readily reversible application of drugs and channel blockers. Electrophysiological and pharmacological identification of channels underlying various components of electrical current will be complemented by immunocytochemical identification of the channels expressed in the neurons. In addition to characterizing the ionic currents that directly drive pacemaking of SCN neurons, we will characterize other currents that are important for regulating its frequency. We will then determine which of these currents change in amplitude, voltage-dependence, or kinetics to produce the diurnal variation in firing frequency. Finally, we will attempt to discover how the molecular clock is linked to electrical pacemaking and determine whether the electrical clock is regulated by alteration in the activity of kinases or other second messenger pathways. Understanding the mechanisms involved in regulating the excitability of SCN neurons will help in understanding the normal function of the nervous system as well as dysregulation of sleep, attention, and hormonal release.