Dynamic Clamp Study of Calcium Channels in Nigral Dopamine Neurons

描述（由申请人提供）：黑质（SN）中的多巴胺（DA）神经元表现出缓慢的内在起搏活动，该活动被突触输入改变，导致对运动和动机行为很重要的规则不规则和突发放电。电压门控性Ca 2+（CaV）通道直接通过传导去极化电流和间接通过激活Ca 2+激活的通道参与SN DA神经元的起搏。已经发现L-、P/Q-和T-型CaV通道对起搏有显著贡献，但仅L-型CaV电流被深入研究。拟议的研究的目标是评估的P/Q-和T-型CaV通道在SNc DA神经元的缓慢起搏器活动的作用，并确定其对起搏的贡献是否介导的去极化电流或二次效应的Ca 2+携带电流。这一目标将通过在急性分离的SN DA神经元中使用药理学和电压、动作电位、电流和动态钳电生理学的组合来实现。动作电位钳位是电压钳位技术的改进形式，其中动作电位或动作电位组用作电压命令，允许人们确定活动期间何时以及有多少特定电流流动。动态钳是电流钳技术的改进形式，其基于对通道行为进行建模的方程来动态地调节响应于膜电位的实时变化而注入的电流，从而允许将虚拟通道添加到活细胞。因此，这些虚拟通道产生模拟天然通道但没有离子选择性的电流。为了实现本申请的目标，这些技术将用于解决以下具体目标：目标1。表征SNc DA神经元中P/Q和T型CaV电流的电压依赖性动力学。目标2.量化在SNc DA神经元中AP和起搏期间流动的P/Q和T型CaV电流的量和时间。目标3：确定去极化电流的相对重要性和Ca 2+内流对P/Q-和T-型CaV电流的SNc DA神经元起搏活性的次级效应。拟议实验的结果将使科学界能够：1。了解这些通道促进正常SNc DA神经元活动的机制。2.了解用于阻断这些通道的药物，例如用于治疗偏头痛，神经性疼痛和癫痫的药物，可能会破坏这些神经元的正常活动。3.开发治疗运动和动力障碍的新药。

DESCRIPTION (provided by applicant): Dopamine (DA) neurons in the substantia nigra (SN) exhibit a slow intrinsic pacemaker activity that is modified by synaptic input resulting in regular irregular, and burst firing important for movement and motivated behaviors. Voltage-gated Ca2+ (CaV) channels contribute to pacing in SN DA neurons directly by conducting depolarizing currents and indirectly by activation of Ca2+-activated channels. The L-, P/Q- and T-type CaV channels have been found to significantly contribute to pacing, but only the L-type CaV current has been studied in depth. The goal of the proposed research is to evaluate the role of the P/Q- and T-type CaV channels in the slow pacemaker activity of SNc DA neurons and determine whether their contribution to pacing is mediated by the depolarizing current or the secondary effects of the Ca2+ carrying the current. This goal will be achieved by using a combination of pharmacology and voltage-, action potential-, current-, and dynamic- clamp electrophysiology in acutely dissociated SN DA neurons. Action potential-clamp is a modified form of the voltage-clamp technique where an action potential or group of action potentials are used as the voltage command allowing one to determine when and how much of a particular current is flowing during activity. Dynamic-clamp is a modified form of the current-clamp technique that dynamically adjusts the current injected in response to real-time changes in the membrane potential based on equations that model channel behavior allowing one to add virtual channels to a living cell. As a result, these virtual channels produce currents that mimic the native channe but without the ion selectivity. To achieve the goal of this application, these techniques will be used to address the following specific aims: Aim 1. Characterize the voltage-dependent kinetics of P/Q- and T-type CaV currents in SNc DA neurons. Aim 2. Quantify the amount and timing of P/Q- and T-type CaV currents flowing during the AP and pacing in SNc DA neurons. Aim 3. Determine the relative importance of the depolarizing current and secondary effects of Ca2+ entry for the pacemaker activity of SNc DA neurons for the P/Q- and T-type CaV currents. The results of the proposed experiments will allow the scientific community to: 1. Understand the mechanism by which these channels contribute to normal SNc DA neuronal activity. 2. Understand how drugs used to block these channels, such as those used to treat migraines, neuropathic pain and epilepsy, might disrupt the normal activity of these neurons. 3. Develop novel drugs to treat movement and motivation disorders.