Dynamic Clamp Study of Calcium Channels in Nigral Dopamine Neurons

描述(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，这种活动被突触输入改变，导致规则的不规则和突发性放电，对运动和动机行为至关重要。电压门控钙通道(Cav)直接通过传导去极化电流参与SN DA神经元的起搏，间接通过激活钙激活通道参与起搏。已发现L通道、P/Q通道和T型通道对起搏有显著贡献，但只有L型通道被深入研究。本研究的目的是评估P/Q和T型Cav通道在SNc DA神经元慢起搏活动中的作用，并确定它们对起搏的贡献是由去极化电流还是携带电流的Ca~(2+)的次要作用介导的。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、电流和动态钳制电生理学的组合来实现。动作电位-钳位是电压钳制技术的一种改进形式，其中动作电位或动作电位组被用作电压指令，从而使人能够确定在活动期间特定电流的流动时间和数量。动态钳位是电流钳制技术的一种改进形式，它根据模拟通道行为的方程动态调整注入的电流，以响应膜电位的实时变化，从而允许将虚拟通道添加到活细胞中。因此，这些虚拟通道产生的电流与自然通道相似，但没有离子选择性。为了实现这一应用的目标，这些技术将被用来解决以下具体目标：目的1.表征SNCDA神经元中P/Q和T型Cav电流的电压依赖动力学。目的2.对SNc DA神经元在AP和起搏过程中流出的P/Q和T型CAV电流的大小和时间进行量化。目的3.确定去极化电流和钙离子内流对P/Q和T型Cav电流的SNc DA神经元起搏活动的相对重要性。拟议的实验结果将使科学界能够：1.了解这些通道对正常的黑质多巴胺神经元活动的作用机制。2.了解用于阻断这些通道的药物，如用于治疗偏头痛、神经性疼痛和癫痫的药物，可能会如何扰乱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.