Dynamic Clamp Study of Calcium Channels in Nigral Dopamine Neurons

黑质多巴胺神经元钙通道的动态钳制研究

• 批准号: 8515780
• 负责人: Kristal Raylone Tucker
• 金额: $ 5.22万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8515780
• 关键词: Action Potentials; Address; Behavior; Calcium Channel; Cells; Communities; Disease; Dopamine; Drug Targeting; Drug usage; Electrophysiology (science); Epilepsy; Equation; Exhibits; Gilles de la Tourette syndrome; Goals; Hodgkin Disease; Horns; Ion Channel; Ions; Kinetics; Life; Measures; Mediating; Membrane Potentials; Mental disorders; Migraine; Modeling; Motivation; Movement; Neurodegenerative Disorders; Neurons; Pacemakers; Parkinson Disease; Pattern; Pharmaceutical Preparations; Pharmacology; Protocols documentation; Relative (related person); Research; Role; Schizophrenia; Second Messenger Systems; Simulate; Substantia nigra structure; Synapses; Techniques; Time; Training; Voltage-Clamp Technics; Work; base; channel blockers; dopaminergic neuron; inhibitor/antagonist; motivated behavior; novel; painful neuropathy; pars compacta; reconstitution; research study; response; second messenger; simulation; virtual; voltage; voltage clamp; voltage gated channel

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.

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器活动，该活动被突触输入修改，导致有规则的不规则和对运动和动机行为重要的突发放电。电压门控Ca2+ (CaV)通道直接通过传导去极化电流和间接通过激活Ca2+激活通道参与SN DA神经元的起搏。研究发现，L型、P/Q型和t型CaV通道对起搏有显著贡献，但对L型CaV电流的研究较为深入。本研究的目的是评估P/Q-和t型CaV通道在SNc DA神经元缓慢起搏器活动中的作用，并确定它们对起搏的贡献是由去极化电流介导的，还是由携带电流的Ca2+的二次效应介导的。这一目标将通过结合药理学和急性分离的SN DA神经元的电压、动作电位、电流和动态钳电生理学来实现。动作电位箝位是电压箝位技术的一种改进形式，它使用一个动作电位或一组动作电位作为电压命令，允许人们确定活动期间某个特定电流的流动时间和强度。动态箝位技术是电流箝位技术的改进形式，它根据模拟通道行为的方程动态调整注入的电流，从而响应膜电位的实时变化，从而允许人们在活细胞中添加虚拟通道。因此，这些虚拟通道产生的电流模仿原生通道，但没有离子选择性。为了实现此应用程序的目标，将使用这些技术来解决以下具体目标：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SNc DA神经元AP和起搏时P/Q和t型CaV电流的数量和时间。目标3。确定去极化电流和Ca2+进入对P/Q和t型CaV电流下SNc DA神经元起搏器活动的次要影响的相对重要性。提出的实验结果将使科学界能够：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. 开发治疗运动和动机障碍的新药。