Mechanisms of Alcohol Withdrawal Seizures: Role of L-type Ca2+ Channels

酒精戒断发作的机制：L 型 Ca2 通道的作用

• 批准号: 8702046
• 负责人: Prosper N'Gouemo
• 金额: $ 38.88万
• 依托单位: GEORGETOWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8702046
• 关键词: Accounting; Alcohol Withdrawal Seizures; Alcohol abuse; Alcohol withdrawal syndrome; Biological Assay; Brain; Brain Stem; Cell Surface Proteins; Cell surface; Cyclic AMP-Dependent Protein Kinases; Data; Dihydropyridines; Electrophysiology (science); Emergency Situation; Epilepsy; Etiology; Exhibits; Gene Expression; Goals; Human; Inferior Colliculus; Knockout Mice; Literature; Medical; Medicine; Messenger RNA; Microinjections; Modeling; Molecular; Molecular Genetics; Molecular Target; Mus; Neurons; Outcome; Pattern; Pharmacology; Phosphorylation; Play; Predisposition; Preparation; Prevention; Principal Investigator; Protein Subunits; Proteins; Public Health; Publishing; RNA; RNA Interference; Rattus; Reporting; Research; Research Proposals; Resistance; Risk; Role; Seizures; Signal Transduction; Slice; Testing; Tonic - clonic seizures; Western Blotting; Withdrawal; Work; alcohol response; base; biophysical properties; channel blockers; crosslink; density; dihydropyridine; effective therapy; improved; in vivo; innovation; knock-down; mRNA Expression; novel therapeutic intervention; novel therapeutics; prevent; programs; protein expression; research study

DESCRIPTION (provided by applicant): Alcohol withdrawal seizures (AWS) are one of the most common medical emergencies, but their underlying mechanisms are poorly understood. The inferior colliculus (IC) is thought to play an important role in initiating acoustically-evoked AWS. In a rat model, epileptiform bursts from IC neurons are critical in initiating acoustically-evoked AWS, and dihydropyridines (L-type Ca2+ channels blockers) suppressed these seizures. The extent to which L-type Ca2+ channels (LTCCs) contribute to generating epileptiform bursts in IC neurons following alcohol withdrawal is unknown. Which of the two LTCC classes (CaV1.2 and CaV1.3) present in IC neurons contribute to AWS is also unknown. Our overall goal is to understand how controlling LTCCs and related Ca2+ signaling can be use to prevent and treat AWS. The objective of this application is to investigate AWS etiology in rats and mice by determining the role of LTCCs in the mechanisms underlying IC neuronal hyperexcitability in response to alcohol withdrawal. Our central hypothesis is that CaV1.3 LTCC activity is required for IC neurons to generate epileptiform bursts that initiate AWS. This hypothesis is based on substantial preliminary data from our rat AWS model, with further support from published reports that: i) blockade of LTCCs suppresses acoustically-evoked AWS, ii) the current density of LTCCs increases markedly in IC neurons following alcohol withdrawal associated with enhanced seizure susceptibility, and iii) LTCCs of the other class, CaV1.2, are not upregulated in IC neurons following alcohol withdrawal. The rationale for the proposed research is that understanding the role of LTCCs in AWS initiation has the potential to improve our ability to prevent and control these seizures. Our central hypothesis will be tested by pursuing three specifics aims: 1) Determine to what extent LTCCs contribute to generating epileptiform bursts and Ca2+ spikes in IC neurons following alcohol withdrawal; 2) Determine to what extent phosphorylation, cell surface protein expression, and mRNA expression of CaV1.3 LTCCs are associated with the enhanced current density in IC neurons following alcohol withdrawal; and 3) Determine if acoustically-evoked AWS can be generated in CaV1.3a1 knockout mice and rats in which CaV1.3a1 subunits are knocked down by short-interfering RNA microinjection into IC neurons. Our approach is innovative because it uses molecular genetics combined with electrophysiology and pharmacology to determine the role of CaV1.3 LTCCs in IC neuronal hyperexcitability and resulting AWS. Our proposed research is significant because the experiments will identify an LTCC- dependent mechanism essential to AWS initiation, fundamentally advancing the field of alcohol abuse and identifying a new molecular target for novel therapeutic approaches to AWS prevention and treatment. PHS 398/2590 (Rev. 06/09) Page Continuation Format Page

描述（由申请人提供）：酒精戒断性癫痫发作 (AWS) 是最常见的医疗紧急情况之一，但对其潜在机制知之甚少。下丘 (IC) 被认为在声诱发 AWS 的启动中发挥着重要作用。在大鼠模型中，IC 神经元的癫痫样爆发对于启动声诱发的 AWS 至关重要，而二氢吡啶（L 型 Ca2+ 通道阻滞剂）可抑制这些癫痫发作。 L 型 Ca2+ 通道 (LTCC) 在酒精戒断后在 IC 神经元中产生癫痫样爆发的程度尚不清楚。 IC 神经元中存在的两种 LTCC 类别（CaV1.2 和 CaV1.3）中哪一种对 AWS 起作用尚不清楚。我们的总体目标是了解如何控制 LTCC 和相关的 Ca2+ 信号传导来预防和治疗 AWS。本申请的目的是通过确定 LTCC 在酒精戒断反应中 IC 神经元过度兴奋机制中的作用来研究大鼠和小鼠的 AWS 病因学。我们的中心假设是 IC 神经元需要 CaV1.3 LTCC 活性来产生启动 AWS 的癫痫样爆发。这一假设基于我们的大鼠 AWS 模型的大量初步数据，并得到已发表报告的进一步支持：i) LTCC 的封锁抑制了声诱发的 AWS，ii) 与酒精戒断相关的 IC 神经元中 LTCC 的电流密度显着增加癫痫易感性增强，以及 iii) 另一类 LTCC，CaV1.2，在酒精戒断后 IC 神经元中的表达并未上调。拟议研究的基本原理是，了解 LTCC 在 AWS 启动中的作用有可能提高我们预防和控制这些癫痫发作的能力。我们的中心假设将通过追求三个具体目标进行检验：1）确定 LTCC 在多大程度上有助于在酒精戒断后 IC 神经元中产生癫痫样爆发和 Ca2+ 尖峰； 2)确定CaV1.3 LTCC的磷酸化、细胞表面蛋白表达和mRNA表达与酒精戒断后IC神经元电流密度增强的相关程度； 3) 确定是否可以在 CaV1.3a1 敲除小鼠和大鼠中产生声诱发 AWS，其中通过将短干扰 RNA 显微注射到 IC 神经元中来敲除 CaV1.3a1 亚基。我们的方法是创新的，因为它使用分子遗传学结合电生理学和药理学来确定 CaV1.3 LTCC 在 IC 神经元过度兴奋和由此产生的 AWS 中的作用。我们提出的研究意义重大，因为这些实验将确定 AWS 启动所必需的 LTCC 依赖性机制，从根本上推进酒精滥用领域的发展，并为 AWS 预防和治疗的新治疗方法确定新的分子靶点。 PHS 398/2590（修订版 06/09） 页面延续 格式页面