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: 8306114
• 负责人: Prosper N'Gouemo
• 金额: $ 30.65万
• 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/8306114
• 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; Property; 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; 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，但其潜在机制知之甚少。下丘（IC）被认为在启动声诱发的AWS中起重要作用。在大鼠模型中，来自IC神经元的癫痫样爆发在启动声学诱发的AWS中是至关重要的，并且二氢吡啶（L-型Ca 2+通道阻断剂）抑制这些癫痫发作。L型钙通道（LTCCs）在酒精戒断后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）中的哪一种有助于AWS也是未知的。我们的总体目标是了解如何控制LTCC和相关的Ca 2+信号传导可以用来预防和治疗AWS。本申请的目的是通过确定LTCCs在响应酒精戒断的IC神经元过度兴奋的机制中的作用来研究大鼠和小鼠的AWS病因。我们的中心假设是，CaV1.3 LTCC活动所需的IC神经元产生癫痫样爆发，启动AWS。这一假设是基于我们的大鼠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）阻断LTCC抑制声诱发的AWS，ii）酒精戒断后IC神经元中LTCC的电流密度显著增加，与癫痫发作易感性增强相关，iii）酒精戒断后IC神经元中另一类LTCC CaV 1.2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以下三个具体目标进行验证：1）确定LTCC在酒精戒断后在IC神经元中产生癫痫样爆发和Ca 2+峰的程度; 2）确定CaV1.3 LTCC的磷酸化、细胞表面蛋白表达和mRNA表达在酒精戒断后IC神经元中与电流密度增加的程度;和3）确定是否可以在CaV1.3a1敲除小鼠和大鼠中产生声学诱发的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。我们提出的研究是重要的，因为这些实验将确定对AWS启动至关重要的LTCC依赖性机制，从根本上推进酒精滥用领域，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（Rev.06/09）