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: 8504892
• 负责人: Prosper N'Gouemo
• 金额: $ 30.1万
• 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/8504892
• 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）：戒酒癫痫发作（AWS）是最常见的医疗紧急情况之一，但其潜在机制知之甚少。次要的（IC）被认为在发起声学诱发的AWS中起着重要作用。在大鼠模型中，IC神经元的癫痫样爆发对于启动声学诱发的AWS至关重要，而二氢吡啶（L型Ca2+通道阻滞剂）抑制了这些癫痫发作。 L型Ca2+通道（LTCC）在戒酒后在IC神经元中产生癫痫样爆发的程度尚不清楚。 IC神经元中存在的两个LTCC类（CAV1.2和CAV1.3）中的哪个也未知。我们的总体目标是了解如何使用控制LTCC和相关的CA2+信号来预防和治疗AWS。该应用的目的是通过确定LTCCS在响应于戒酒的IC神经元过度兴奋性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活性是IC神经元产生启动AWS的癫痫样爆发所必需的。该假设基于我们大鼠AWS模型的大量初步数据，并在发表的报告中进一步支持：i）LTCC的封锁抑制了声学诱发的AWS，ii）IC在与IC神经元相关的IC神经元中LTCC的当前密度显着增加。提高癫痫发作的癫痫发作易感性，iii）cav1.2的LTCC在戒酒后没有上调IC神经元。拟议研究的理由是，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的来检验：1）确定LTCC在何种程度上有助于在戒酒后在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表达在何种程度上与戒酒后IC神经元的电流密度增强有关； 3）确定在CAV1.3A1敲除小鼠和大鼠中是否可以产生声学诱发的AWS，其中CAV1.3A1亚基通过将RNA微分注射短在IC神经元中敲低。我们的方法具有创新性，因为它使用分子遗传学与电生理学和药理学相结合来确定CAV1.3 LTCC在IC神经元过度兴奋性和由此产生的AWS中的作用。我们提出的研究很重要，因为这些实验将确定对AWS启动必不可少的LTCC依赖性机制，从根本上促进酒精滥用领域，并确定用于预防AWS预防和治疗的新型治疗方法的新分子靶标。 PHS 398/2590（修订版06/09）页面延续格式页面