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

描述（由申请人提供）：酒精戒断发作（AWS）是最常见的医疗紧急情况之一，但其潜在机制尚不清楚。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）抑制了这些发作。l型Ca2+通道（ltcc）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 （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Ca2+信号可以用来预防和治疗AWS。本应用程序的目的是通过确定ltcc在酒精戒断反应中IC神经元高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的活性是IC神经元产生癫痫样脉冲启动AWS所必需的。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：1)ltcc的阻断抑制声诱发的AWS， 2)酒精戒断后IC神经元中ltcc的电流密度显著增加，与癫痫易感性增强相关，3)酒精戒断后IC神经元中其他类型的ltcc CaV1.2没有上调。这项研究的基本原理是，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来检验：1)确定酒精戒断后ltcc在多大程度上促进IC神经元产生癫痫样爆发和Ca2+峰值；2)确定CaV1.3 ltcc的磷酸化、细胞表面蛋白表达和mRNA表达在多大程度上与酒精戒断后IC神经元电流密度增强相关；3)在敲除CaV1.3a1亚基的小鼠和敲除CaV1.3a1亚基的大鼠中，通过短干扰RNA显微注射到IC神经元中，确定是否可以产生声诱发的AWS。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神经元高兴奋性和导致的AWS中的作用。我们提出的研究具有重要意义，因为实验将确定对AWS启动至关重要的LTCC依赖机制，从根本上推进酒精滥用领域，并为预防和治疗AWS的新治疗方法确定新的分子靶点。小灵通398/2590 （Rev. 06/09）页延续格式页

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