Effect of ethanol on Bergmann glia Ca2+ dynamics during motor behavior

描述（由申请人提供）：在被滥用的物质中，酒精滥用的发生率最高，对西方社会的社会经济影响最大。乙醇是对小脑毒性最大的物质之一，可严重导致共济失调运动行为。小脑皮层中的电路使自适应运动反应利用伯格曼胶质（BG）和神经元元素之间的密切相互作用。BGs可以通过广泛的、协调的Ca2+瞬态（“耀斑”）响应运动，可能影响神经元的兴奋性。值得注意的是，并不是每个运动事件都会导致耀斑，为了了解它们的功能，有必要揭示将耀斑与行为环境联系起来并调节其强度的细胞机制。乙醇使GABAA受体增敏并减少兴奋性离子通道的激活。慢性乙醇暴露导致这些神经化学通路的反应性适应，从而使小脑和大脑相当正常地运作。从乙醇中退出后，这些适应会导致严重的震颤和其他症状。尽管许多膜受体在神经胶质细胞和神经元中共享，但目前尚不清楚乙醇是否对BGs有影响。为了阐明这个重要的问题，拟议的研究将使用转基因小鼠，这些小鼠在所有bg中选择性地表达遗传编码的Ca2+指示剂GCaMP3，当动物在线性跑步机上行走或休息时，将用双光子成像分析Ca2+反应。这种方法提供了前所未有的机会，在几个月的时间过程中以细胞分辨率研究BG功能。我们的初步研究表明，当运动与警觉性增强的状态同时发生时，就会产生耀斑，这是由动物侧面的一股气流引起的。初步的体内药理学研究支持这一假设，表明耀斑需要α - 1肾上腺素受体的激活。此外，我们发现与运动相关剂量的急性乙醇暴露可可逆地抑制耀斑。本提案的总体目标是验证急性乙醇暴露对耀斑的抑制与共济失调运动行为相对应的假设，以及在慢性乙醇暴露的戒断期间，耀斑的增强与震颤相对应的假设。1)研究不同剂量急性乙醇注射对耀斑和运动协调（旋转杆、平衡木）的影响。在急性小脑切片中，乙醇对BG AMPA受体电流和去甲肾上腺素诱导的Ca2+升高的影响将被研究。2)，在慢性乙醇暴露期间和停药时监测耀斑和震颤（EMG）。如果在停药期间耀斑增强，将确定药理学上将耀斑重置为正常强度是否会减少震颤。在完成所提议的研究后，BG耀斑在正常小脑运动控制和乙醇影响下的作用将被深入定义。这些研究有可能引领人们走向新的、更具体的治疗乙醇戒断的方法。

DESCRIPTION (provided by applicant): Alcohol abuse has the highest prevalence and the greatest socioeconomic impact on western societies among the abused substances. Ethanol is one of the most toxic substances for the cerebellum, acutely leading to ataxic motor behavior. The circuitry in the cerebellar cortex that enables adaptive motor responses utilizes a close interaction between Bergmann glia (BG) and neuronal elements. BGs can respond to locomotion with widespread, coordinated Ca2+ transients ("flares") possibly influencing the excitability of neurons. Remarkably, not every locomotion event leads to flares, and in order to understand their function it is necessary to uncover the cellular mechanisms that link flares to behavioral context and modulate their intensity. Ethanol sensitizes GABAA receptors and reduces activation of excitatory ion channels. Chronic ethanol exposure leads to adaptation of the responsiveness of these neurochemical pathways which allows the cerebellum and brain to operate fairly normal. Withdrawal from ethanol unmasks these adaptations leading to severe tremor and other symptoms. It is not known whether ethanol has an effect on BGs despite many membrane receptors being shared among glia and neurons. To shed light on this important question, the proposed studies will use transgenic mice which express the genetically encoded Ca2+ indicator GCaMP3 selectively in all BGs, and Ca2+ responses will be analyzed with two-photon imaging while the animal is walking or resting on a linear treadmill. This approach offers the unprecedented opportunity to investigate BG function with cellular resolution over the time course of months. Our preliminary studies revealed that flares are initiated when locomotion coincides with a state of increased alertness, induced by an air puff to the flank of the animal. Preliminary in vivo pharmacological investigations support this hypothesis, suggesting that flares require activation of alpha1-adrenoceptors. Furthermore, we found that acute ethanol exposure at locomotion-relevant dosages suppresses flares reversibly. The overall goal of this proposal is to test the hypothesis that the inhibition of flares by acute ethanol exposure corresponds with ataxic motor behavior, and that during withdrawal from chronic ethanol exposure, enhancement of flares corresponds with tremor. 1), the effect of different dosages of acute ethanol injections on flares and motor coordination (rotarod, balance beam) will be investigated. In acute cerebellar slices the effect of ethanol on BG AMPA receptor currents and norepinephrine induced Ca2+ elevations will be investigated. 2), flares and tremor (EMG) will be monitored during chronic ethanol exposure and upon withdrawal. If flares are enhanced during withdrawal, it will be determined whether pharmacologically resetting flares to normal intensity will reduce tremor. Upon completion of the proposed studies the role of BG flares during normal cerebellar motor control and under the influence of ethanol will be defined in depth. The studies have the potential to lead the way towards novel, more specific treatments of ethanol withdrawal.