The effects of alcohol on cerebellar synaotic transmission and plasticity

描述（由申请人提供）：大脑运动系统的可塑性允许运动协调的精细调整，这种调整可以在很长一段时间内发展，但也可以在几分钟内发生。快速运动学习依赖于小脑浦肯野细胞兴奋性突触输入的突触可塑性。突触增益的变化，如长时程增强（LTP）和长时程抑制（LTD）在平行纤维（PF）和爬行纤维（CF）突触到浦肯野细胞提供了小脑运动学习的细胞基础。已知酒精会干扰突触传递和可塑性，但迄今为止尚未研究过酒精对小脑突触可塑性的急性影响，尽管众所周知，酒精消费的急性后果包括运动协调和微调运动的能力受损。在这里，我们建议研究急性乙醇应用对小脑突触可塑性的影响，在大鼠脑切片使用全细胞膜片钳记录以及显微荧光成像技术。除了我们为准备这项应用而进行的初步实验外，我们以前没有与酒精相关的研究经验。然而，我们已经表征了小脑突触可塑性的细胞机制（例如Hansel等人，2001年; Jvrntell和Hansel，2006年）。国家酒精滥用和酒精中毒研究所（NIAAA）的财政支持申请是出于我们的希望，我们现在手头有必要的经验和工具来研究乙醇如何干扰小脑学习机制。我们进入酒精研究领域的第一步将通过与C.F. Valenzuela（美国阿尔伯克基的新墨西哥州大学），他在这一领域有着长期的经验。在此，我们提出三个具体目标：a）检测乙醇对小脑突触传递和突触可塑性的急性作用，B）检测乙醇对浦肯野细胞中NMDA受体、代谢型谷氨酸受体和电压依赖性钙通道的作用，和c）使用钙成像技术，结合体细胞-树突膜片钳技术，监测乙醇对浦肯野细胞树突中树突钙峰的时空图的影响。这些树突状钙峰对小脑可塑性至关重要，可能是由电压门控钙电流和NMDA受体触发的。两者都被描述为其他类型神经元中的乙醇靶点，因此树突状钙峰可能也会受到乙醇的影响。我们的建议具有创新性和探索性，因为我们计划使用成像（超高速钙成像）和膜片钳技术（一个体细胞和两个树突位置的三重修补）的新组合，以能够表征树突钙峰活性，这在酒精研究中尚未完成。我们认为，这种类型的研究对于更好地了解酒精消费急性效应的细胞基础非常重要。其中之一是运动协调能力受损，这导致与酒精有关的交通事故造成的死亡率很高。 公共卫生相关性：小脑是参与运动协调和运动学习微调的大脑结构。这些过程可以受损的后果，急性饮酒，但急性酒精对小脑突触传递和可塑性小脑网络内的影响尚未研究到目前为止。在这里，我们建议研究酒精对小脑突触可塑性的形式的影响，以及这种影响的细胞原因，使用电生理记录技术和荧光钙成像技术在大鼠脑切片。

DESCRIPTION (provided by applicant): Plasticity in brain motor systems allows fine adjustments of motor coordination that can develop over long periods of time, but can also occur within minutes. Rapid motor learning rests on synaptic plasticity at excitatory synaptic inputs to cerebellar Purkinje cells. Synaptic gain changes such as long-term potentiation (LTP) and long-term depression (LTD) at parallel fiber (PF) and climbing fiber (CF) synapses onto Purkinje cells provide a cellular basis for cerebellar motor learning. Alcohol is known to interfere with synaptic transmission and plasticity, but acute alcohol effects on cerebellar synaptic plasticity have not been studied so far, although it is well known that acute consequences of alcohol consumption include the impairment of motor coordination and the ability to fine-tune movements. Here, we propose to examine the effects of acute ethanol application on cerebellar synaptic plasticity in rat brain slices using whole-cell patch-clamp recordings as well as microfluorometric imaging techniques. Aside from our preliminary experiments performed in preparation for this application, we have no previous experience in alcohol-related research. However, we have characterized cellular mechanisms underlying cerebellar synaptic plasticity (e.g. Hansel et al., 2001; Jvrntell and Hansel, 2006). This application for financial support from the National Institute on Alcohol Abuse and Alcoholism (NIAAA) is motivated by our hope that we now have the required experience and tools at hand to study how ethanol interferes with cerebellar learning mechanisms. Our first steps into the field of alcohol research will be facilitated by a collaboration with the laboratory of Prof. C.F. Valenzuela (University of New Mexico at Albuquerque, USA), who has long-standing experience in this field. Here, we suggest three specific aims: a) to examine acute ethanol effects on cerebellar synaptic transmission and synaptic plasticity, b) to examine ethanol effects on NMDA receptors, metabotropic glutamate receptors and voltage-dependent calcium channels in Purkinje cells and c) to use calcium imaging techniques, in combination with somato-dendritic patch-clamp techniques, to monitor ethanol effects on the spatio-temporal map of dendritic calcium spikes in Purkinje cell dendrites. These dendritic calcium spikes, which are crucial for cerebellar plasticity, are likely triggered by voltage-gated calcium currents and NMDA receptors. Both have been described as ethanol targets in other types of neurons, and therefore dendritic calcium spikes might be affected by ethanol as well. Our proposal has an innovative and exploratory character, as we plan to use a novel combination of imaging (ultra high-speed calcium imaging) and patch-clamp techniques (triple patching with one somatic and two dendritic locations) to be able to characterize dendritic calcium spike activity, which has not been done so far in alcohol research. We believe that this type of research is important to better understand the cellular basis of acute effects of alcohol consumption. One of these is impaired motor coordination, which contributes to the high rate of deaths caused by alcohol-related traffic accidents. PUBLIC HEALTH RELEVANCE: The cerebellum is a brain structure involved in the fine-adjustment of motor coordination and in motor learning. These processes can be impaired as a consequence of acute alcohol consumption, and yet acute alcohol effects on cerebellar synaptic transmission and on plasticity within cerebellar networks have not been studied so far. Here, we propose to examine alcohol effects on forms of cerebellar synaptic plasticity as well as the cellular causes for such effects using electrophysiological recording techniques and fluorometric calcium imaging techniques in rat brain slices.