Alcohol and cerebellar circuits

描述(申请人提供)：急性和长期接触乙醇(Etoh)会导致小脑功能障碍，导致步态、平衡和协调能力的改变，这是导致美国大量伤亡的原因。最近的证据表明，乙醇影响执行功能，这可能是额小脑回路中断的结果。然而，我们对乙醇在小脑中的作用机制的了解还处于初级阶段。来自脑干和脊髓的兴奋性输入通过苔藓纤维进入颗粒细胞的小脑皮质。这些神经元也接受高尔基细胞的抑制性输入，高尔基细胞是主要的颗粒细胞层神经元间亚型。高尔基体细胞依次接受苔藓纤维的前馈兴奋性输入、颗粒细胞轴突的反馈兴奋性输入和分子层中间神经元的抑制性输入。我们的主要假设是，急性乙醇暴露通过减少颗粒和高尔基细胞上的谷氨酸能和增加GABA能传递而损害颗粒层回路的正常功能。具体目的#1是研究乙醇对颗粒细胞谷氨酸能传递的影响。在之前的资助期间，我们确定乙醇增加了颗粒细胞的紧张性和相性GABA能输入，而不影响AMPA受体介导的自发谷氨酸能传递。利用急性小脑切片制备和膜片钳电生理技术，我们现在将研究乙醇对苔藓状纤维到颗粒细胞突触的NMDA受体功能和长时程增强的影响。我们将通过苔藓纤维激活的感觉样模式来研究它对颗粒细胞激活的影响；对于这些研究，单个神经元的记录将由[苔藓纤维激活区域]的自发荧光光学成像来补充。具体目标#2是进一步表征乙醇对高尔基体细胞的影响。在之前的资金支持期间，我们证明了乙醇促进高尔基体细胞的激活，并建议对这种效应的机制进行表征。[基于计算机模拟和实验研究的结合，我们建议将IA、Na/K泵和持续的Na电流表征为Etoh效应的潜在介体]。我们还将研究乙醇对高尔基细胞GABA能和/或谷氨酸能输入的影响。这些急性切片的研究将与乙醇对这些神经元功能的急性影响的体内电生理实验相补充。在具体目标#3中，我们将使用数据驱动的计算神经科学方法来研究乙醇对颗粒细胞层整体网络活动的影响。这些多学科的研究将显著增加我们对乙醇对小脑颗粒细胞层的急性影响的了解，为确定治疗乙醇诱导的小脑功能障碍的新靶点奠定基础。

DESCRIPTION (provided by applicant): Acute and long-term ethanol (EtOH) exposure produces cerebellar dysfunction, leading to alterations in gait, balance and coordination that are responsible for a large number of injuries and deaths in the United States. Recent evidence indicates that EtOH affects executive functions and this could be a consequence of disruptions in frontocerebellar circuitry. However, our understanding of the mechanism of action of EtOH in the cerebellum is still in its infancy. Excitatory input from the brain stem and spinal cord enters the cerebellar cortex at the granule cells via the mossy fibers. These neurons also receive inhibitory input from the Golgi cells, which are the major granule cell layer interneuronal subtype. Golgi cells, in turn, receive feedforward excitatory input from mossy fibers, feedback excitatory input from granule cell axons and inhibitory input from molecular layer interneurons. Our overarching hypothesis is that acute EtOH exposure impairs the normal functioning of granule layer circuitry by decreasing glutamatergic and increasing GABAergic transmission at both granule and Golgi cells. Specific Aim #1 is to characterize the effect of EtOH on glutamatergic transmission at granule cells. During the previous funding period, we determined that EtOH increases tonic and phasic GABAergic input to granule cells without affecting spontaneous glutamatergic transmission mediated by AMPA receptors. Using the acute cerebellar slice preparation and patch-clamp electrophysiological techniques, we will now study the effect of EtOH on NMDA receptor function and long- term potentiation at mossy fiber-to-granule cell synapses. We will investigate its effects on granule cell activation by sensory-like patters of mossy fiber activation; for these studies, single-neuron recordings will be complemented by autofluorescence optical imaging of [the mossy fiber area of activation]. Specific Aim #2 is to further characterize the effects of EtOH on Golgi cells. During the previous funding period, we demonstrated that EtOH increases Golgi cell firing and propose to characterize the mechanism responsible for this effect. [Based on a combination of computer modeling and experimental studies, we propose to characterize IA, Na+/K+ pump and persistent Na+ currents as potential mediators of EtOH's effect]. We will also investigate EtOH's effect on GABAergic and/or glutamatergic input to Golgi cells. These studies with acute slices will be complemented with in vivo electrophysiological experiments of the acute effects of EtOH on the function of these neurons. In Specific Aim #3, we will investigate the effect of EtOH on network activity in the granule cell layer as a whole using a data-driven computational neuroscience approach. These multidisciplinary studies will significantly increase our understanding of the acute effects of EtOH on the cerebellar granule cell layer, forming the basis for the identification of new targets for therapeutic interventions against EtOH-induced cerebellar dysfunction.