Regulation of release probability by tonic presynaptic inhibition

描述(申请人提供)：对我们理解突触功能至关重要的是概率释放(P)的概念，它对应于突触前动作电位导致单个释放部位的囊泡融合的概率。关于改变突触初始p的机制，我们知道的很多，但关于设置和维持突触初始p的机制，我们知道的要少得多。突触前钙通道的密度和分布、释放机制的钙敏感性以及各种突触前信号级联等因素被认为是影响初始p的因素。然而，目前尚不清楚这些因素中哪些在特定突触中最重要，以及这些因素如何相互作用来设定初始p。这一建议将解决关于初始p调节的一个问题：强直突触前抑制对设定初始p是否重要？这个项目将检验这样一个假设，即多个突触前受体的紧张性抑制会导致初始p的大幅降低。初步数据表明，小脑平行纤维-浦肯野细胞(PF-PC)突触上的多个突触前G蛋白偶联受体(GPCRs)共同作用，使初始p减少两倍以上。这与单独使用单个突触前GPCRs对初始p的极小或可忽略的影响形成了对比。这项申请提出了实验，以表征GPCR介导的紧张性突触前抑制，并测试其潜在的生理学意义。使用的主要技术是(1)全细胞电压钳记录，(2)在脑片上的大量突触前钙成像，(3)在体内的局部场电位记录，(4)在体内的大量突触前钙成像。在初步实验的基础上，这项建议将集中在突触前腺苷(A1)受体、内源性大麻素(CB1)受体、GABAB受体和代谢性谷氨酸4型(MGlu4)受体的作用。第一个和第二个特定目标将表征强直GPCR介导的抑制的程度，并寻找可能发生这种抑制的突触前机制。第三个目标将测试紧张性突触前抑制的潜在生理学相关性。这个项目有望为内源性信号分子对释放概率的调节提供相当大的洞察力，并有可能暗示这种调节可能发生的活性依赖机制。这样的洞察力也可能提供对运动学习调节机制的洞察。 与公共卫生相关：这个项目将研究通过信号分子在小脑中持续调节突触传递，这些分子自然存在于大脑中。这一结果有望对运动学习的机制产生重要影响，并可能解释大麻类药物对大脑功能产生重要影响的大麻素相关现象。

DESCRIPTION (provided by applicant): Crucial to our understanding of synaptic function is the concept of the probability release (p), which corresponds to the probability that a presynaptic action potential results in vesicle fusion at a single release site. Much is known about the mechanisms that change p from its initial value, but much less is known about the mechanisms that set and maintain the initial p of a synapse. Factors such as the density and distribution of presynaptic calcium channels, calcium sensitivity of the release machinery, and various presynaptic signaling cascades are thought to influence initial p. However, it is not understood which of these factors are most important at specific synapses and how these factors may interact to set initial p. This proposal will address one question regarding the regulation of initial p: is tonic presynaptic inhibition important for setting initial p? This project will test the hypothesis that tonic inhibition by multiple presynaptic receptors produces a large reduction in initial p. Preliminary data suggests that multiple presynaptic G-protein coupled receptors (GPCRs) at the cerebellar parallel fiber-Purkinje cell (PF-PC) synapse work together to produce a greater than two-fold reduction in initial p. This contrasts with the very small or negligible effects of individual presynaptic GPCRs on initial p in isolation. This application proposes experiments to characterize GPCR-mediated tonic presynaptic inhibition and to test for its potential physiological significance. The primary techniques used will be (1) whole-cell voltage clamp recordings, (2) bulk presynaptic calcium imaging in slices, (3) local field potential recordings in vivo, and (4) bulk presynaptic calcium imaging in vivo. Based on preliminary experiments, this proposal will focus on the effects of presynaptic adenosine (A1) receptors, endcannabinoid (CB1) receptors, GABAB receptors, and metabotropic glutamate type 4 (mGlu4) receptors. The first and second specific aims will characterize the extent of tonic GPCR-mediated inhibition and search for presynaptic mechanisms by which this inhibition may occur. The third aim will test for potential physiological relevance of tonic presynaptic inhibition. This project is expected to provide considerable insight into the regulation of release probability by endogenous signaling molecules and has the potential to implicate activity-dependent mechanisms by which this regulation may occur. Such insights are also likely to provide insight into mechanisms regulating motor learning. PUBLIC HEALTH RELEVANCE: This project will study persistent regulation of synaptic transmission in the cerebellum by signaling molecules naturally present in the brain. The results are expected to have important implications for mechanisms of motor learning and may explain cannabinoid-related phenomena important to the effects of marijuana on brain function.