Regulation of release probability by tonic presynaptic inhibition

描述（由申请人提供）：对于我们理解突触功能至关重要的是概率释放（p）的概念，其对应于突触前动作电位导致单个释放位点处囊泡融合的概率。人们对改变p值的机制了解很多，但对设定和维持突触初始p值的机制了解甚少。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会影响初始p。然而，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以设置初始p。强直性突触前抑制对初始p值设定重要吗？该项目将测试多个突触前受体的紧张性抑制产生初始p的大幅度降低的假设。初步数据表明，小脑平行纤维-浦肯野细胞（PF-PC）突触处的多个突触前G蛋白偶联受体（GPCR）共同作用产生大于两个的突触前G蛋白偶联受体（GPCR）。这与单独的突触前GPCR对初始p的非常小或可忽略的影响形成对比。本申请提出了实验来表征GPCR介导的强直性突触前抑制，并测试其潜在的生理意义。所使用的主要技术将是（1）全细胞电压钳记录，（2）切片中的整体突触前钙成像，（3）体内局部场电位记录，以及（4）体内整体突触前钙成像。基于初步的实验，该建议将集中在突触前腺苷（A1）受体，endcannabinoid（CB 1）受体，GABAB受体和代谢型谷氨酸4型（mGlu 4）受体的影响。第一个和第二个具体目标将表征紧张性GPCR介导的抑制的程度，并寻找这种抑制可能发生的突触前机制。第三个目标是测试强直性突触前抑制的潜在生理相关性。该项目预计将提供相当大的洞察内源性信号分子的释放概率的调节，并有可能牵连的活动依赖性机制，这种调节可能发生。这些见解也可能提供对调节运动学习的机制的见解。

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.