Calcium regulation of spontaneous release of GABA

描述（由申请人提供）：突触传递通过从囊泡释放神经递质和随后激活突触后电压变化发生。它是脑功能各个方面的基础，与神经系统疾病有关。突触传递的关键步骤，囊泡融合，依赖于Ca2+通过突触前电压门控Ca2+通道（VGCCs）进入，该通道由动作电位激活。然而，尽管经过多年的研究，在没有动作电位的情况下，vgc在调节自发囊泡融合中的作用尚不清楚。VGCCs与皮层神经元中抑制性神经递质GABA的自发释放机制紧密耦合，多个VGCCs协同触发这些突触的自发释放。此外，Ca2+激活Ca2+感应受体（CaSR）促进自发的GABA释放。本提案的目的是提高对Ca2+在调节神经递质释放中的作用的理解，特别是它在不同类型的突触和不同形式的释放中是如何不同的。总的假设是自发性和动作电位依赖性的GABA释放是由Ca2+通过不同的机制调节的。这一假设将在新皮质神经元培养中进行测试，使用全细胞电压钳记录抑制性突触后电流来检测GABA的释放和Ca2+成像来监测端内Ca2+的变化。目的1是确定vgc如何调节GABA的释放。本提案的第一个实验将使用Ca2+螯合剂EGTA和BAPTA来测试钙从vgc到囊泡释放机制的扩散距离是否不同于自发GABA释放。该目的下一个实验将使用Ca2+成像来确定细胞外Ca2+浓度的变化是否会导致gaba能神经元的端内Ca2+浓度的变化。本实验的最后一个目的是研究VGCC协同自发释放GABA的机制。这可能通过两种可能的机制发生：(i)通过多通道联动的生理耦合或（ii）随机同步门控。这两种可能性都将通过测量VGCC在VGCC敲除小鼠培养的神经元中自发释放GABA的协同性来进行研究，这些小鼠转染了缺失c端尾的突变VGCC，并进行计算机建模和蒙特卡罗模拟。目的2将确定CaSR如何调节自发的GABA释放。这些实验将使用药理学工具和细胞内Ca2+成像来确定casr信号通路中的参与者。总之，这一建议将提供Ca2+在调节突触传递中的作用的理解。了解Ca2+影响神经递质释放的机制将有助于提高对突触功能的理解，以及当传递在疾病状态下被破坏时。

DESCRIPTION (provided by applicant): Synaptic transmission occurs through the release of neurotransmitter from vesicles and the subsequent activation of a postsynaptic voltage change. It underlies every aspect of brain function and is relevant to neurological diseases. The key step in synaptic transmission, vesicle fusion, is dependent on Ca2+ entry through presynaptic voltage-gated Ca2+ channels (VGCCs) that are activated by an action potential. However, despite many years of study, the role of VGCCs in regulating spontaneous vesicle fusion in the absence of an action potential is not clear. VGCCs are tightly coupled to the release machinery for spontaneous release of the inhibitory neurotransmitter GABA in cortical neurons, and multiple VGCCs cooperate in triggering spontaneous release at these synapses. Additionally, activation of the Ca2+-sensing receptor (CaSR) by Ca2+ facilitates spontaneous GABA release. The goal of this proposal is to improve understanding of the role of Ca2+ in regulating neurotransmitter release, specifically how it is different at different types of synapse and for different forms of release. The overall hypothesis is that spontaneous and action potential-dependent release of GABA are regulated by Ca2+ via distinct mechanisms. This hypothesis will be tested in neocortical neuronal cultures using whole-cell voltage-clamp recordings of inhibitory postsynaptic currents to detect release of GABA and Ca2+ imaging to monitor changes in intraterminal Ca2+. Aim 1 is to determine how VGCCs regulate GABA release. The first experiment in this proposal will use Ca2+ chelators, EGTA and BAPTA, to test if the diffusion distance for calcium from VGCCs to the vesicle release machinery for activity-evoked release of GABA is different from that observed for spontaneous GABA release. The next experiment in this aim will use Ca2+ imaging to determine if changes in the extracellular Ca2+ concentration produce changes in the intraterminal Ca2+ concentration in GABAergic neurons. The final experiment in this aim will investigate the mechanism underlying VGCC cooperativity for spontaneous GABA release. This could occur through two possible mechanisms: (i) physiological coupling through linkage of multiple channels or (ii) stochastic synchronized gating. Both of these possibilities will be investigated by measuring VGCC cooperativity for spontaneous GABA release in cultured neurons from VGCC knock-out mice that are transfected with mutant VGCCs, lacking their C-terminal tails, and computer modeling and Monte Carlo simulation. Aim 2 will determine how the CaSR regulates spontaneous GABA release. These experiments will use pharmacological tools and intracellular Ca2+-imaging to determine the players in the CaSR-signaling pathway. Together, this proposal will provide an understanding of the role of Ca2+ in regulating synaptic transmission. Understanding the mechanism by which Ca2+ influences release of neurotransmitter will contribute to an improved understanding of synaptic function in general and when transmission is disrupted in disease states.