Calcium regulation of spontaneous release of GABA

GABA 自发释放的钙调节

• 批准号: 8775607
• 负责人: Courtney L Williams
• 金额: $ 4.27万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8775607
• 关键词: Action Potentials; Affect; Agonist; Amyotrophic Lateral Sclerosis; Brain; Buffers; C-terminal; Calcium; Calcium-Sensing Receptors; Cells; Chelating Agents; Communication; Computer Simulation; Coupled; Coupling; Data; Diffusion; Disease; Egtazic Acid; Enzymes; Epilepsy; Excitatory Synapse; Glutamate Decarboxylase; Glutamates; Goals; Heart; Image; Inhibitory Synapse; Knockout Mice; Knowledge; Link; Measures; Membrane; Monitor; Monte Carlo Method; Mutant Strains Mice; Nerve; Nervous system structure; Neuroglia; Neurons; Neurotransmitters; Parkinson Disease; Pathway interactions; Phospholipase C; Physiological; Play; Process; Property; Proteins; Receptor Activation; Receptor Signaling; Regulation; Research; Role; Signal Pathway; Signal Transduction; Source; Synapses; Synaptic Transmission; Tail; Testing; Training; Vesicle; cell type; extracellular; gamma-Aminobutyric Acid; improved; inhibitor/antagonist; innovation; insight; mutant; neocortical; nervous system disorder; neurotransmission; neurotransmitter release; patch clamp; postsynaptic; presynaptic; public health relevance; receptor; research study; synaptic function; therapy development; tool; transmission process; voltage; voltage clamp

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.

描述(申请人提供)：突触传递通过从小泡释放神经递质和随后激活突触后电压变化而发生。它是大脑功能的各个方面的基础，与神经疾病有关。突触传递的关键步骤是囊泡融合，它依赖于由动作电位激活的突触前电压门控钙通道(VGCC)的钙离子内流。然而，尽管多年来的研究，在缺乏动作电位的情况下，VGCC在调节自发性囊泡融合中的作用尚不清楚。VGCC与皮质神经元自发释放抑制性神经递质GABA紧密相连，多个VGCC协同触发这些突触的自发释放。此外，钙离子激活钙敏感受体(CaSR)促进GABA的自发释放。这项建议的目的是提高对钙离子在调节神经递质释放中的作用的理解，特别是在不同类型的突触和不同形式的释放中它是如何不同的。总的假设是，自发的和动作电位依赖的GABA的释放是由钙离子通过不同的机制调节的。这一假说将在新皮质神经元培养中得到验证，使用抑制突触后电流的全细胞电压钳记录来检测GABA的释放，并通过钙成像来监测终端内钙的变化。目的1是确定VGCC如何调节GABA的释放。这个方案中的第一个实验将使用钙离子螯合剂EGTA和BAPTA来测试钙从VGCC到囊泡释放机制的扩散距离是否与观察到的自发释放GABA的距离不同。这一目标的下一个实验将使用钙成像来确定细胞外钙浓度的变化是否会导致GABA能神经元内钙浓度的变化。这一目标的最终实验将探索VGCC协同自发释放GABA的机制。这可以通过两种可能的机制发生：(I)通过多个通道的链接的生理耦合或(Ii)随机同步门控。这两种可能性都将通过测量VGCC对培养的神经元自发释放GABA的协同性进行研究，这些细胞是从VGCC基因敲除小鼠的培养神经元中自发释放GABA的，这些突变的VGCC基因敲除小鼠缺乏C-末端，并通过计算机建模和蒙特卡罗模拟进行研究。目标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钙成像来确定CaSR信号通路中的参与者。综上所述，这一建议将有助于理解钙离子在调节突触传递中的作用。了解钙离子影响神经递质释放的机制将有助于更好地了解突触功能，以及在疾病状态下传递中断的情况。