The Role of IP3 and Ryanodine Receptors in Ethanol-enhanced GABA Release

描述(由申请人提供)：历史上，乙醇在GABA能突触中的作用研究主要集中在突触后机制上。最近的数据挑战了这一观点，证明乙醇也增加了许多大脑区域自发和诱发的GABA释放。然而，人们对乙醇促进GABA释放的机制知之甚少。神经递质的释放可以通过细胞内钙水平的变化来改变，而乙醇可以增加细胞内的钙水平。因此，钙依赖机制可能在乙醇促进GABA释放中起作用。肌醇-1，4，5-三磷酸受体(IPSR)和兰尼定受体(RyRs)的激活可以通过从体内钙库释放钙来影响神经递质的释放。初步数据显示，IP3R抑制剂可干扰乙醇引起的小脑浦肯野神经元(CPN)微小抑制性突触后电流(MlPSC)频率的增加，这表明乙醇促进GABA的释放依赖于内部钙库的钙释放。我将通过证实抑制IP3R减少乙醇促进的GABA释放，并确定RyRs是否参与乙醇促进的GABA释放(目标1)来探索这一想法。神经调节剂可以改变乙醇促进的GABA释放；一个例子是大麻素(CB)受体激动剂，它可以防止乙醇诱导的CPN中mlPSC频率的增加。CB受体是G蛋白偶联受体，通过G-α-I连接的第二信使发挥作用；因此，我将确定乙醇是否也通过第二信使改变IPSR和RYR。IP3R和RyR上都有蛋白激酶A(PKA)的磷酸化位点，乙醇和CBS都对PKA起调节作用。因此，乙醇可能通过激活PKA来影响IPSR和/或RyR(目标2)。乙醇也会改变磷脂酶(PLC)的活性。由于PLC产生IPS并导致蛋白激酶C(PKC)的激活，乙醇促进GABA释放的机制可能涉及通过产生IPS和/或由PKC磷酸化IP3R来激活IP3R(目标3)。为了深入探索CPN的这些目的，我将分析mlPSC频率，这是评估神经递质释放变化的最直接方法。我还将使用配对脉搏比(PPR)，它可以检测到发生在神经递质释放部位更远的影响。总而言之，我将探索酒精对大脑中GABA释放的影响的机制，这是一种酒精可以影响行为的方式。这一重要问题的答案可能有助于科学界理解乙醇的这种严重后果是如何导致酒精中毒的。

DESCRIPTION (provided by applicant): Historically, research on the actions of ethanol at the GABAergic synapse has focused on postsynaptic mechanisms. Recent data has challenged this view by demonstrating that ethanol also increases both spontaneous and evoked GABA release in many brain regions. However, little is yet known about the mechanism through which ethanol acts to enhance GABA release. Neurotransmitter release can be altered by changes in intracellular calcium levels, and intracellular calcium levels are increased by ethanol. Therefore, it seems plausible that calcium dependent mechanisms could play a role in ethanol-enhanced GABA release. Activation of inositol-1,4,5-trisphosphate receptors (IPSRs) and ryanodine receptors (RyRs) can influence neurotransmitter release by releasing calcium from internal calcium stores. Preliminary Data show that an IP3R inhibitor interferes with the ethanol-induced increase in miniature Inhibitory Postsynaptic Current (mlPSC) frequency in cerebellar Purkinje neurons (cPNs), suggesting that ethanol-enhanced GABA release depends on calcium release from internal calcium stores. I will explore this idea by confirming that IP3R inhibition reduces ethanol-enhanced GABA release and by determining whether RyRs are involved in ethanol-enhanced GABA release (Aim 1). Neuromodulators can alter ethanol-enhanced GABA release; one example being a cannabinoid (CB) receptor agonist, which prevents the ethanol-induced increase in mlPSC frequency in cPNs. The CB receptors are G protein-coupled receptors that act through G-alpha-i-linked second messengers; therefore, I will determine if ethanol is also acting through second messengers to alter the IPSRs and RyRs. There are protein kinase A (PKA) phosphorylation sites on both the IP3R and RyR, and ethanol and CBs both regulate PKA. Therefore, ethanol might influence IPSRs and/or RyRs through activation of PKA (Aim 2). Ethanol also alters phospholipase (PLC) activity. Because PLC generates IPS and leads to activation of protein kinase C (PKC), the mechanism of ethanol-enhanced GABA release could involve activation of the IP3R through production of IPS and/or phosphorylation of the IP3R by PKC (Aim 3). To thoroughly explore these Aims in cPNs, I will analyze mlPSC frequency, which is the most direct way to assess changes in neurotransmitter release. I will also use paired-pulse ratio (PPR), which can detect effects that occur more distal to the neurotransmitter release site. Overall, I will explore mechanisms that could account for the effect of ethanol on GABA release in the brain, a means by which ethanol can affect behavior. An answer to this important issue could assist the scientific community in understanding how this acute consequence of ethanol contributes to alcoholism.