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

描述（由申请人提供）：历史上，对乙醇在gaba能突触中的作用的研究主要集中在突触后机制。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，我们对乙醇促进GABA释放的机制知之甚少。神经递质释放可因细胞内钙水平的变化而改变，而乙醇可使细胞内钙水平升高。因此，钙依赖机制可能在乙醇增强的GABA释放中发挥作用似乎是合理的。肌醇-1,4,5-三磷酸受体（IPSRs）和红嘌呤受体（RyRs）的激活可以通过从体内钙储存中释放钙来影响神经递质的释放。初步数据显示，一种IP3R抑制剂干扰了乙醇诱导的小脑浦肯野神经元（cPNs）中微型抑制性突触后电流（mlPSC）频率的增加，表明乙醇增强的GABA释放依赖于内部钙储存的钙释放。我将通过证实IP3R抑制减少乙醇增强的GABA释放，并确定ryr是否参与乙醇增强的GABA释放来探索这一观点（目的1）。神经调节剂可以改变乙醇增强的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连接的第二信使起作用；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上均存在蛋白激酶A （PKA）磷酸化位点，乙醇和CBs均可调控PKA。因此，乙醇可能通过激活PKA来影响ipsr和/或RyRs （Aim 2）。乙醇也改变磷脂酶（PLC）的活性。由于PLC产生IPS并导致蛋白激酶C （PKC）的激活，乙醇增强GABA释放的机制可能涉及通过IPS的产生和/或PKC对IP3R的磷酸化激活IP3R （Aim 3）。为了深入探讨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。我还将使用成对脉冲比（PPR），它可以检测到发生在神经递质释放位点远端的影响。总的来说，我将探索可以解释乙醇对大脑中GABA释放的影响的机制，这是乙醇可以影响行为的一种手段。这个重要问题的答案可以帮助科学界理解乙醇的严重后果是如何导致酗酒的。

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.