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

描述（由申请人提供）：历史上，乙醇对GABA能突触作用的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，表明乙醇也增加了许多脑区的自发和诱发GABA释放。然而，很少有人知道乙醇通过何种机制，以提高GABA的释放。神经递质的释放可以通过细胞内钙水平的变化而改变，并且细胞内钙水平通过乙醇而增加。因此，钙依赖性机制可能在乙醇增强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）和兰尼碱受体（RyRs）的激活可以通过从内部钙储存中释放钙来影响神经递质的释放。初步数据表明，IP 3R抑制剂干扰乙醇诱导的小脑浦肯野神经元（cPN）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释放依赖于从内部钙储存中释放钙。我将通过确认IP 3R抑制减少乙醇增强的GABA释放和确定RyR是否参与乙醇增强的GABA释放来探索这个想法（目的1）。神经调节剂可以改变乙醇增强的GABA释放;一个实例是大麻素（CB）受体激动剂，其防止cPN中乙醇诱导的mlPSC频率增加。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。在IP 3R和RyR上都有蛋白激酶A（PKA）磷酸化位点，乙醇和CB都调节PKA。因此，乙醇可能通过激活PKA影响IPSR和/或RyR（目的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）的活化，因此乙醇增强的GABA释放的机制可能涉及通过产生IPS和/或通过PKC磷酸化IP 3R来活化IP 3R（Aim 3）。为了彻底探索cPN中的这些目标，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部位远端的效应。 总的来说，我将探索乙醇对大脑中GABA释放的影响的机制，这是乙醇影响行为的一种方式。对这个重要问题的回答可以帮助科学界了解乙醇的这种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.