丙泊酚和依托咪酯对小脑皮层PC、MLI活动及突触可塑性的影响机制

The anesthatized action of propofol and etomidate (ET) are mainly involved in facilitatition of presynaptic GABA release and activation postsynaptic GABAA receptor. Meanwhile, the GABAA receptors play a critical role in cerebellar circuitry that control PC to output the commands, and involve synaptic transmission and plasticity, suggesting that anesthesia with propofol and ET affect cerebellar neuronal activity, synaptic transmission and pasticity, but the mechanisms of propofol and ET on cerebellar neuronal activity, synaptic transmission and pasticity are currently unclear. In addition, we previously found that propofol inhited the spontaneous activity of PCs via both GABAA and glycine receptors, indicating that propofol and ET affect cerebellar neuronal activity and synaptic plasticity is not simply dependent GABAA receptors. However, the effective mechanisms of propofol on cerebellar cortical neuronal activity and synaptic transmission in living animals are currently unclear. Therefore, applicant will use patch-clamp recording and neuropharmacology methods to study the effective mechanisms of propofol and ET on spontaneous activity, membrane potential and currents, synaptic transmission of cerebellar PCs, molecular layer interneurons (MLI), granule cells and Golgi cells, and to investigate their effects on parallel fiber (PF)-PC and PF-MLI long-term synaptic plasticity in vitro in mice. We aimed to understand the mechanisms of propofol and ET affect neuronal activity, synaptic transmission and long-term plasticity in cerebellar cortex, and to clarify the possible mechanisms of anesthesia with propofol and ET affect cerebellar motor learning. This study will make sence for understanding the effective mechanisms of propofol on central nervous system.

丙泊酚与依托咪酯（ET）的麻醉机制主要与促进突触前GABA释放和活化突触后GABAA受体活性有关,而GABAA受体在小脑皮层神经环路中起重要作用，控制浦肯野细胞（PC）指令输出，参与突触传递及可塑性，提示丙泊酚与ET麻醉可影响神经元活动、突触传递及可塑性，但其影响机制尚不明确。另外，申请人前期研究发现丙泊酚通过活化GABAA和甘氨酸受体抑制PC自发性活动，表明丙泊酚和ET对小鼠小脑神经元活动及突触传递的影响可能不单纯依赖于GABAA受体，因此，本研究拟应用电生理和药理学手段，在小脑切片上研究丙泊酚和ET对小脑皮层PC、MLI、颗粒和Golgi细胞膜电位和膜电流的影响，探讨其对平行纤维(PF)-PC和PF-MLI突触长时程可塑性的影响，明确丙泊酚与ET对小脑皮层神经元活动、突触传递及长时程可塑性影响的受体、通道及分子机制，阐明丙泊酚与ET麻醉对小脑运动学习影响的机制。

本项目主要应用电生理学、组织化学和药理学方法在小脑离体切片和活体小鼠上研究了丙泊酚激活突触前GABAB受体，通过PKA信号通路完成对离体小鼠小脑PF-PC突触传递的抑制作用，丙泊酚对小鼠小脑爬行纤维-浦肯野细胞突触传递的影响机制，丙泊酚对小鼠小脑爬行纤维-浦肯野细胞长时程突触可塑性的影响机制，依托咪酯对小鼠小脑浦肯野细胞自发性简单峰电位放电活动的影响机制以及依托咪酯对小鼠小脑颗粒细胞层触觉刺激诱发场电位反应的影响机制，阐明了丙泊酚与依托咪酯麻醉对小脑运动学习影响的机制。具体结果如下：.（1）发现GABAA受体与甘氨酸受体在被阻断时，丙泊酚可减少PF-PC兴奋性突触后电流（EPSC）的振幅大小，并显著增加配对脉冲比率（PPR）。丙泊酚的这种抑制作用是可逆的，并具有浓度依赖性，其半数有效浓度为4.7uM。本研究中发现GABAB受体的阻断剂saclofen（10uM）消除了丙泊酚对PF-PC突触的EPSC振幅值与PPR值的影响，而NMDA受体的阻断剂D-APV（50uM）并未实现对丙泊酚作用的消除。并且GABAB受体激动剂baclofen也诱发了类似丙泊酚作用的PF-PC突触的EPSC振幅值减小与PPR值增大的现象。除此之外，蛋白激酶A（PKA）KT5720也成功阻断了丙泊酚对PF-PC突触EPSC振幅值的增大效应与PPR值的减小效应。.（2）发现在GABA能受体活性缺失条件下，异丙酚可通过激活NMDA受体增强小鼠小脑皮质CF-PC突触传递。低频刺激小脑爬行纤维可诱导出mGluR1依赖的CF-PC长时程抑制，而异丙酚可以通过激活突触后的NMDA受体来增强小鼠小脑CF-PC的长时程抑制。.（3）依托咪酯通过活化GABAA和甘氨酸受体来抑制小鼠小脑浦肯野细胞自发性简单峰电位放电活动，导致放电频率显著降低。在阻断GABAA受体活性的情况下，依托咪酯通过活化小鼠小脑颗粒细胞层 CB1受体，并通过PKA信号通路抑制感觉刺激诱发的苔藓纤维-颗粒细胞的突触传递。