Mechanisms of Anesthesia Mediated Neurotoxicity

描述(申请人提供)：我们最近的初步研究表明，常用的全身麻醉药(GAS)，异氟醚和异丙酚，通过激活InsP3R钙通道2+开放，通过mTOR依赖的途径调节自噬，这有助于GAS对细胞存活或死亡的影响。此次R01更新的首要目标是通过激活InsP3Rs来研究GAS对自噬活动的作用机制，以及随后对细胞生存或死亡、突触完整性和功能以及认知功能的作用。因此，我们假设GAS通过InsP3 R的剂量和时间依赖的激活来调节自噬，其大小决定了神经元存活、突触完整性和功能以及随后的认知功能的命运。我们建立了三个特定的目标(SA)来研究这一假说：SA1)。我们将确定GA介导的InsP3R亚型激活在自噬调节中的作用。利用单通道膜片钳技术，在不同InsP3R亚型水平的细胞培养中，我们将表征和比较四种气体(异丙酚、异氟醚、七氟醚和地氟烷)对InsP3R三种亚型的激活效力，并将膜片钳数据与麻醉剂引起的胞浆、线粒体和内质网钙离子浓度的变化联系起来。我们将研究GAS通过mTOR依赖途径，在不同细胞培养或不同基础自噬水平和InsP3R活性的不同海马区或大脑皮层中，通过不同的自噬调节位点和控制来研究GAS对自噬的影响和机制。Sa2)。我们将确定GA调节的自噬在细胞凋亡和突触完整性中的作用。我们将在不同自噬水平和InsP3R基线活性的小鼠的相同细胞培养和脑区同时研究GAS对自噬和凋亡的影响，在存在或不存在各种自噬和凋亡刺激或抑制物的情况下。我们将通过测量原代神经元培养或自噬或InsP3R-1缺陷小鼠的海马区和皮质中突触前突触蛋白和突触后标记的变化来研究GAS对突触完整性的影响。SA3)。我们将确定GA调节的自噬对突触和认知功能的作用。我们将研究GAS对自噬或InsP3R-1缺陷小鼠的基本突触传递、海马片长时程增强(LTP)和认知的长期影响，以及在存在或不存在各种自噬和凋亡刺激和抑制物的情况下。我们将把这些GAS对突触和认知功能的影响与它们对InsP3R激活、2+内质网钙释放、自噬活性以及相关的细胞凋亡和突触完整性的影响联系起来。

DESCRIPTION (provided by applicant): Our recent preliminary studies suggest that the commonly used general anesthetics (GAs), isoflurane and propofol, regulate autophagy via mTOR dependent pathway by activation of InsP3R Ca channel 2+ opening, which contribute to GAs effect on cell survival or death. The overarching goal of this R01 renewal is to investigate the mechanisms of GAs on autophagy activity via activation of InsP3Rs and the subsequent role on cell survival or death, synapse integrity and function and cognitive function. Thus, we hypothesize that GAs modulate autophagy through a dose- and time-dependent activation of InsP3 R, and the magnitude determines the fate of neuronal survival, synaptic integrity and function, and subsequent cognitive function. We have set up three specific aims (SA) to investigate this hypothesis: SA1). We will determine the role of GA-mediated activation of InsP3R subtypes on modulation of autophagy. Using the single channel nuclear patch-clamp technique in cell cultures with various levels of each subtype of InsP3R, we will characterize and compare the activating potency of four GAs (propofol, isoflurane, sevoflurane and desflurane) on each of the three subtypes of InsP3Rs, and correlate patch-clamp data with anesthetics-evoked changes in cytosolic, mitochondrial and ER Ca2+ concentrations. We will study the effects and mechanisms of GAs on autophagy via mTOR dependent pathway using various stimulators and inhibitors and controls of various autophagy regulatory sites in different cell cultures or hippocampus or cortex with different levels of baseline autophagy and InsP3R activity. SA2). We will determine the role of GA-modulated autophagy on apoptosis and synaptic integrity. We will study the effects of GAs on autophagy and apoptosis simultaneously in the same cell cultures and brain regions of mice with varying levels of autophagy and InsP3R baseline activity, in the presence or absence of various autophagy and apoptosis stimulators or inhibitors. We will study the GAs effects on synapse integrity by measuring changes of presynaptic synapsins and postsynaptic markers in primary neuronal cultures or hippocampus and cortex of mice with deficient autophagy or InsP3R-1. SA3). We will determine the role of GA-modulated autophagy on synaptic and cognitive function. We will study the long-term effects of GAs on basic synaptic transmission, long term potentiation (LTP) in hippocampal slices, and cognition in mice with deficient autophagy or InsP3R-1, in the presence or absence of various autophagy and apoptosis stimulators and inhibitors. We will correlate these GAs effects on synapse and cognitive function to their effects on activation of InsP3R, Ca release from the 2+ ER, autophagy activity and related apoptosis and synapse integrity.