SELECTIVE ANESTHETIC ACTIONS ON NMDA RECEPTORS

挥发性麻醉剂通过作用于许多神经系统而产生平衡的麻醉。虽然早期的研究将这一系列作用归因于磷脂膜的非特异性流化，但最近的工作指出了麻醉剂对离子通道的特异性作用。挥发性麻醉剂选择性地阻断NMDA敏感的谷氨酸受体与其遗忘、镇痛、催眠和神经保护作用有关。我们的实验室和其他人最近的工作表明，挥发性麻醉剂和正醇对nmda受体功能的影响不同，这取决于所研究的大脑区域。此外，我们有初步数据显示，挥发性麻醉剂对NMDA受体的作用受到NMDA -1亚基外显子5剪接的影响。本研究将利用单细胞RT-PCR技术研究各种挥发性麻醉药对大鼠脑内天然NMDA受体电生理特性的影响，并将这些影响与受体亚基组成联系起来。我将使用全细胞贴片记录NMDA诱导的电流，这些电流来自急性解离神经元和原代培养中维持的神经元，以研究异氟醚对NMDA受体功能的影响。记录NMDA介导的电流后，将从单个细胞中提取细胞质，分析每个细胞中存在的NMDA受体亚单位的mrna。这一特定目标将扩展我们的初步数据，将外显子5剪接的存在与麻醉效力的增加联系起来，并将这项工作扩展到其他NMDAR-1和NMDAR-2亚型。这一目的将检验一个假设，即异氟醚对NMDA受体功能的影响取决于该受体的亚基组成。Specific Aim II将比较乙醚、异氟烷、安氟烷、氟烷和水合氯醛对NMDA引发的全细胞电流的影响，并将这些反应与特定NMDA受体亚基mrna的存在或缺失联系起来。这一特定目的将检验挥发性麻醉药共同依赖于亚基组成的假设。特异性目的III将研究挥发性麻醉剂对HEK-293细胞中表达的NMDA受体的影响，这些细胞转染的NMDA受体亚基组合被发现会影响天然受体的麻醉效力。本目标将通过实验验证来自具体目标I和II的相关数据。

Volatile anesthetics produce a balanced anesthesia by acting on a number of neural systems. While early studies attributed this array of actions to a nonspecific fluidization of phospholipid membranes, more recent work has pointed to specific actions of anesthetics on ion channels. Selective blockade of NMDA- sensitive glutamate receptors by volatile anesthetics has been linked to their amnesic, analgesic, hypnotic and neuroprotective action. Recent work by our laboratory and others has shown that both volatile anesthetics and n-alcohols have differing effects on NMDA-receptor function depending on the brain area studied. Further, we have preliminary data showing that the effects of volatile anesthetics on the NMDA receptor are influenced by the presence of the exon-5 splice in the NMDAR-1 subunit the receptor. The proposed studies will examine the effect of various volatile anesthetics on the electrophysiological properties of native NMDA receptors in rat brain and correlate those effects with the subunit composition of the receptors using single-cell RT-PCR. Specific Aim I will use whole-cell patch recording of NMDA- induced currents from acutely dissociated neurons and neurons maintained in primary culture to examine the effect of isoflurane on NMDA receptor function. Following recording of the NMDA- mediated currents, cytoplasm will be extracted from individual cells to analyze the mRNAs for NMDA receptor subunits present in each cell. This specific aim will extend our preliminary data linking the presence of the exon-5 splice to increased anesthetic potency and extend this work to other NMDAR-1 and NMDAR-2 isoforms. This aim will test the hypothesis that the effect of isoflurane on NMDA receptor function depends upon the subunit composition of that receptor. Specific Aim II will compare the effects of ethyl ether, isoflurane, enflurane, halothane and chloral hydrate on whole- cell currents elicited by NMDA and correlate those responses with the presence or absence of specific NMDA receptor subunit mRNAs. This specific aim will test the hypothesis that volatile anesthetics share a common dependence on subunit composition. Specific Aim III will examine the effect of volatile anesthetics on NMDA receptors expressed in HEK-293 cells transfected with combinations of NMDA receptor subunits found to influence anesthetic potency in native receptors. This Aim will verify experimentally, the correlational data from Specific Aims I and II.