SELECTIVE ANESTHETIC ACTIONS ON NMDA RECEPTORS

挥发性麻醉剂通过作用于许多神经系统来产生平衡麻醉。 虽然早期的研究将这一系列的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。 挥发性麻醉药对NMDA敏感的谷氨酸受体的选择性阻断与其遗忘、镇痛、催眠和神经保护作用有关。 我们实验室和其他实验室最近的工作表明，挥发性麻醉剂和n-醇对NMDA受体功能有不同的影响，这取决于所研究的大脑区域。此外，我们有初步的数据表明，挥发性麻醉剂对NMDA受体的影响是由存在的NMDAR-1亚基受体的外显子-5剪接。 拟议的研究将检查各种挥发性麻醉剂对大鼠脑中天然NMDA受体的电生理特性的影响，并使用单细胞RT-PCR将这些影响与受体的亚基组成相关联。具体目的I将使用全细胞膜片记录急性分离神经元和原代培养神经元的NMDA诱导电流，以检查异氟烷对NMDA受体功能的影响。 在记录NMDA介导的电流后，从单个细胞中提取细胞质以分析每个细胞中存在的NMDA受体亚基的mRNA。 这一特定目标将扩展我们的初步数据，将外显子5剪接的存在与增加的麻醉效力联系起来，并将这项工作扩展到其他NMDAR-1和NMDAR-2亚型。 该目的将检验异氟烷对NMDA受体功能的影响取决于该受体亚基组成的假设。具体目标II将比较乙醚、异氟烷、恩氟烷、氟烷和水合氯醛对NMDA引起的全细胞电流的影响，并将这些反应与是否存在特异性NMDA受体亚基mRNA相关联。这一特定目标将检验挥发性麻醉剂对亚基组成具有共同依赖性的假设。具体目标III将检查挥发性麻醉剂对HEK-293细胞中表达的NMDA受体的影响，该细胞转染有影响天然受体中麻醉剂效力的NMDA受体亚基组合。 本目标将通过实验验证具体目标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.