VOLATILE ANESTHETICS AND NEURONAL CALCIUM

描述（由申请人提供）：挥发性麻醉剂（VAs）介导的不动似乎主要是由于脊髓水平兴奋性神经传递的抑制。神经元通过调节细胞内Ca2+浓度和区室化，将电和化学刺激转化为有意义的生理信号。作用于特定Ca2+调节位点的化学物质已被证明可以改变VAs的MAC。在细胞水平上，VAs已被证明可以抑制神经元细胞中电压依赖性的Ca2+通道和Ca2+瞬态。我们假设挥发性麻醉剂通过调节Ca2+通道和信号传导抑制脊髓神经元产生不动。我们将通过比较树形VAs、异氟烷、氟烷和F3与两种结构相关的分子（非固定化剂（NIMs）） F6和F8的作用来验证这一假设。对电压依赖性（L型和n型）和配体（谷氨酸）激活的Ca2+通道、谷氨酸介导的神经元兴奋性和突触体谷氨酸释放的影响将进行探讨。最初的研究将涉及人类SH-SY5Y神经母细胞瘤细胞作为模型系统，以确定药物作用的预期靶点。我们还将研究原代培养的背根神经节神经元（DRG）和脊髓腹角(VH)-运动神经元以及突触体，这些都是从成年大鼠脊髓中分离出来的。具体目标将决定VAs和nim(1)在质膜L型和n型电压依赖性Ca2+通道阻断方面是否存在差异；（2a）不同的阻断能力-谷氨酸激活的阳离子Ca2+通道；（2b）改变谷氨酸唤起细胞质Ca2+瞬态的敏感性，以及对谷氨酸激活的嗜离子性和/或代谢性受体的作用；和（2c）对脊髓突触体谷氨酸的突触前释放及其对Ca2+的依赖性的影响不同。全细胞和膜片电压和电流钳，荧光方法包括成像测量细胞内Ca2+，质膜电位，谷氨酸释放将被采用。这些研究的结果将对神经元中的Ca2+信号传导产生重要的分子见解，并阐明VA对Ca2+信号传导的影响与VA介导的麻醉的不动方面的相关性。

DESCRIPTION (provided by applicant): Immobility mediated by volatile anesthetics (VAs) appears to result largely from depression of excitatory neurotransmission at the spinal cord level. Neurons transform electrical and chemical stimuli into meaningful physiological signals by the regulation of intracellular Ca2+ concentration and compartmentalization. Chemicals that act at particular Ca2+ regulatory sites have been shown to alter the MAC of VAs. At the cellular level, VAs have been shown to inhibit voltage-dependent Ca2+ channels and Ca2+ transients in neuronal cells. We hypothesize that Volatile anesthetics produce immobility by inhibiting spinal cord neurons through the modulation of Ca2+ channels and signaling. We will test this hypothesis by comparing the effects of tree VAs, isoflurane, halothane, and F3, with that of two structurally related molecules that fail to produce immobility (non-immobilizers (NIMs)), F6 and F8. Effects on voltage-dependent (L- and N-type) and ligand (glutamate)-activated Ca2+ channels, glutamate-mediated neuronal excitability, and glutamate release from synaptosomes will be explored. Initial studies will involve human SH-SY5Y neuroblastoma cells as a model system to identify prospective targets of drug action. We will also study dorsal root ganglion neurons (DRG), and spinal ventral horn (VH)- motor neurons in primary culture as well as synaptosomes, all isolated from the adult rat spinal cord. The specific aims will determine whether VAs and NIMs (1) differ in their block of plasma membrane L- and N-type voltage dependent Ca2+ channels; (2a) differ in their block of capacitative- glutamate activated cationic Ca2+ channels; (2b) change the sensitivity of glutamate for evoking cytoplasmic Ca2+ transients, and action on glutamate-activated ionotropic and/or metabotropic receptors; and (2c) differ in their effect on the presynaptic release of glutamate from spinal cord synaptosomes and its dependence on Ca2+. Whole cell and patch voltage- and current-clamp, and fluorescence methodologies including imaging for measuring intracellular Ca2+, plasma membrane potential, and glutamate release will be employed. The results of these studies will yield important molecular insights into Ca2+ signaling in neurons and clarify the relevance of VA effects on Ca2+ signaling to the immobility aspect of VA-mediated anesthesia.