VOLATILE ANESTHETICS AND NEURONAL CALCIUM

描述(申请人提供)：由挥发性麻醉剂(VAS)介导的静止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钙离子浓度和区划，将电刺激和化学刺激转化为有意义的生理信号。作用于特定的钙调节部位的化学物质已被证明会改变VAS的MAC。在细胞水平上，VAS可抑制神经元细胞的电压依赖性钙通道和钙瞬变。我们假设，挥发性麻醉剂通过调节钙通道和信号来抑制脊髓神经元，从而产生不动。我们将通过比较树状VAS、异氟烷、氟烷和F3与两个不能产生固定化的结构相关分子(非固定剂(NIM))F6和F8的效果来检验这一假设。对电压依赖(L和N型)和配体(谷氨酸)激活的钙通道、谷氨酸介导的神经元兴奋性和谷氨酸从突触体释放的影响将被探索。最初的研究将以人类SH-SY5Y神经母细胞瘤细胞为模型系统，以确定预期的药物作用靶点。我们还将研究原代培养的背根神经节神经元(DRG)、脊髓腹角(VH)运动神经元以及突触体，这些神经元都是从成年大鼠脊髓中分离出来的。这些具体目标将决定VAS和NIMs是否(1)阻断质膜L和N型电压依赖性钙通道；(2a)阻断容量-谷氨酸激活的阳离子钙通道；(2b)改变谷氨酸引起胞浆钙瞬变的敏感性，以及对谷氨酸激活的离子和/或代谢性受体的作用；以及(2c)它们对脊髓突触体谷氨酸突触前释放及其对钙的依赖性的不同。将使用全细胞和膜片电压和电流钳，以及荧光方法，包括成像测量细胞内钙、质膜电位和谷氨酸释放。这些研究的结果将对神经元中的钙信号产生重要的分子见解，并阐明VA对钙信号的影响与VA介导的麻醉的静止方面的相关性。

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.