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.
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