Isoflurane Alters Presynaptic Endoplasmic Reticulum Calcium Dynamics in Wild-Type and Malignant Hyperthermia-Susceptible Rodent Hippocampal Neurons

Abstract

Volatile anesthetics reduce excitatory synaptic transmission by both presynaptic and postsynaptic mechanisms which include inhibition of depolarization-evoked increases in presynaptic Ca2+concentration and blockade of postsynaptic excitatory glutamate receptors. The presynaptic sites of action leading to reduced electrically evoked increases in presynaptic Ca2+concentration and Ca2+-dependent exocytosis are unknown. Endoplasmic reticulum (ER) of Ca2+release via ryanodine receptor 1 (RyR1) and uptake by SERCA are essential for regulation intracellular Ca2+and are potential targets for anesthetic action. Mutations in sarcoplasmic reticulum release channels mediate volatile anesthetic-induced malignant hyperthermia (MH), a potentially fatal pharmacogenetic condition characterized by unregulated Ca2+release and muscle hypermetabolism. However, the impact of MH mutations on neuronal function are unknown. We used primary cultures of postnatal hippocampal neurons to analyze volatile anesthetic-induced changes in ER Ca2+dynamics using a genetically encoded ER-targeted fluorescent Ca2+sensor in both rat and mouse wild-type neurons and in mouse mutant neurons harboring theRYR1T4826I MH-susceptibility mutation. The volatile anesthetic isoflurane reduced both baseline and electrical stimulation-evoked increases in ER Ca2+concentration in neurons independent of its depression of presynaptic cytoplasmic Ca2+concentrations. Isoflurane and sevoflurane, but not propofol, depressed depolarization-evoked increases in ER Ca2+concentration significantly more in mouseRYR1T4826I mutant neurons than in wild-type neurons. TheRYR1T4826I mutant neurons also showed markedly greater isoflurane-induced reductions in presynaptic cytosolic Ca2+concentration and synaptic vesicle exocytosis. These findings implicate RyR1 as a molecular target for the effects of isoflurane on presynaptic Ca2+handling.Significance StatementDespite their essential clinical roles, the molecular and cellular mechanisms of action of general anesthetics are not fully understood. Malignant hyperthermia is a potentially fatal pharmacogenetic disorder that leads to dysregulation of intracellular Ca2+handling in response to triggering by volatile anesthetics. While research on malignant hyperthermia has focused on skeletal muscle effects, much less is known about its neuronal effects. We identify neuronal endoplasmic reticulum Ca2+regulation as a novel target for volatile anesthetic action and as a potential target in malignant hyperthermia. While depression of CNS electrical activityin vivoby anesthesia has been observed in another model of malignant hyperthermia, our study reveals fundamental presynaptic mechanisms of volatile anesthetics with implications for the development of more selective anesthetics and for prevention and treatment of malignant hyperthermia.