title: Simulated ischaemia induces Ca2+-independent glutamatergic vesicle release through actin filament depolymerization in area CA1 of the hippocampus.
abstract: Transient, non-catastrophic brain ischaemia can induce either a protected state against subsequent episodes of ischaemia (ischaemic preconditioning) or delayed, selective neuronal death. Altered glutamatergic signalling and altered Ca(2+) homeostasis have been implicated in both processes. Here we use simultaneous patch-clamp recording and Ca(2+) imaging to monitor early changes in glutamate release and cytoplasmic [Ca(2+)] ([Ca(2+)](c)) in an in vitro slice model of hippocampal ischaemia. In slices loaded with the Ca(2+)-sensitive dye Fura-2, ischaemia leads to an early increase in [Ca(2+)](c) that precedes the severe ischaemic depolarization (ID) associated with pan necrosis. The early increase in [Ca(2+)](c) is mediated by influx through the plasma membrane and release from internal stores, and parallels an early increase in vesicular glutamate release that manifests as a fourfold increase in the frequency of miniature excitatory postsynaptic currents (mEPSCs). However, the increase in mEPSC frequency is not prevented by blocking the increase in [Ca(2+)](c), and the early rise in [Ca(2+)](c) is not affected by blocking ionotropic and metabotropic glutamate receptors. Thus, the increase in [Ca(2+)](c) and the increase in glutamate release are independent of each other. Stabilizing actin filaments with jaspamide or phalloidin prevented vesicle release induced by ischaemia. Our results identify several early cellular cascades triggered by ischaemia: Ca(2+) influx, Ca(2+) release from intracellular stores, actin filament depolymerization, and vesicular release of glutamate that depends on actin dynamics but not [Ca(2+)](c). All of these processes precede the catastrophic ID by several minutes, and thus represent potential target mechanisms to influence the outcome of an ischaemic episode.
authors.affiliation: Department of Behavioral Neuroscience, Oregon Health and Science University, 505 NW 185th Avenue, Beaverton, OR 97006, USA.
authors.forename: Adriana L
authors.initials: AL
authors.lastname: Andrade
authors.forename: David J
authors.initials: DJ
authors.lastname: Rossi
mesh_terms: D008841:Actin Cytoskeleton / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D002545:Brain Ischemia / Q000503:physiopathology*; D056547:CA1 Region, Hippocampal / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D002118:Calcium / Q000502:physiology*; D002614:Chelating Agents / Q000494:pharmacology; D047630:Depsipeptides / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004594:Electrophysiology; D019706:Excitatory Postsynaptic Potentials / Q000502:physiology; D005456:Fluorescent Dyes; D016257:Fura-2 / Q000031:analogs & derivatives; D005971:Glutamates / Q000502:physiology*; D066298:In Vitro Techniques; D018408:Patch-Clamp Techniques; D010590:Phalloidine / Q000494:pharmacology; D011108:Polymers; D017729:Presynaptic Terminals / Q000502:physiology; D017966:Pyramidal Cells / Q000166:cytology / Q000378:metabolism / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013572:Synaptic Vesicles / Q000502:physiology*
chemical_list: D002614:Chelating Agents; D047630:Depsipeptides; D005456:Fluorescent Dyes; D005971:Glutamates; D011108:Polymers; C057531:jasplakinolide; C049925:fura-2-am; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D010590:Phalloidine; D004533:Egtazic Acid; D002118:Calcium; D016257:Fura-2