title: Seizure-induced cell death produced by repeated tetanic stimulation in vitro: possible role of endoplasmic reticulum calcium stores.
abstract: Seizures may cause brain damage due to mechanisms initiated by excessive excitatory synaptic transmission. One such mechanism is the activation of death-promoting intracellular cascades by the influx and the perturbed homeostasis of Ca2+. The neuroprotective effects of preventing the entry of Ca2+ from voltage-dependent Ca2+ channels, NMDA receptors, and non-NMDA receptors, is well known. Less clear is the contribution to excitotoxicity of Ca2+ released from endoplasmic reticulum (ER) stores. We produced epileptiform discharges in combined entorhinal cortex/hippocampus slices using repeated tetanic stimulation of the Schaffer collaterals and assessed cell death after 1, 3, or 12-14 h with gel electrophoresis of genomic DNA and immunohistologically using terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine 5'-triphosphate (dUTP) nick end labeling (TUNEL) staining. We manipulated ER Ca2+ stores using two conventional drugs, dantrolene, which blocks the Ca2+ release channel, and thapsigargin, which blocks sarco-endoplasmic reticulum Ca2+-ATPases resulting in depletion of ER Ca2+ stores. To monitor epileptogenesis, and to assess effects attributable to dantrolene and thapsigargin on normal synaptic transmission, extracellular potentials were recorded in stratum pyramidale of the CA1 region. Repeated tetanic stimulation reliably produced primary afterdischarge and spontaneous epileptiform discharges, which persisted for 14 h, the longest time recorded. We did not observe indications of cell death attributable to seizures with either method when assessed after 1 or 3 h; however, qualitatively more degraded DNA always was observed in tetanized slices from the 12- to 14-h group compared with time-matched controls. Consistent with these data was a significant, fourfold, increase in the percentage of TUNEL-positive cells in CA3, CA1, and entorhinal cortex in tetanized slices from the 12- to 14-h group (16. 5 +/- 4.4, 33.7 +/- 7.1, 11.6 +/- 2.1, respectively; means +/- SE; n = 7) compared with the appropriate time-matched control (4.1 +/- 2.2, 7.3 +/- 2.0, 2.8 +/- 0.9, respectively; n = 6). Dantrolene (30 microM; n = 5) and thapsigargin (1 microM; n = 4) did not affect significantly normal synaptic transmission, assessed by the amplitude of the population spike after 30 min of exposure. Dantrolene and thapsigargin also were without effect on the induction or the persistence of epileptiform discharges, but both drugs prevented seizure-induced cell death when assessed with gel electrophoresis. We suggest that Ca2+ entering a cell from the outside, in addition to the Ca2+ contributed from ryanodine-sensitive stores (i.e., Ca2+-induced Ca2+ release), may be necessary for seizure-induced cell death.
authors.affiliation: Bloorview Epilepsy Research Laboratory, University of Toronto, Toronto, Ontario M5T 2S8, Canada.
authors.forename: M R
authors.initials: MR
authors.lastname: Pelletier
authors.forename: J S
authors.initials: JS
authors.lastname: Wadia
authors.forename: L R
authors.initials: LR
authors.lastname: Mills
authors.forename: P L
authors.initials: PL
authors.lastname: Carlen
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D016923:Cell Death; D003620:Dantrolene / Q000494:pharmacology; D004558:Electric Stimulation; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D018728:Entorhinal Cortex / Q000473:pathology / Q000503:physiopathology / Q000648:ultrastructure; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006624:Hippocampus / Q000473:pathology / Q000503:physiopathology / Q000648:ultrastructure; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D066298:In Vitro Techniques; D008297:Male; D018408:Patch-Clamp Techniques; D051381:Rats; D017208:Rats, Wistar; D012640:Seizures / Q000378:metabolism / Q000473:pathology* / Q000503:physiopathology; D019284:Thapsigargin / Q000494:pharmacology
chemical_list: D002121:Calcium Channel Blockers; D004791:Enzyme Inhibitors; D019284:Thapsigargin; D003620:Dantrolene; D002118:Calcium