Development of an in vitro model of neuronal activity induced excitotoxicity using photoconductive stimulation.
Loss of the ability to regulate calcium is a central event leading to neuronal cell death during a wide range of pathological conditions including stroke and seizure. Here we present a new dissociated hippocampal cell culture model of acute electrical activity which incorporates the photoconductive stimulation of neuronal networks grown on silicon wafers. This technology allows precise modeling of user defined neuronal activity patterns, and the study of their effect on neuronal physiology. Here, seizure-like conditions were created by continuous stimulation, causing hundreds of neurons to fire synchronously at 50Hz for 4min. This stimulation protocol induced cell death as monitored by propidium iodide staining. The number of dead cells per stimulation region increased from 3.6+/-2.1 preceding stimulation to 81+/-21 30min following stimulation. Excitotoxicity primarily affected excitatory rather than inhibitory neurons, and was preceded by an increase in intracellular calcium as well as changes in the mitochondrial membrane potential, as measured by a tetramethylrhodamine methyl ester (TMRM) assay. Cyclosporin A (CsA), a mitochondrial permeability transition pore (PTP) blocker, was effective in preventing cell death. We propose that photoconductive stimulation is a useful tool for investigating the pathogenesis of excitotoxicity in vitro.
Pavlov
Evgeny
E
Department of Physiology and Biophysics, Dalhousie University, Halifax, NS, Canada.
Gutiérrez
R Carolina
RC
Zhang
Yuan
Y
Kertesz
Audrey C
AC
Hung
Johanna
J
Espina
Ferdinand Joseph
FJ
Colicos
Michael A
MA
D000818:Animals; D017209:Apoptosis; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D016572:Cyclosporine / Q000494:pharmacology; D008027:Light; D008954:Models, Biological; D009474:Neurons / Q000378:metabolism* / Q000502:physiology / Q000528:radiation effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D012640:Seizures / Q000378:metabolism; D013997:Time Factors
D016572:Cyclosporine; D002118:Calcium