1. Vivian L. Smith Center for Neurologic Research, Department of Neurosurgery, University of Texas-Houston Health Science Center, Houston, Texas, U.S.A.
2. ^*Center for Traumatic Brain Injury Studies, Department of Neuroscience, University of Florida, Gainesville, Florida, U.S.A.
3. ^†Department of Neuroscience Therapeutics, Parke-Davis Pharmacological Research, Division of Warner-Lambert Company, Ann Arbor, Michigan, U.S.A.
4. ^‡Institute of Pharmacology, Policlinico Universitario, University of Messina, Messina, Italy

Correspondence: Ronald L. Hayes, Center for Traumatic Brain Injury Studies, Department of Neuroscience, University of Florida, P. O. Box 100244, 100 Newell Dr., L1-100F, Gainesville, FL 32610-0244, U.S.A.

Received 21 June 1999; Revised 11 November 1999; Accepted 17 November 1999.

Abstract

Studies examined the phenotypic characteristics of glutamate-induced cell death and their relationship to calpain and caspase-3 activation. Cell viability was assessed by fluorescein diacetate and propidium iodide staining and lactate dehydrogenase release. Calpain and caspase-3 activity was inferred from signature proteolytic fragmentation of -spectrin. Characterization of cell death phenotypes was assessed by Hoechst 33258 and DNA fragmentation assays. Exposure of septohippocampal cultures to 1.0, 2.0, and 4.0 mmol/L glutamate induced a dose-dependent cell death with an LD₅₀ of 2.0 mmol/L glutamate after 24 hours of incubation. Glutamate treatment induced cell death in neurons and astroglia and produced morphological alterations that differed from necrotic or apoptotic changes observed after maitotoxin or staurosporine exposure, respectively. After glutamate treatment, cell nuclei were enlarged and eccentrically shaped, and aggregated chromatin appeared in a diffusely speckled pattern. Furthermore, no dose of glutamate produced evidence of internucleosomal DNA fragmentation. Incubation with varying doses of glutamate produced calpain and caspase-3 activation. Calpain inhibitor II (N-acetyl-Leu-Leu-methionyl) provided protection only with a narrow dose range, whereas carbobenzoxy-Asp-CH₂-OC(O)-2,6-dichlorobenzene (Z-D-DCB; pan-caspase inhibitor) and MK-801 (N-methyl-D-aspartate receptor antagonist) were potently effective across a wider dose range. Cycloheximide did not reduce cell death or protease activation.