Novel characteristics of glutamate-induced cell death in primary septohippocampal cultures: Relationship to calpain and caspase-3 protease activation

Studies examined the phenotypic characteristics of glutamate-induced cell d eath and their relationship to calpain and caspase-3 activation. Cell viabi lity was assessed by fluorescein diacetate and propidium iodide staining an d lactate dehydrogenase release. Calpain and caspase-3 activity was inferre d from signature proteolytic fragmentation of alpha-spectrin. Characterizat ion of cell death phenotypes was assessed by Hoechst 33258 and DNA fragment ation assays. Exposure uf septohippocampal cultures to 1.0. 2.0, and 4.0 mm ol/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 s taurosporine 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 do ses of glutamate produced calpain and caspase-3 activation. Calpain inhibit or II (N-acetyl-Leu-Leu-methionyl) provided protection only with a narrow d ose range, whereas carbobenzoxy-Asp-CH2-OC(O)-2,6-dichlorobenzene (Z-D-DCB; pan-caspase inhibitor) and MK-801 (N-methyl-D-aspartate receptor antagonis t) were potently effective across a wider close range. Cycloheximide did no t reduce cell death or protease activation.