EVIDENCE OF APOPTOSIS IN PRIMARY NEURONAL CULTURES AFTER HEAT-SHOCK

We investigated the effects of 30-min heat shock on survival, DNA degr adation, and nuclear morphology of primary rat cortical and hippocampa l neurones. In cell cultures which were grown for 8 days in vitro (DIV ), only a small portion of neurones showed apoptotic morphology after heat shock of 45 degrees C and typical DNA laddering was not detectabl e, despite the fact that nearly 50% of the neurones died within 24 h. The majority of the neurones presumably died by necrosis, as indicated by random DNA degradation. In neuronal cultures grown for 15 DIV, hea t shock, however, resulted in DNA laddering, occurrence of apoptotic b odies and DNA strand breaks, typical of apoptosis. In these cultures, about 50% of the neurones showed apoptotic morphology following exposu re to 45 degrees C in TUNEL and acridine orange staining, whereas glia were not affected in vitality. In addition we were interested whether the highly inducible member of the heat-shock protein family, HSP72, would be induced in apoptotic cells. Double staining for HSP72 and TUN EL revealed concomitant HSP72 induction and occurrence of DNA degradat ion only in very few neurones in 15-DIV cultures, which were growing a djacent to astrocytes. A clear association of the degenerative process and HSP72 expression, therefore, could not be established. These resu lts demonstrate that environmental stress, such as heat shock, can ind uce apoptotic death in aged primary cultured neurones. The differentia tion state and/or the presence of glial cell elements in the cultures appears to be an important factor for the occurrence of apoptotic feat ures in cultured neurones. (C) 1997 Elsevier Science B.V.