Toxicity of tunicamycin to cultured brain neurons: Ultrastructure of the degenerating neurons

Our previous study has shown that tunicamycin irreversibly downregulates th e expression of GABA(A)R and causes cell death in cultured brain neurons by biochemical and light microscopic methods. In this study, we examined mech anisms underlying the degeneration of the neurons mainly employing electron microscopic analysis. Cultured neurons derived from embryonic chicken brai ns were incubated with 5 mu g/ml of tunicamycin (TM) for 24 h, followed by continual incubation or removal of TM for additional 3 h or 24 h. Neurons t reated with TM for 24 h showed dilated rough endoplasmic reticulum (rER), n uclear envelope and components of Golgi apparatus, in addition to the degra nulation of rER and disaggregation of ribosomal rosettes. In neurons subjec ted to the prolonged incubation, some ribosomes reattached to the membranes of rER; the polyribosomes reappeared, and the swelling of Golgi apparatus subsided. However, the distention of rER persisted, and an uncommon spindle -like structure appeared in the perikarya. This structure is implicated to involve the neuronal degeneration. Moreover, extracellular cell debris was increased with time of incubation. The ratio of the light neurons, defined as containing lower cytoplasmic matrix density than the untreated control, decreased from 28% at 3 h to 3% at 24 h after the removal of TM, and 45% at further 3 h to 6% at further 24 h incubation of TM, whereas dense neurons only appeared in the two 24 h groups, as 44% and 34%. The light neurons res emble necrotic cells, but the dense neurons exhibit distinct morphological features from necrosis and apoptosis. The gel electrophoresis assay reveale d the absence of DNA fragmentation in all cultures. In addition, whole cell recordings exhibited a 40% decrease of the GABA-elicited current in the ne urons exposed to TM for 24 h. The results indicate irreversible toxicity of chronic TM treatment to the neurons and suggest differential mechanisms fo r the neuronal death among various populations of cells. It is evident that the N-glycosylation plays a critical role for neuronal survival. (C) 1999 Wiley-Liss, Inc.