Correlation between anti-ubiquitin immunoreactivity and region-specific neuronal death in N-methyl-D-aspartate-treated rat hippocampal organotypic cultures.

Neuronal degeneration appears to be associated with changes in anti-ubiquitin immunoreactivity (UIR). To elucidate the relationship between the two events, we examined the time course of changes in UIR in pyramidal neurons of hippocampal organotypic cultures following exposure to an excitotoxin, N-methyl-D-aspartate (NMDA). In nontreated cultures, weak UIR was confined to the nucleus. Exposure to 100 microM NMDA for 15 min induced degeneration of pyramidal neurons, within 24 h, in the CA1 and CA3c regions. In these neurons, the nuclear UIR was reduced, and instead, UIR developed in the cytoplasm. In response to the same procedure, CA3a,b pyramidal neurons showed slight shrinkage but otherwise virtually normal morphological features. Little perikaryal (cytoplasmic) UIR developed in CA3a,b neurons. Both degeneration and perikaryal UIR were observed in CA3a,b neurons, however, when the culture was exposed to 300 microM NMDA. Immunoblot analysis showed that changes in the amount of a ubiquitin protein conjugate (24 kDa), presumably ubiquitinated histone, are similar to those of nuclear UIR in the same time course. We propose that the changes in the expression of nuclear and perikaryal ubiquitinated proteins represent some process closely related to neuronal death.