Developmental changes in the inducibility of fos-like immunoreactivity in primary embryonic spinal cord cultures

The immediate early gene (IEG) transcription factor c- fos coordinates changes in the pattern of long term gene expression and, therefore, it may be involved in mediating epigenetic control during neurodevelopment. We used pharmacological treatments mimicking various environmental and intracellular signals and assessed the inducibility of fos-like immunoreactivity (LIR) at various stages of neurodifferentiation in a primary embryonic spinal cord culture system by immunohistochemistry. Constitutive fos LIR exclusively found in neurons, was driven by the onset and extent of spontaneous electrical activity, as it was blockable by tetrodotoxin (TTX) at all developmental stages. Phorbol myristate 13 acetate (PMA) increased the number of fos-LIR cells equally effectively at all stages, but the predominant cellular localization of fos-LIR changed through ontogeny. The effect of veratridine, kainate and serum-derived factors in significantly inducing fos-LIR was restricted to the earliest developmental stage (4 days in vitro; DIV) investigated; whereas forskolin, the GABA A antagonist picrotoxin and NMDA failed to induce fos-LIR at this stage, but increased the number of fos-LIR neurons at later stages. Dihydropyridine agonists of the voltage-sensitive calcium channels (VSCC) raised the number of fos-LIR neurons and also prevented TTX-mediated down-regulation; whereas antagonists markedly reduced fos-LIR at all ages. Either type of NMDA antagonists (AP5 and MK801) and the GABA A agonist muscimol significantly reduced fos-LIR at all ages. These findings demonstrate that the inducibility of fos-LIR is substantially different in embryonic neurons than in adult ones and that inducibility by various first and second messengers is dependent on the developmental stage.