Developmental-specific regulation of transmitter phenotypic genes was studied during neuronal differentiation. Developing primary cell culture systems and transgene animals were employed as model systems. Spontaneous neuronal activity was shown to be the key epigenetical factor inducing the expression of enkephalin and VIP genes. Tetrodotoxin blocks the expression of these genes in a reversible and developmental specific manner. Neuronal activity is mediated through NMDA receptors and also by voltage-gated calcium channels converging on a yet unknown intracellular calcium-dependent pathway. experiments with cycloheximide showed that the activity-dependent expression of the enkephalin gene requires intact protein biosynthesis, indicating the need for de novo synthesis of activity-dependent trans-factors. Transfecting the enkephalin hybrid gene in primary developing spinal cord neurons showed that activity-dependent, embryonic-type of expression is conferred to the far 5' upstream regulatory region, distinct from the -190 bp region required for governing adult-type of expression. Both the constitutive and induced expression of the transcription factor c-fos was shown to be developmentally regulated showing declining sensitivity to soluble factors and increasing sensitivity to neuronal activity, which peaks during the """"""""critical period"""""""". The mouse VIP chromosomal gene was isolated, characterized and transgene experiments demonstrated that the correct tissue specific expression of the VIP gene requires the intactness of at least 12 kb of 5' upstream regulatory elements. Stereotactic injection of tetradotoxin fully mimicked the effects of septohippocampal axotomy showing temporal up-regulation of galanin expression in septal cholinergic neurons. Nuclear translocation of the classical alpha- and the novel delta-protein kinase C isoenzymes, rather than the classical cAMP induction was identified as intracellular signaling following subnanomolar VIP stimulation of cortical astrocytes.
