Calcium-dependent regulation of the enkephalin phenotype by neuronal activity during early ontogeny

Genetic components of the neuronal phenotype are regulated by epigenetic factors--trophic molecules and neuronal activity--during neurodifferentiation. Developing neurons in dissociated cultures of embryonic mouse spinal cord show spontaneous electrical activity after one week in culture. We now report that the blockade of this spontaneous electrical activity for two days with tetrodotoxin (TTX) causes virtually complete down-regulation of preproenkephalin A gene transcripts in embryonic spinal cord cultures. This TTX-induced down-regulation is fully reversed upon reinitiation of neuronal activity (removal of TTX from cultures). This reversible, tetrodotoxin-induced down-regulation of enkephalin mRNA is confined to a restricted period of early neurodevelopment (days 7 to 21 in culture). Since depolarization triggers calcium entry through voltage-activated calcium channels, we have investigated the involvement of calcium in the mechanism of this activity- and age-dependent regulation of preproenkephalin A expression. The selective activation of the L-type of voltage-sensitive calcium channels by a dihydropyridine derivative [(+) 202-791] prevented this TTX-induced down-regulation without reducing methionine enkephalin secretion. This effect was observed only when the drug was applied to electrically active cultures, prior to the addition of TTX. Simultaneous application of (+) 202-791 and TTX, or pretreatment with TTX, failed to prevent TTX-induced down-regulation. Thus, activity-dependent phenotypic plasticity of met-enkephalinergic neurons in spinal cord is: 1) maximum at an early age of neuronal development (less than 10 days in culture) and becomes less apparent in old cultures (greater than 30 days); 2) reversible throughout; and 3) mediated by calcium entry through L-type channels.