Development and activity-dependent expression of neuronal marker proteins in organotypic cultures of rat visual...

We are interested in activity-dependent mechanisms which govern the structural and functional maturation of neurons in the visual cortex. We have asked whether the expression of neuronal markers microtubule-associated proteins tau, MAP-2, synaptophysin (p38), and the growth-associated protein GAP-43 are dependent on cortical afferents or spontaneous activity. As a model system we have employed organotypic monocultures of rat visual cortex (OTCs, isolated from subcortical structures) in comparison with visual cortex in vivo (innervated by thalamic and other afferents) at different postnatal ages. We know from previous work that the OTCs, like the cortex in vivo, display a high rate of spontaneously generated action potentials. Therefore, as a third objective, we have analysed OTCs grown as monocultures under chronic blockade of spontaneous action potentials. Protein expression was detected by protein blots and/or immunohistochemistry. The proteins examined in this study are expressed in OTCs, even when grown under activity blockade. However, the pattern of expression differs from the cortex in vivo. Tau is expressed much weaker in OTCs than in cortex in vivo. The expression of the major band of about 50 kDa increases over time in vivo and in OTCs. Smaller isoforms of tau are dramatically downregulated, and larger (adult) isoforms do not appear within 35 days in vitro (DIV). Under activity blockade the expression of tau reaches a maximum by 21 DIV and decreases dramatically, so that the protein is hardly detectable by 47 DIV. MAP-2-immunoreactive proteins are localized in somata and dendrites, but also persist in axons. The expression in OTCs of p38 and GAP-43 correlates well with the expression observed in vivo. Synaptophysin (p38) occurs with a similar time course and amount in OTCs as in cortex in vivo. Synaptic boutons appear in all layers, and specialized terminal elements have been observed. Activity blockade slightly affects the p38 expression, although the late postnatal decline in p38 immunoreactivity observed on protein blots from cortex in vivo and in normal OTCs appears more accentuated in activity-blocked OTCs. The GAP-43 expression is prominent from birth onwards in vivo and in OTCs. However, in normal OTCs GAP-43 is not declining as it is in vivo, although it is downregulated in activity-blocked OTCs. As a major finding we report that neuronal markers which are normally expressed in immature neurons and axons during the period of differentiation and structural plasticity are continuously expressed in OTCs, suggesting that a monocultured cortex retains the ability for growth and structural changes longer than the cortex in vivo.