New organotypic model to culture the entire fetal rat spinal cord

We have developed a system for culturing separately the entire longitudinally-cut ventral and dorsal parts of the whole spinal cord of 12-14 day rat embryos, either on polylysine-collagen mixture or co-cultured with human muscle. At the time of explantation and throughout the period of culturing (up to 21 days) choline acetyltransferase (ChAT) activity in dorsal macroexplants (D-MEs) was negligible (2% of that in the ventral macroexplants (V-MEs)). During culturing neuronal survival was evaluated by measurements of ChAT and enolase. The densities and lengths of neuronal processes outgrowing from the MEs were measured in living and fixed cultures. In the latter, immunocytochemical and cytochemical stainings, allowing visualization of neurites, were evaluated by computerized video image analysis. The ChAT activity in the MEs cultured under our two experimental conditions differed only during the first few days of culturing, being significantly higher in the V-MEs co-cultured with muscle as compared to those cultured on a polylysine-collagen mixture. After 3 weeks of culturing, ChAT activity was not statistically different. However, the co-cultured muscle fibers definitely influenced: (a) neurite outgrowth, (b) the expression of neurofilament proteins and (c) the ability to maintain cultured MEs long-term. This new system allows study of motor neurons and the influence on them of putative growth, survival and maturation factors in a tissue culture milieu that more closely resembles in vivo conditions than other culture systems. Our established parameters can now serve as a basis for such studies.