A simple method for quantifying changes in neuronal populations in primary cultures of dissociated rat brain.

A simple method which allows easy quantification of neuronal and glial sub-populations in primary neuronal tissue culture is described. The method takes advantage of the fact that the staining of neuronal and glia nuclei with Hoechst 33258 is distinctive and characteristic of the cell type. Double-staining experiments show that only cells with nuclear staining characteristic of neurones are immunopositive for neuronal markers. There is no obvious difference between neurones from different regions of the brain nor any apparent differences in nuclear staining of neurones of different sizes. This method is particularly useful for the quantification of neurones in cultures where neurones are growing on glial beds or where the neurones are growing in clusters. Further, it eliminates the need to double-stain cultures with neuronal markers in order to calculate neuronal sub-populations. We have used this technique to examine the effect of time in vitro on the proportion of calbindin D28K-positive neurones in striatal cultures. We show that with increasing time in culture, there is a significant increase in the percentage of neurones which express calbindin D28K. This supports the suggestion that calbindin D28K may be important for promoting the survival of neurones in which it is expressed.