Calretinin-containing neurons in rat cerebellar granule cell cultures

Using an antiserum against calretinin, a calcium-binding protein, we discovered two distinct neuronal cell types that stain intensely in enriched cerebellar granule cells. One neuronal cell type resembles unipolar brush cells, whereas the other resembles Lugaro cells. During early culture times, these calretinin-positive neurons are most numerous but represent less than one percent of the total neuronal population. In cultured cells, calretinin mRNA levels peak at day three in vitro, followed by a rapid decline to undetectable levels by day six in vitro. However, calretinin-immunoreactive neurons are observed up to 29 days in vitro. Excitotoxic concentrations of glutamate receptor agonists failed to elicit an excitotoxic response on the intensely staining calretinin-positive neurons, whereas greater than 95% of the cerebellar granule cells were susceptible to the excitotoxic actions of the glutamate receptor agonists. To distinguish between the two possibilities that calretinin-positive neurons either do not express glutamate receptors or they are not susceptible to the excitotoxic effects of glutamate receptor agonists, we performed immunocytochemistry using glutamate receptor antibodies to detect the presence of receptor protein. We found that the AMPA/kainate glutamate receptor (GluR2R3) colocalized with calretinin, suggesting that calretinin-immunoreactive neurons express the AMPA/kainate receptor; cerebellar granule cells, which are known to express this receptor, were also immunoreactive for the GluR2R3 receptor.