In situ hybridization histochemistry of Spot 35 protein, a calcium-binding protein, in the rat brain

Using in situ hybridization, we analyzed the localization of mRNA for Spot 35 protein (Spot 35), a calcium-binding protein of the EF-hand type, in the rat cerebellum at various developmental stages. A cDNA fragment corresponding to part of the 3'-noncoding region was 35S-labeled and used as a hybridization probe. Autoradiographic signals for Spot 35 mRNA were detected in all the Purkinje cells, but not in any other neurons or glial cells in the adult rat cerebellum. There was no significant difference in signal intensity among individual cells. The signals were observed exclusively in Purkinje cell bodies, but not in their processes, in striking contrast to previous immunohistochemical studies in which Spot 35 protein was demonstrated in both cell bodies and processes. In the time-course study, signals for Spot 35 mRNA were detected in Purkinje cell bodies weakly at embryonic day 19, thereafter more intensely at more developed stages and most intensely at postnatal days 30 and 60 (adulthood). The signal intensities of individual cells were similar at each of these developmental stages except for the very early stages at which signals were weak and slightly variable among cells. These findings, especially that of the characteristic coordinated expression of Spot 35 mRNA at given stages, should prove useful in studies of degenerative diseases in the cerebellum in experimental animals and man. A weak expression of Spot 35 mRNA in some of non-Purkinje cells was also noted.