Detection of Purkinje cell loss following drug exposures to developing rat pups using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis for calbindin-D28k mRNA expression.

A technique is described that allows for the identification and quantification of Purkinje cell loss in cerebellum subsequent to developmental toxic exposures. This technique relies upon the extensively validated findings that the Purkinje cell is the only site of expression in the cerebellum of the calcium binding protein calbindin-D28k. Thus, analysis of mRNA expression specific to this protein by comparison to matched controls provides a reliable means of determining whether cell loss has occurred. Purkinje cell loss was induced in rat pups by ethanol exposure on postnatal day (PN) 4 or valproic acid administration to pregnant dams on gestational day 13. Analysis was conducted on PN5 or PN10 and the results compared to parallel groups of pups where the Purkinje cells were counted by traditional means. When compared to matched control rat pups the decrease in calbindin-D28k mRNA expression indicates Purkinje cell loss regardless of whether the cell loss was induced by prenatal valproic acid or postnatal ethanol exposure. The availability of a biochemical alternative to histological cell counting allows for more detailed analyses of the mechanisms of Purkinje cell death induced by these two toxicants, including analyses of the early alterations in signal transduction proteins.