In situ detection of activated caspase-3 in apoptotic granule neurons in the developing cerebellum in slice cultures and in vivo

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Some granule neurons naturally undergo apoptosis in the external granular layer (EGL) of the postnatally developing cerebellum. In the present study, we examined the involvement of caspase-3 in this apoptosis using an organotypic slice culture system of postnatal rat cerebellum and an antibody specific for the active form of caspase-3 (p20/17). Double staining by immunohistochemistry against p20/17 and in situ nick-end labeling showed that p20/17 was present in some of the apoptotic EGL neurons. A similar staining pattern was also observed in the postnatal cerebellum in vivo. Double positive cells were observed more frequently when T7 DNA polymerase was used for the DNA fragmentation labeling in place of terminal deoxynucleotidyl transferase, by which apoptotic cells at earlier stages were thought to be labeled. Taken together, whereas caspase-3 was shown to be activated in some of the apoptotic EGL neurons in the developing cerebellum, activation of caspase-3 in some apoptotic EGL neurons may occur before they become positive on DNA fragmentation labeling. In addition, there may be another mechanism of EGL neuron apoptosis that is independent of caspase-3.