Blockade of the NMDA receptor increases developmental apoptotic elimination of granule neurons and activates caspases in the rat cerebellum

Elimination of neurons produced in excess naturally occurs during brain dev elopment through programmed cell death. Among the many survival factors aff ecting this process, a role for neurotransmitters acting on specific recept ors has been suggested. We have performed an in vivo pharmacological blocka de of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors, using the competitive NMDA receptor antagonist CGP 39551 at developmental stages corresponding to those at which a survival dependence on the stimulation o f this receptor has been demonstrated for cerebellar granule neurons explan ted in culture (typically from postnatal day 7 to postnatal day 11 or 13). We were able to demonstrate an increased level of DNA fragmentation in the cerebellum of the treated rats. At the P11 stage, in particular, the fragme nted DNA extracted from the cerebellum of CGP 39551-treated pups showed a c lear laddering of nucleosomal fragments after agarose-gel electrophoresis. Accordingly, in situ TUNEL technique showed a remarkable increase of cells positive for nucleosomal DNA fragmentation, particularly in the inner granu lar layer of the cerebellum of treated rats at P11 stage. Therefore, the na tural rate of apoptotic elimination of cerebellar granule neurons is consid erably enhanced under conditions of pharmacological blockade of the NMDA re ceptor, thus demonstrating, for the first time in vivo, a clear survival de pendence of these neurons upon the stimulation of the NMDA receptor. Concom itantly with the increased rate of apoptotic elimination of granule neurons , the activity of two death proteases of the caspase family, in particular of caspase 3 and caspase 1 at a lower extent, was remarkably increased in t he cerebellum of the treated rats. On the contrary, a marker related to the normal differentiation process of granule neurons, the enzyme ornithine de carboxylase, was strongly decreased in its activity in the cerebellum of tr eated rat pups.