Proliferation and migration of granule cells in the developing rat cerebellum: cisplatin effects.

We evaluated the relationship among proliferation, death and migration of granule cells in lobules VI-VIII of vermis, in comparison with lobule III, during cerebellar development. To this aim, a single injection of cisplatin, i.e., a cytostatic agent that is known to induce death of proliferating granule cells, was given to 10-day-old rats. Histochemical markers of proliferating (PCNA immunoreaction) and apoptotic (TUNEL staining) cells were used; the variations of the external granular layer (EGL) thickness were evaluated in parallel. After PCNA and TUNEL reactions, evident changes of the whole EGL were found on PD11 (1 day after treatment), when a reduction of the thickness of this layer was found in treated rats, mainly in consequence of the high number of apoptotic cells in all the cerebellar lobules. On PD17 (7 days after treatment), a thick layer of proliferating cells was observed in lobules VI-VIII of treated rats, while the peculiar pattern of the normal development showed a thin EGL. At the same time, in treated rats, the number of apoptotic cells in EGL was low. In all developmental stages of treated rats, after GFAP immunoreaction, glial fibers appeared twisted, thickened, and with an irregular course; intensely labeled end-feet were present. The damage of radial glia suggests an alteration of migratory processes of granule cells, which is also evidenced by the decreased thickness of the premigratory zone of the EGL. Injured radial glia fibers were restricted to lobules VI-VIII and they persisted at PD30, leading to the presence of ectopic granule cells in the molecular layer, as we previously described.

Signal molecules and receptors in the differential development of cerebellum lobules. Acute effects of cisplatin on nitric oxide and glutamate systems in Purkinje cell population.

Three functionally correlated parameters, nitric oxide (NO), glutamate and NMDA receptors were analyzed through enzymehistochemical and immunohistochemical reactions. A single injection of cisplatin (cisPt) was administered to 10-day-old rats in order to study how Purkinje cells differentiation may be early changed by a mild injury due to the drug during postnatal cerebellar histogenesis. In comparison with age-matched control rats, a correlated decreasing expression of nitric oxide synthase (NOS), glutamate and NMDAR1 was observed in the Purkinje cells of lobules VI-VIII 6 h after the treatment. Moreover, at 24 h after cisPt, the expression of glutamate, NMDAR1 and nicotinamide adenine dinucleotide phosphate-diaphorase (NADPHd) reactivity was further decreased. In the same period, the ionotropic receptor GluR2 evidenced a less developed dendrite of Purkinje neurons in the top of lobules. In addition, the metabotropic receptor mGluR1alpha revealed unstained areas in the molecular layer, which was entirely stained in control rats; on PD11 this altered pattern was observed in all the lobules and in both the outer and the inner parts. Findings show the importance of NO-glutamate interactions via NMDAR1 in the crucial phases of Purkinje cells differentiation and their involvement on Purkinje neurons dendrite branching as demonstrated by the patterns of the other glutamate receptors. Changes were discussed in relation to an important critical event of Purkinje cell differentiation, i.e. regression of perisomatic spines and elimination of climbing fiber synapses on the somata. Finally, lobules VI-VIII appear to be the most vulnerable ones when cisplatin treatment is administered at 10 days of life, which demonstrates that at this stage some critical developmental changes occur in these lobules and that slower/damaged dendritic tree development is different in the outer versus the inner regions of the lobules.