Autoradiographic characterization of [ 3H] l-glutamate binding sites in developing mouse cerebellar cortex

Abstract

Postnatal changes of [ 3H] l-glutamate binding sites in mouse cerebellum were studied by in vitro autoradiography. These sites were already present at birth, their density globally increased until postnatal day 25, and at all ages it was higher when Cl − and Ca 2+ were present in the incubation buffer. At birth, these binding sites were diffused through the whole cerebellar mass, but became distinctly concentrated in the molecular and the internal granular layers by postnatal day 10. From this age on, binding site sensitivity to ions and glutamate analogues takes a different course in each layer. The external granular layer and the white matter never displayed significant amounts of binding. In the molecular layer the Cl −/Ca 2+ effect increased during ontogeny until, in adults, the ion-dependent binding was threefold higher than the ion-independent binding. Quisqualate-sensitive sites accounted for 80% of the total binding sites already at postnatal day 15, while displacement by α-amino-3-hydroxy-methyl-4-isoxazolepropionic and ibotenic acids attained the maximum (68%) at postnatal day 60.N-Methyl- d-aspartate displaced glutamate binding (50%) only in the presence of Cl − and Ca 2+. Starting from postnatal day 15, binding site density in the molecular layer of lobules VIb and VII of the vermis was lower than in other lobules. In the internal granular layer, the Cl −/Ca 2+ effect observed in young animals decreased during development. These transient binding sites were sensitive to quisqualic and ibotenic acid. In adults, the majority of glutamate binding sites were ion-independent and mainly sensitive to d,l-amino-5-phosphovaleric acid andN-methyl- d-aspartate. Throughout development and in both layers, sites displaced by kainate were present at low density and sites displaced by d,l-2-amino-4-phosphonobutyric acid were not detected. The localized postnatal changes of the [ 3H] l-glutamate binding sites were correlated with the events occurring during growth and maturation of cerebellar structures. The increase of the Cl −/Ca 2+-dependent binding in the molecular layer is simultaneous with the growth of Purkinje cell dendrites and of parallel fibres and with the formation of the synapses between them. This suggests that these binding sites are localized in these synapses. The changing pattern of sensitivity to different agonists during development might correspond to the maturation of these synapses. The low density of [ 3H] l-glutamate binding in the molecular layer of lobules VIb and VII probably indicates the presence of specific nerve projections to these areas. The decrease of ion-dependent and quisqualate-sensitive binding sites in the internal granular layer is concurrent with developmental modifications of the mossy fibre synapses with granule cell dendrites in the glomeruli. This is another example of developmentally regulated transient binding sites of this type, similar to those described in the globus pallidus and in the deep cerebellar nuclei. Despite the temporal correlation between neuronal maturation and the [ 3H] l-glutamate binding site accumulation in the developing cerebellum, it cannot be excluded that binding sites in glial cells also contributed to the described developmental patterns.