Expression of GABA A/benzodiazepine receptor α1-subunit mRNA and [ 3H]flunitrazepam binding sites during postnatal development of the mouse cerebellum

Abstract

Previous studies have shown that the α subunit of the GABA A receptor contains the flunitrazepam binding site. In the present study, in situ hybridization and receptor autoradiography were used to examine the temporal and spatial relationships between α 1 subunit mRNA and [ 3H]flunitrazepam binding sites in the developing mouse cerebellum. A [ 35S]cRNA probe was used to study the expression of GABA A/benzodiazepine receptor α 1 subunit mRNA by in situ hybridization. At postnatal day (P) 1, a diffuse band of labeling was observed in the molecular/Purkinje cell layer; subsequently, this band became progressively more concentrated and restricted to the interface between the granular and molecular layers. By P5–P7, high intensity labeling was clearly associated with Purkinje cells. Clusters of grains became visible over basket and stellate cells in the molecular layer between P11 and P13; the internal granule cell layer and the deep cerebellar nuclei showed an increasingly strong hybridization signal during postnatal development. The external germinal layer was devoid of labeling throughout its existence. The developmental distribution of [ 3H]flunitrazepam binding sites was studied by receptor autoradiography. Cerebellar labeling was detectable at birth, with the highest levels present over the deep cerebellar nuclei, and relatively low levels equally distributed over the molecular and Purkinje cell layers. Cerebellar cortical grain density increased gradually during postnatal weeks 1 and 2, with the molecular, Purkinje and granule cell layers remaining essentially equally labeled. Between P11 and P15, the labeling over the molecular layer increased dramatically, reaching the high adult levels by P20. As with the in situ hybridization studies, there was a complete absence of [ 3H]flunitrazepam binding sites in the external germinal layer throughout development. These results indicate that, in the Purkinje cell, the production of mRNA and the synthesis of the α 1 subunit occur prior to the formation of afferent inhibitory synapses, suggesting that GABA A/benzodiazepine receptor expression precedes, and is independent of GABAergic synaptic input.