Developmental regulation of GABA_B receptors and downstream molecules in the mouse brain.

Metabotropic GABA (GABA_B) receptors have modulatory functions on neuronal excitability and neurotransmitter release. To fulfil these functions, GABA_B receptors form macromolecular signaling complexes with G proteins, effectors, and other associated proteins. Here we investigated the postnatal development of GABA_B receptors (GABA_B1 and GABA_B2 subunits) in mouse brain, focusing on potential similarities in the spatial and temporal expression pattern of their associated proteins Ca_V2.1, Gα_o, Gβ5, and RGS7, using histoblots, immunofluorescence, and immunoelectron microscopic techniques. At all ages analyzed, histoblot showed that the six proteins were widely expressed in the brain, with mostly an overlapping pattern throughout postnatal development. In the hippocampus, immunoelectron microscopy and quantitative analysis of immunoparticles for GABA_B1, GABA_B2, Gα_o, Gβ5, and RGS7 revealed their progressive enrichment around excitatory synapses on dendritic spines of CA1 pyramidal cells toward P15. At presynaptic sites, GABA_B receptors colocalize with Ca_V2.1, Gα_o, Gβ5, and RGS7 in the active zone and extrasynaptic membranes of axon terminals, establishing synapses on dendritic spines of CA1 pyramidal cells. In the cerebellum, double immunofluorescence at P7 and P10 revealed the colocalization of GABA_B1 and Ca_V2.1 in the whole dendritic tree of developing Purkinje cells. Immunoelectron microscopy at P15 showed that GABA_B1, GABA_B2, Ca_V2.1, Gα_o, Gβ5, and RGS7 are distributed along the dendritic surface of Purkinje cells, enriched close to excitatory synapses in spines. Altogether, these data suggest that macromolecular complexes composed of GABA_B1/GABA_B2/Ca_V2.1/Gα_o/Gβ5/RGS7 are pre-assembled during key stages of postnatal development in hippocampal and cerebellar neurons.