• clustering;
  • cortex;
  • development;
  • hippocampus;
  • GABAA receptors;
  • synaptogenesis


We compared the expression and co-expression of α1, α2, α3, and α5-subunit protein clusters of the γ-aminobutyric acid (GABA)A receptor in the neocortex and hippocampus of rat at postnatal days (PND) 5–10 and 30–40 in order to understand how inhibitory receptors reorganize during brain maturation. The size, intensity, density and pattern of co-localization of fluorescently tagged subunit clusters were determined in deconvolved digital images using a novel 2D cross-correlational analysis. The cross-correlation analysis allowed an unbiased identification of GABAA receptor subunit clusters based on staining intensity. Cluster size increased through development; only the α2 clusters in dentate gyrus (DG) decreased in size. α5-subunit cluster density either increased or decreased with maturation depending on the brain region. For the other subunits, the cluster density remained rather constant, with noted exceptions (increase in α2 clusters in cortical layer 5 but a decrease of α3 clusters in hilus). The co-localization of α1-subunit with the others was unique and not correlated to overall changes in subunit abundance between developmental époques. So, although α2-subunit expression went up in the DG, the clusters became less co-localized with α1. In contrast, α5-subunit clusters became more co-localized with α1 as the α5-subunit expression declined in cortex and CA1. The co-localization of α3 with α1 also became greater in layer 6. In the adult brain not all clustering was associated with synapses, as many α-subunit clusters did not co-localize with synaptophysin. Overall, these data indicate that the regulation of GABAA receptor clustering is both synaptic and extrasynaptic, presumably reflecting complex cellular trafficking mechanisms.