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Periglomerular (PG) cells in the rodent olfactory bulb are heterogeneous anatomically and neurochemically. Here we investigated whether major classes of PG cells use γ-aminobutyric acid (GABA) as a neurotransmitter. In addition to three known subtypes of PG cells expressing tyrosine hydroxylase (TH), calbindin D-28k (CB), and calretinin (CR), we identified a novel PG cell population containing the GABAA receptor α5 subunit. Consistent with previous studies in the rat, we found that TH-positive cells were also labeled with antibodies against GABA, whereas PG cells expressing CB or the α5 subunit were GABA-negative. Using GAD67-GFP knockin mice, we found that all PG cell subtypes expressed GAD67-GFP. Calretinin labeled the major fraction (44%) of green fluorescent protein (GFP)-positive cells, followed by TH (16%), CB (14%), and the α5 subunit (13%). There was no overlap between these neuronal populations, which accounted for ≈85% of GAD67-GFP-positive cells. We then demonstrated that PG cells labeled for TH, CB, or CR established dendrodendritic synapses expressing glutamic acid decarboxylase (GAD) or the vesicular inhibitory amino acid transporter, VGAT, irrespective of their immunoreactivity for GABA. In addition, CB-, CR-, and TH-positive dendrites were apposed to GABAA receptor clusters containing the α1 or α3 subunits, which are found in mitral and tufted cells, and the α2 subunit, which is expressed by PG cells. Together, these findings indicate that all major subtypes of PG cells are GABAergic. In addition, they show that PG cells provide GABAergic input to the dendrites of principal neurons and are interconnected with other GABAergic interneurons, which most likely are other PG cells. J. Comp. Neurol. 502:990–1002, 2007. © 2007 Wiley-Liss, Inc.

Clustering of gamma aminobutyric acid (GABA)A receptors to postsynaptic sites requires the presence of both the γ2 subunit and gephyrin. Here, we analyzed by double-immunofluorescence staining the colocalization of gephyrin and major GABAA-receptor subtypes distinguished by the subunits α1, α2, α3, or γ2 in adult rat brain. By using confocal laser scanning microscopy, GABAA-receptor subunit staining revealed brightly stained clusters that were colocalized with gephyrin-positive clusters of similar size and distribution in several brain regions, including cerebellum, hippocampus, thalamus, and olfactory bulb. In addition, a diffuse staining was observed for GABAA-receptor subunits in the neuropil, presumably representing extrasynaptic receptors. Overall, only few gephyrin-positive clusters were not colocalized with GABAA-receptor subunit clusters. Electron microscopic analysis in cerebellar cortex confirmed the selective postsynaptic localization of gephyrin. High-resolution images (voxel size, 50 × 50 × 150 nm) were restored with an iterative image deconvolution procedure based on a measured point-spread function to analyze the colocalization between GABAA-receptor subunits and gephyrin in individual clusters. This analysis revealed a considerable heterogeneity in the micro-organization of these presumptive GABAergic postsynaptic sites. For instance, whereas gephyrin- and γ2 subunit-positive clusters largely overlapped in the cerebellar molecular layer, the colocalization was only partial in glomeruli of the granule cell layer, where small gephyrin clusters typically were “embedded” in larger GABAA-receptor clusters. These findings show that gephyrin is associated with a majority of GABAA-receptor subtypes in brain, and document the usefulness of image deconvolution for analyzing the structural organization of the postsynaptic apparatus by fluorescence microscopy. J. Comp. Neurol. 420:481–498, 2000. © 2000 Wiley-Liss, Inc.

The synaptic organization of the accessory olfactory bulb (AOB) was studied in the rat with antibodies against the excitatory neurotransmitter glutamate (Glu) and the inhibitory neurotransmitter γ-aminobutyric acid (GABA). To a large extent, the immunoreactivity patterns produced by the two antibodies were complementary. Glu-like immunoreactivity (-LI) was observed in the glomerular neuropil, in the mitral cells, and in large neurons located in the periglomerular region. Immunogold electron microscopy revealed particularly high levels of Glu-LI in the axon terminals of vomeronasal neurons. GABA-LI was present in granule and periglomerular cells and in their processes. The dendritic spines of granule cells, which were presynaptic to mitral cells, were strongly labelled by the antiserum against GABA. Labelling of serial semithin sections showed that the GABA-positive and Glu-positive neurons of the periglomerular region are generally distinct, and colocalization of Glu and GABA occurred only in a few cells. These results are consistent with electrophysiological studies indicating that the synaptic organization of the AOB is similar to that of the main olfactory bulb. In both systems, Glu is the neurotransmitter used by primary afferents and output neurons, whereas GABA is involved in the circuits underlying lateral and feed-back inhibition. J. Comp. Neurol. 408:61–72, 1999. © 1999 Wiley-Liss, Inc.