Layer-specific variation in intracellular GABA immunoreactivity in the neuronal organization of the mouse main olfactory bulb.

The main olfactory bulb (MOB) hosts diverse γ-aminobutyric acid (GABA)ergic interneurons that play critical roles in sensory processing. Previous studies have reported variability in GABA immunoreactivity among cells in the granule cell layer (GCL) of the mouse MOB. However, the degree of intracellular GABA content across MOB layers and its relationship with molecular subtypes remain unclear. In this study, we conducted a quantitative analysis of GABA immunoreactivity in individual cells across all MOB layers using confocal laser scanning and electron microscopy. We found striking variability in the GABA immunoreactivity intensity among individual neurons across all layers, not limited to the GCL. Electron tomography with immunogold labeling confirmed more precisely substantial differences in intracellular GABA content, particularly in the glomerular layer and GCL. Mitral cells exhibited minimal GABA immunoreactivity, consistent with their excitatory phenotype. Co-labeling of molecular markers, including tyrosine hydroxylase, calbindin, calretinin, parvalbumin, and secretagogin, revealed that variability in GABA levels is common across all interneuron subtypes. Furthermore, a comparative analysis of the depth-dependent distribution of cells within the GCL and their GABA immunoreactivity demonstrated that more superficial regions contain stronger immunoreactive cells. This likely reflects the function of interneurons that regulate the olfactory output of tufted cells, which are projection neurons located in the superficial layers. To investigate the contribution of GABA-synthesizing enzymes, we examined the expression of glutamate decarboxylase 67 (GAD67) using GAD67-GFP knock-in mice. Although GFP intensity, reflecting GAD67 promoter activity, varied among cells throughout the MOB, it did not correlate clearly with GABA immunoreactivity. This finding suggests that GAD67 expression alone does not fully explain the observed intracellular GABA variability. Our findings show the molecular and cellular heterogeneity of GABAergic neurons in the MOB. Further integrative studies are needed to elucidate the mechanisms regulating GABA content and their functional relevance.