GABAergic and Glutamatergic Phenotypes of Neurons Expressing Calcium-Binding Proteins in the Preoptic Area of the Guinea Pig

Abstract

The mammalian preoptic area (POA) has large populations of calbindin (CB), calretinin (CR) and parvalbumin (PV) neurons, but phenotypes of these cells are unknown. Therefore, the question is whether neurons expressing CB, CR, and/or PV are GABAergic or glutamatergic. Double-immunofluorescence staining followed by epifluorescence and confocal microscopy was used to determine the coexpression patterns of CB, CR and PV expressing neurons with vesicular GABA transporters (VGAT) as specific markers of GABAergic neurons and vesicular glutamate transporters (VGLUT 2) as specific markers of glutamatergic neurons. The guinea pig was adopted as, like humans, it has a reproductive cycle with a true luteal phase and a long gestation period. The results demonstrated that in the guinea pig POA of both sexes, ~80% of CB+ and ~90% of CR+ neurons coexpress VGAT; however, one-fifth of CB+ neurons and one-third of CR+ cells coexpress VGLUT. About two-thirds of PV+ neurons express VGAT, and similar proportion of them coexpress VGLUT. Thus, many CB+, CR+ and PV+ neurons may be exclusively GABAergic (VGAT-expressing cells) or glutamatergic (VGLUT-expressing cells); however, at least a small fraction of CR+ cells and at least one-third of PV+ cells are likely neurons with a dual GABA/glutamate phenotype that may coexpress both transporters.

The Co-Expression Pattern of Calcium-Binding Proteins with γ-Aminobutyric Acid and Glutamate Transporters in the Amygdala of the Guinea Pig: Evidence for Glutamatergic Subpopulations

“…Similar excitatory cortical PV neurons were also reported in the mouse forebrain [59]. The cells co-expressing PV and VGLUT2 were also observed in the preoptic area [50], which has many embryological relationships with the "striatum-pallidum-like" amygdala.…”

“…Interestingly, neurons containing CB and VGLUT3 were identified in the cerebral cortex and hippocampus [79]. The next issue is that in the brain, there are neurons that co-express both VGAT and VGLUT2 [50,80] and may co-release both GABA and glutamate at the same synapses [81]. The striatal efferent may contain both glutamate and GABA [82], and the hippocampal axon terminals contain both VGLUT2 synaptic vesicles and vesicles that carry VGAT [83].…”

“…The mouse antibodies directed to calcium-binding proteins (anticalbindin; 300, anti-parvalbumin; P3088, and anti-calretinin; 6B 3 ) were positively evaluated by Western blotting of the guinea pig brain homogenates and immunohistochemistry in brain sections from knock-out mice proving their specificity to their targets [96,97]. The rabbit antibodies against VGAT (AB5062P) and VGLUT2 (135 402) were frequently applied in different studies [33,50,[98][99][100][101][102], and they were also positively verified by Western blotting of the mice brain extracts and immunohistochemistry in brain sections from knock-out mice [103,104]. The specificity of secondary antibodies was controlled by the omission and/or replacement of all primary antisera by non-immune sera or PBS.…”

“…The first definitive marker of glutamatergic neurons was vesicular glutamate transporters [41,42]. Furthermore, the specificity of VGLUT2 in the detection of glutamatergic somata has been established via previous research employing qualified methods [41,[45][46][47][48][49][50].…”