Immunofluorescently labeling glutamic acid decarboxylase 65 coupled with confocal imaging for identifying GABAergic somata in the rat dentate gyrus-A comparison with labeling glutamic acid decarboxylase 67

As gamma-aminobutyric acid (GABA) is synthesized by two isoforms of glutamic acid decarboxylase (GAD), namely, GAD(65) and GAD(67), immunohistochemically targeting either isoform of GAD is theoretically useful for identifying GABAergic cell bodies. In practice, targeting GAD(67) remains to be a popular choice. However, identifying GABAergic cell bodies with GAD(67) immunoreactivity in the hippocampal dentate gyrus, especially in the hilus, is not without pitfalls. In the present study, we compared the characteristics of GAD(65) immunoreactivity to GAD(67) immunoreactivity in the rat dentate gyrus and examined perikaryal expression of GAD(65) in four neurochemically prevalent subgroups of interneurons in the hilus. Experiments were done in normal adult Sprague-Dawley rats and GAD(67)-GFP knock-in mice. Horizontal hippocampal slices cut from the ventral portion of hippocampi were immunofluorescently stained and scanned using a confocal microscope. Immunoreactivity for both GAD(67) and GAD(65) was visible throughout the dentate gyms. Perikaryal GAD(67) immunoreactivity was denser but variable in terms of distribution pattern and intensity among cells whereas perikaryal GAD(65) immunoreactivity displayed similar distribution pattern and staining intensity. Among different layers of the dentate gyrus, GAD(67) immunoreactivity was densest in the hilus despite GAD(65) immunoreactivity being more intense in the granule cell layer. Co-localization experiments showed that GAD(65), but not GAD(67), was expressed in all hilar calretinin (CR)-, neuronal nitric oxide synthase (nNOS)-, parvalbumin (PV)- or somatostatin (SOM)-positive somata. Labeling CR, nNOS, PV, and SOM in sections obtained from GAD(67)-GFP knock-in mice revealed that a large portion of SOM-positive cells had weak GFP expression. In addition, double labeling of GAD(65)/GABA and GAD(67)/GABA showed that nearly all of GABA-immunoreactive cells had perikaryal GAD(65) expression whereas more than one-tenth of GABA-immunoreactive cells lacked perikaryal GAD(67) immunoreactivity. Inhibition of axonal transport with colchicine dramatically improved perikaryal GAD(65) immunoreactivity in GABAergic cells without significant augmentation to be seen in granule cells. Double labeling GAD(65) and GAD(67) in the sections obtained from colchicine-pretreated animals confirmed that a portion of GAD(65)-immunoreactive cells had weak or even no GAD(67) immunoreactivity. We conclude that for confocal imaging, immunofluorescently labeling GAD(65) for identifying GABAergic somata in the hilus of the dentate gyrus has advantages over labeling GAD(67) in terms of easier recognition of perikaryal labeling and more consistent expression in GABAergic somata. Inhibition of axonal transport with colchicine further improves perikaryal GAD(65) labeling, making GABAergic cells more distinguishable. (C) 2014 Elsevier B.V. All rights reserved.