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

Abstract As γ-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 gyrus. 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.