Metabolic Activity in Antigenically Identified Neurons: a Double Labeling Method for High-resolution 2-deoxyglucose and Immunohistochemistry

The 2-deoxyglucose (2DG) histochemical method of Sokoloff et al. ((1977) J. Neurochem., 28: 897-916) has been widely used to map metabolic activity in the brain, but lacks sufficient resolution to view individual neurons. Modifications of the original method have achieved cellular resolution with emulsion autoradiography and tissue apposed directly to the autoradiogram, but the ability to discriminate clearly between populations of neurons identified by antigenic markers has been lacking. A method is presented which makes it possible, for the first time, to study the metabolic activity of every immunohistochemically labeled neuron in a neural system of interest. The procedure combines a high-resolution 2-deoxyglucose technique and immunostaining for glutamate decarboxylase (GAD) or other antigens. The method stabilizes label during immunohistochemical processing by the addition of glycogen to all solutions that come into contact with tissue after sectioning. The postulated effect of the added glycogen is to limit diffusion or net efflux of unfixed macromolecules which contain 2-deoxyglucose label. When used with a GAD antibody to label putative GABAergic inhibitory interneurons, the procedure allows a direct assessment of the extent of inhibition in the circuitry of cerebral cortex. A detailed protocol for this double-labeling procedure is presented, along with examples of the labeling patterns obtained in the whisker-related barrel field of rodent somatosensory cortex.