High-resolution 2-deoxyglucose autoradiography in quick-frozen slabs of neonatal rat olfactory bulb

We have used rapid freezing and freeze-substitution fixation to permit electron microscopic study of [3H]2-deoxyglucose autoradiographs. The techniques minimize diffusion of label into processing fluids and, by inference, migration of label within tissue. Slabs of olfactory bulbs from 12-day-old rats were quick-frozen after one hour of exposure to physiological olfactory stimuli. In light microscopic autoradiographs at low magnification, the neuropil of individual olfactory glomeruli appeared uniformly labeled with different levels of labeling in different glomeruli. At higher magnification, glomerular neuropil labeling consisted of small unlabeled regions surrounded by label clusters, suggesting greater deoxyglucose uptake by olfactory nerve terminals as compared with their postsynaptic dendrites. Periglomerular neurons were labeled differentially. Some microglia and glia precursor cells were heavily labeled in all bulbar laminae. The ultrastructure of cells and neuropil in all bulbar laminae was well-preserved. Cell processes and organelles could be identified in both stained sections and unstained electron microscopic autoradiographs. These experiments demonstrate the feasibility of combining quick-freezing with freeze substitution, in order to extend the resolution of studies using diffusable tracers such as 2-deoxyglucose. The results suggest that this is a promising method for assessing several controversies concerning deoxyglucose incorporation and neuronal and glial metabolism.