New high-resolution 2-deoxyglucose method featuring double labeling and automated data collection

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Abstract
A new approach to high-resolution 2-deoxy-D-glucose (2DG) emulsion-autoradiography which combines improved retention of 2DG labeling, staining with immunohistochemical and other specific markers, and automated data collection and analysis of local silver grain and stain densities is described. The Durham et al. (J. Neurosci. 1:519–526, '81) procedure for fixation of 2DG with periodate-lysine-paraformaldehyde (PLP, McLean and Nakane: J. Histochem. Cytochem. 22:1077–1083, '74) was adapted to increase retained label roughly tenfold. Phenobarbital anesthesia is induced 45 minutes after 2DG injection. Barbiturate anesthesia increases brain glycogen (Nelson et al.: J. Neurochem. 15:1271–1279, '68) and presumably increases the incorporation of intracellular 2DG from 2DG-6P into brain glycogen and other molecules (Nelson et al.: J. Neurochem. 43:949–956, '84; Pentreath et al.: Neuroscience 7:759–767, '82). Iodoacetate is added to cold fixative to prevent glycogen breakdown (Cammermeyer and Fenton: Histochemistry 76:339–356, '82). This high-resolution 2DG protocol is directly compatible with many other neuroanatomical techniques. We demonstrate 2DG emulsion autoradiography combined with cytochrome oxidase (CO) histochemistry, markers for axonal pathway tracing, plastic embedding for semithin sections, and immunohistochemical staining for glutamate decarboxylase (GAD). The method should be compatible with antibodies for other antigens and with other neuroanatomical stains. To collect the data directly from microscope slides, a computer-controlled microscope was integrated with image-processing software to eliminate the need for manual counting and scoring of autoradiograms. Regions of interest are scanned automatically at high resolution to map regional labeling and/or stain density. There is excellent correspondence between computer-enhanced two-dimensional maps of the data and the original autoradiograms. Automated counts for five specimens were compared to counts of labeled cells by trained observer. The correlation between the two sets of measurements is high (r =. 93). Automated data collection has been generalized to measure regional stain densities on the autoradiographed sections for direct comparison with silver grain density. The method is extremely flexible, especially since new image-processing strategies can be developed in software to extract the desired information from materials labeled by other methods (e.g., HRP). The combination of experimental and data collection strategies generates two- or three-dimensional “maps” of 2DG labeling, histochemical stain, etc., over a brain area of interest and allows direct comparison of these different maps. To our knowledge this is the first report of quantitative reconstructions of 2DG labeling patterns on the basis of large and systematically scanned data samples from emulsion autoradiograms.