Radioautographic method for quantifying regional monoamine innervations in the rat brain. Application to the cerebral cortex

Abstract

Conditions leading to selective and complete labeling of the noradrenaline (NA) and serotonin (5-HT) innervations in rat cerebral cortex were sought by incubating 200-μm-thick whole hemisphere slices with various combinations of tritiated monoamines and uptake blockers at different concentrations in the presence of a monoamine oxidase inhibitor. After fixation with glutaraldehyde, post-fixation with osmium tetroxide and flat-embedding in Epon, 4-μm-thick sections of the entire slices were radioautographed by dipping in nuclear emulsion. As previously reported, dopamine (DA) terminals could be specifically visualized and counted following incubation with 1 μm [ 3H]DA and 5 μM desipramine (DMI) with or without 5 μM citalopram (CITAL). The number of NA terminals could thus be obtained by subtracting DA varicosities from the total number of sites labeled in adjacent slices incubated without DMI but in presence of CITAL to eliminate some interspecific labeling of 5-HT terminals. NA terminals could also be identified exclusively and counted after labeling with 1 μM [ 3H]NA in the presence of 10 μM benztropine. 5-HT terminals were specifically detected after incubation with 1 μM [ 3H]5-HT in the presence of 10 μM non-radioactive NA. The labeled varicosities were counted in areas FR1 and PAR1 of the frontal and the parietal neocortex, respectively, with the aid of a microcomputer-based image analysis system. DA varicosities were concentrated mainly in layer VI of these regions and were more numerous in the frontal than the parietal area. NA terminals were equally distributed in the two regions but approximately twice as numerous in layer I than subjacent layers. The 5-HT innervation also showed a comparable overall density in the two cortical regions but with a differing intracortical distribution. In the frontal area. 5-HT terminals were slightly more concentrated in layer 1 (1.3-fold) than underlying layers where they were rather uniformly distributed. In the parietal area, layer I was again the most densely innervated (1.8 times the average), but a second zone of higher density (1.5 times average) was present in the outer part of layer V. The remaining layers showed lower numbers of 5-HT terminals than in the frontal region. To obtain absolute estimates of these innervation densities, the number of detected varicosities was assessed experimentally as a function of radioautographic exposure time and of histological section thickness, and their ‘equivalent circle diameter’ was measured in electron microscope radioautographs. After appropriate corrections for incomplete detection at the exposure times selected for counting and for the thickness of sections used, stereological extrapolation of the results yielded figures of 0.7 and 0.3 × 10 6 DA varicosities per mm 3 in layer VI of the frontal and the parietal cortex, respectively. Averages of 0.9 × 10 6 NA and 4.4 × 10 6 5-HT varicosities per mm 3 of cortex were obtained for both regions. Correlation of these estimates with currently available data on the total number of cortical nerve cell bodies and axon terminals, micromeasurements of regional monoamine contents, and B max values for various monoamine uptake blockers in the cortex exemplify the advantages of obtaining absolute values for the direct comparison of the different monoamine innervations with one another or with other quantifiable parameters relevant to the understanding of their function.