Differential quenching and limits of resolution in autoradiograms of brain tissue labeled with3H-,125I- and14C-compounds

Problems in interpretation of autoradiograms generated by ligand binding in brain tissue may be caused by two types of technical limitations: the differential absorption of 3H-generated emissions within the tissue (differential quenching) and the reduced resolution when 125I and 14C are used as isotopes. In the course of our ongoing receptor binding studies in primate brain, we have examined these methodological problems using neocortex of adult rhesus monkey as an example of a complex multilayered brain structure. We have compared: (1) film images produced by brain sections mounted on 3H- and 14C-labeled plastic; (2) autoradiograms of sections labeled with pairs of similar compounds containing 3H, 125I or 14C; and (3) autoradiograms of normal and defatted brain sections. The results indicate that differential absorption of 3H-generated emissions presents a genuine problem for film autoradiography of neocortex of adult monkey when 3H-compounds are used. Particularly significant attenuations of 3H-generated emissions are associated with sublayer IVb of primary visual cortex (Brodmann's are 17) and layers III (deep strata), V and VI of primary motor cortex (Brodmann's area 4). This study provides the necessary corrections for autoradiographic measurements. We also found a loss of resolution associated with use of 125I and 14C, a result that poses a significant problem for analysis of fine laminar patterns of the neocortex in adult monkeys. The use of isotopes with high energy emissions tends to decrease the variations in optical densities within the autoradiograms of cortical sections. Thus, the variations in optical density of autoradiograms of cortices labeled with 125I- and 14C-compounds may not represent the true distribution of these compounds.