Computer-assisted video analysis of [ 3H]spiroperidol binding autoradiographs

Abstract

A computer-based video image processing system is described that quantifies the binding of the neuroleptic drug, [ 3H]spiroperidol, to rat forebrain sections. Adjacent sections were incubated in buffer containing [ 3H]spiroperidol or [ 3H]spiroperidol plus 1 μM (+)-butaclamol and exposed to tritium-sensitive film to produce autoradiographs of total binding or non-butaclamol-displaced (‘non-specific’) binding. The image processing system digitized each autoradiograph, attenuated geometric distortion and unevenness of background illumination, and reassigned the digitized image intensities (gray values) to be a linear function of fmol [ 3H]spiroperidol bound per mg protein by using a calibration curve generated from 3H-containing tissue standards. An image of butaclamol-displaced (‘specific’) [ 3H]spiroperidol binding was produced by subtracting the linearized image of non-specific binding from the superimposed image of total binding. An image of percent specific [ 3H]spiroperidol binding was obtained by dividing the image of specific binding by the superimposed image of total binding. The computer-derived images, which could be displayed in gray tones or pseudocolor-coded, revealed that the greatest amounts of specific [ 3H]spiroperidol binding (1000–3000 fmol/mg protein: 60–80% specifically bound) were located in layers 5A and 5C of the neocortex, the claustrum, and in the caudate-putamen, where a lateral-to-medial binding gradient occurred. [ 3H]Spiroperidol was bound to a lesser extent (400–1000 fmol/mg protein; 31–51% specifically bound) to the medial nucleus accumbens septi or olfactory tubercle and without measurable specificity to the lateral septum, anterior commissure, corpus callosum, or superficial neocortex. These procedures are particularly useful for the quantitative and visual analysis of autoradiographs in which [ 3H]ligand binding is associated with more than a single site.