Quantitative receptor autoradiography using [3H]ultrofilm: application to multiple benzodiazepine receptors

Radiohistochemical methods utilized to label drug and neurotransmitter receptors in vitro at the light microscopic level are quantitative. In this report, the response of tritium-sensitive sheet film ([3H]Ultrofilm) was characterized under conditions used for the light microscopic localization of neurotransmitter receptors by in vitro autoradiographic techniques. Radioactive standards containing varying concentrations of tritium were prepared from brain tissue and exposed to [3H]Ultrofilm for varying lengths of time, and the response of the film was measured by microdensitometry. A 1n optical density versus 1n radioactivity plot provided a useful linear relationship. Relationships established between radioactivity concentrations, exposure times and optical density were utilized to establish appropriate exposure conditions for several [3H]ligands in brain sections. The accuracy and pharmacological relevance of these methods were tested by studying the regional distribution of multiple benzodiazepine (BZ) receptors, and by analyzing the inhibitory potency of the triazolopyridazine, CL218,872, and methyl-beta-carbolinecarboxylate, two agents which discriminate between type 1 and 2 BZ receptors. The results obtained compared favorably with results previously obtained in tissue homogenates by biochemical methods. Overall, these results have practical implications. For example, quantitative autoradiography can be used to generate accurate kinetic and pharmacological displacement curves for small tissue areas. Also, since the film response curve is not linear with exposure (radioactivity X time), autoradiographs of the same sections can have relatively different density patterns depending on the time of exposure.