Abstract

The receptor occupancy required to produce cannabinoid effects in the central nervous system was determined in both a neurochemical and a behavioral assay for cannabinoid actions. In the neurochemical experiments, performed on superfused rat hippocampal slices, electrically evoked [³H]acetylcholine release was inhibited by the cannabinoid agonist, WIN 55212 to 2 with an EC₅₀ of 0.005 μM and maximum effect of 79%. In parallel experiments examining binding of the radiolabeled CB1 antagonist [¹³¹I]AM 281 {N-(morpholin-4-yl)-5-(4-[¹³¹I]iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide} to living hippocampal slices, WIN 55212 to 2 inhibited [¹³¹I]AM 281 binding with an EC₅₀ of 1.3 μM. From these two sets of data it was determined that 50% of maximal inhibition of [³H]acetylcholine release in hippocampal slices occurs at a receptor occupancy of only 0.13% and 95% of maximal inhibition at a receptor occupancy of 7.5%, suggesting the presence of a receptor reserve that is large compared with other G protein-coupled receptor systems in the central nervous system. In behavioral experiments, WIN 55212 to 2 inhibited spontaneous locomotor activity in mice with an ED₅₀ of 0.3 mg/kg, i.v.. In in vivo binding experiments using [¹³¹I]AM 281, WIN 55212 to 2 failed to produce significant inhibition of radiotracer binding in the mouse brains, except at very high doses (10 mg/kg or greater, i.v.). By contrast, the CB1 antagonist SR 141716A (10 mg/kg, i.p.), completely abolished specific [¹³¹I]AM 281 binding. These experiments suggest that behavioral effects of cannabinoids, like neurochemical effects, are produced at very low receptor occupancy.