Serotonin binding in mouse brains. Some methodological aspects

The binding of (3H)5-hydroxytryptamine ([3H]5-HT, serotonin) to crude homogenates of brains from three different strains of mice has been studied. The strains, C57/BL, DBA and BALB did not show significant differences in the binding characteristics, with Kd values around 6-7 nM and Bmax 270-310 fmoles/mg protein. Various methodological aspects were investigated and found to be important for the binding assay. The presence of ascorbic acid (5.7 mM) thus caused a significant increase in Bmax by 30% without any change in the Kd values. This increase seemed to be due to a decrease in the non-specific binding rather than to an increase in the total binding of serotonin. The presence of a specific MAO-A inhibitor, clorgyline (10 microM), during the assay, resulted in a significant reduction of Bmax by 20% without any change in the affinity for serotonin, when tissue which had been frozen was used. Less than 2% of added serotonin was metabolized during the binding procedure in the absence of clorgyline. Thus, the decrease in binding capacity caused by clorgyline, may be due to a non-competitive blocking effect of the serotonin binding structure. These results indicate that the use of MAO inhibitors in (3H)5-HT binding assays on frozen tissue, rather than being necessary, might negatively affect the result. Neither storage of the brains at -70 degrees nor postmortem storage of the animals for 60 hours at 4 degrees resulted in obvious changes in the (3H)5-HT binding characteristics.