A microassay for measuring synaptosomal ³H-dopamine and ³H-metabolite release

A modified synaptosomal superfusion apparatus is described which uses less than 10 micrograms of tissue per replicate sample and facilitates the routine separation of ³H-DA, ³H-DOPAC, and ³H-HVA. A flow rate of 1.5 ml/min allows superfusion without the use of reuptake or monoamine oxidase inhibitors. Superfusate samples are collected directly onto alumina columns for the separation of ³H-DA and its acid metabolites. Total recovery of authentic ³H-DA applied via superfusion was 87.63(1.10) percent [Mean(SEM)]. Contamination of the acetic acid eluate fraction, containing 80.98(1.15)% of the total DA, by DOPAC and HVA was less than 0.1%. To illustrate the utility of this technique, the relative proportions of ³H-DA and ³H-metabolites released from synaptosomes by 6 mM potassium and 1 μM reserpine were compared.