A microassay for measuring synaptosomal super(3)H-dopamine and super(3)H-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 tritiated dopamine ( super(3)H-DA), super(3)H-dihydroxypheny-acetic acid ( super(3)H-DOPAC), and super(3)H-homovanillic acid ( super(3)H-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 super(3)H-DA and its acid metabolites. Total recovery of authentic super(3)H-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 super(3)H-DA and super(3)H-metabolites released from synaptosomes by 6 mM potassium potassium and 1 mu M reserpine were compared.