Trans-striatal dialysis coupled to reverse phase high performance liquid chromatography with electrochemical detection: a new method for the study of the in vivo release of endogenous dopamine and metabolites
A method for the estimation in rats of the in vivo release and metabolism of dopamine (DA) is described. The method is based on the dialysis principle and consists of inserting transversally in the striatum a thin (0.2 mm) dialysis tube (Amicon Vitafiber) which is then perfused with Ringer. The Ringer, flowing at a constant rate of 2 microliters/min in the dialysis tube, extracts low molecular weight substances from the surrounding tissue by way of simple diffusion along a concentration gradient. At the distal end of the dialysis tube, the Ringer is collected every 10 to 20 min and directly injected into a high performance liquid chromatographer (HPLC) equipped with reverse phase octadecyl sulfate columns which separate DA and its metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). These substances are then quantitatively estimated by oxidative electrochemical detection. The basal output of DA is 0.3 pmol/20 min, whereas the outputs of DOPAC and HVA are 60 and 20 pmol/20 min, respectively. In basal conditions the output of DA, DOPAC, and HVA is stable over at least 10 hr. Histological examination of the track left by the dialysis probe in rats after 10 hr of continuous dialysis reveals very little damage and normal neuronal morphology in the vicinity of the dialysis tube. Increase of the K+ concentration in the Ringer to 30 mM produced a sharp, reversible increase of DA output. Both the basal and K+-stimulated release were Ca++ dependent, because omission of Ca++ abolished basal and K+-stimulated DA release. Electrical stimulation of the nigrostriatal DA neurons in the medial forebrain bundle sharply increased DA output. Amphetamine sulfate in low doses (1.0 mg/kg, i.v.) produced a 9-fold increase in DA release and decreased DOPAC and HVA output. alpha-Methyl tyrosine (150 mg/kg, i.v.) reduced within 2 hr DA release to 15% of basal values and in parallel also decreased the output of DOPAC and HVA. Reserpine (5 mg/kg, i.p.) reduced DA release but in a slower fashion than alpha- methyl tyrosine and increased DOPAC and HVA. Pargyline (75 mg/kg, i.p.) produced a 4-fold increase of DA release, while it rapidly brought to zero DOPAC and HVA output. gamma-Butyrolactone (700 mg/kg, i.p.) rapidly and lastingly reduced DA, DOPAC, and HVA output. The biochemical and histological results obtained indicate that the method is suitable to estimate in the rat the changes in the release o f endogenous DA and its metabolites which take place in vivo under administration of centrally acting drug.