In vivo output of dopamine and metabolites from the rat caudate nucleus as estimated with push-pull perfusion on-line with HPLC-EC in unrestrained, conscious rats. I. Chromatographic and biological validation.

Abstract

In the present experiment we used push-pull perfusion (PPP) on-line with high performance liquid chromatography with electrochemical detection (HPLC-EC) to measure the concentration of neuroactive substances collected in perfusates from the caudate nucleus (CN) of conscious, freely moving rats. To validate the suitability of such an approach, both chromatographic and biological procedures were used. The chromatographic performances of four pure standards, dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindole acetic acid (5-HIAA) were examined under the conditions of the experiment and the release of these four chemicals by amphetamine (AMPH) locally infused into the CN or systemically administered to conscious rats used as an index of biological validation. Distinct dose-response curves were obtained for each standard injected into the HPLC-EC singly or mixed together in perfusion medium (modified Krebs-Ringer's Phosphate, KRP , pH 7.4). Moreover, each standard in the chromatogram appeared as a well-defined elution band with a different retention time. The brain perfusate samples did not contain factors interfering with the normal operation of the HPLC-EC or measurement of the concentration of added standards. The recovery of pure DA, DOPAC, 5-HIAA and HVA added to the perfusate samples was 97, 87, 98 and 114%, respectively. No decrements in peak heights were observed in the chromatograms when a 1-ng dose mixture of the four standards dissolved in medium and maintained at 4 degrees C was injected into the HPLC-EC at regular intervals for a 60-min period after initial preparation.(ABSTRACT TRUNCATED AT 250 WORDS)