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

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 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°C was injected into the HPLC-EC at regular intervals for a 60-min period after initial preparation. In addition, similar concentration levels of DA (8.03 ± 0.72 vs. 8.51 ± 0.62 ng/mg) were detected in male rat corpus striatum (CS) fragments extracted with either perfusion medium or with 0.1 VHCIO4; however, higher levels of DOPAC were found using perfusion medium than 0.1 N NClO₄ (1.85 ± 0.16 vs. 1.15 ± 0.15 ng/mg). To validate biologically the preparation, AMPH was infused either locally into the CN or administered systemically. Infusion of this psychostimulant directly into the CN of a diestrous rat evoked a clear dose-response in DA output (from a basal level of 10–15 pg/min to a maximum of 150 pg/min) without modifying HVA or 5-HIAA output. However, an apparent decrease in DOPAC output immediately following the 10^–6 M dose of AMPH was noticed. When two consecutive doses of AMPH (10^–3 M) were infused into the CN of a diestrous rat at 2-hour intervals, similar increases in DA output were recorded. The basal and AMPH-stimulated output of DA from the CN of proestrous and diestrous rats were also estimated by measuring the concentration of this amine in perfusate samples with an already validated radioenzymatic assay (REA). Comparable data was obtained (5–15 pg/min, DA basal output), corroborating the authenticity of DA determinations by HPLC-EC. In addition, when AMPH was administered intraperitoneally the expected changes in behavior and DA output were also observed in a fully awake diestrous rat. Therefore, the present data based on chromatographic and biological validations indicate that the PPP on-line with HPLC-EC can be used advantageously to examine the effect of drugs and hormones in the in vivo output of several neuroactive substances from the CN of conscious, freely moving rats. Moreover, it will be possible to correlate behavioral events with in vivo output of biological amines.