In vivo determination of endogenous biogenic amines in rat brain using HPLC and push-pull cannula

Combining the methods of push-pull cannulation with those of high performance liquid chromatography (HPLC), we have measured the content of a number of biogenic amines in the perfusate of freely moving rats. In an initial study, the lateral hypothalamus (LH) was chronically implanted with a push-pull cannula and was perfused with 0.9% NaCl. Fifteen minute samples were collected through the push-pull cannula (flow rate: 25 μl/min) and aliquots of 200 μl were injected into the HPLC without any extraction or prepurification procedure. Simultaneous determination of the levels of 5-hydroxytryptophan (5-HTP), 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), norepinephrine (NE) and dopamine (DA) in the perfusate was accomplished by means of HPLC with electrochemical detection. The HPLC system utilized a C-18 reverse phase column coupled with a glassy carbon detector. Results indicate that this combination of push-pull perfusions and HPLC assay can provide a simple, rapid, and sensitive technique for the in vivo simultaneous determination of the compounds released in discrete brain areas. In preliminary studies in which these methods were used, 50 mg/kg D,L-5-HTP was injected subcutaneously (SC) into rats implanted with push-pull cannulae and working on a variable interval (VI 1) schedule of reinforcement. Increases in 5-HTP, 5-HT and 5-HIAA were measured during the period of behavioral depression following 5-HTP administration. This technique could provide a useful tool in the assessment of neurochemical changes in brain during ongoing steady-state behaviors or during the disruption of behavior following administration of drugs, precursors, or other perturbations.