IDENTIFICATION AND QUANTIFICATION OF MONOAMINERGIC NEUROMODULATORS IN THE SUB-CORTICAL REGION OF CAT VISUAL CORTEX BY MICROBORE HPLC-ED AND PROTEIN ASSAY

Abstract

Removal of retinal input from a restricted region of adult visual cortex leads to a substantial reorganisation of the retinotopy within the deprived zone. To investigate the role of the total (intra- and extracellular) concentration of the monoaminergic neuromodulators in the sub-cortical region of cat visual cortex, which is possibly involved in this reorganisation mechanism, a method for identification and quantification of noradrenaline, dopamine, serotonin, and their major metabolites, 3,4-dihydroxyphenylacetic acid, 4-hydroxy-3-methoxyphenylacetic acid, 5-hydroxyindole-3-acetic acid in small amounts of brain extracts has been developed and validated. Control or retinal lesion cats were killed with pentobarbital; the brains were quickly frozen, and 200 μm cryostat sections were cut.

Under visual control through a surgical microscope, the cortical tissue was first separated from the underlying white matter. Three pieces of tissue measuring 2 × 4 mm², containing the six cortical layers, were sampled out from the different cortical regions subserving different parts of the visual field (central and peripheral portion of area 17). After homogenising these samples in 80 μL, 0.01M HCl which included 0.01% cysteine as antioxidant, 10 μL of the supernatant was injected directly onto the microbore HPLC system, separated on a microbore column (150 × 1 mm i.d.; ODS), and the components detected electrochemically at a potential of +0.75V. The flow rate of mobile phase through the column was 44 μL/min.

The specificity, recovery, analytical precision, calibration curves, and detection limits for each neurotransmitter were determined. In order to express the total neuromodulator concentration as pg/μg protein, the pellets obtained after centrifugation were used for protein determination. A modified protein assay with non-linear regression equation data processing is also described.

ACKNOWLEDGMENTS

We are grateful to Luc Grauwels and Ria Vanlaer for their valuable technical assistance, and to Steve Raiguel for the critical review of the manuscript. This work was supported by grants of the Queen Elisabeth Medical Foundation, the Fund for Scientific Research-Flanders, Belgium, and the Belgian program of Inter-University Poles of Attraction, initiated by the Belgian State, Prime Minister's Office, Science Policy Programming (IUAP 22/P4). Lutgarde Arckens was supported as a postdoctoral fellow of the Fund for Scientific Research-Flanders (Belgium, F.W.O.). Ying Qu was supported by a grant funded by the scholarship from the Queen Elisabeth Medical Foundation.