Comparative single and double immunolabelling with antisera against catecholamine biosynthetic enzymes: criteria for the identification of dopaminergic, noradrenergic and adrenergic structures in selected rat brain areas

Abstract

Immunodetection of catecholamine biosynthetic enzymes is frequently used for the visualization of central nervous catecholaminergic systems. Because of the method's limited specificity for the different catecholamines, interpretation of the results often presents difficulties. To determine criteria for the identification of dopaminergic, noradrenergic, and adrenergic afferents to the rat amygdaloid complex, comparative immunolabelling for tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), and phenylethanolamine-N-methyl-transferase (PNMT) was carried out using single- and double-labelling for fluorescence, light- and electron microscopy. The observations were complemented by findings in brainstem and hypothalamic areas. The results indicated that TH-labelling detected preferentially dopaminergic afferents in the lateral central and intercalated amygdaloid nuclei. DBH-labelling detected noradrenergic axons in nuclei lacking PNMT-immunoreactive fibres, and PNMT was a marker for adrenergic axons in the entire complex. For nuclei with combined dense dopaminergic, noradrenergic and/or adrenergic innervation, morphological and immunolabelling characteristics were described which, to a certain extent, enabled identification of the different afferents in anti-TH or anti-DBH-preparations. Using a monoclonal TH-antiserum, noradrenergic and adrenergic axons displayed weaker immunoreactivity than dopaminergic ones, and possessed characteristic morphological features. TH-immunoreactivity in noradrenergic axons differed depending on their origin, and showed intra-axonal compartmentalization. The present study provides a basis for the use of the detection of biosynthetic enzymes in future investigations into the ultrastructure and connectivity of the catecholaminergic amygdala innervation.