Studies on dopamine-, tyrosine hydroxylase-and aromatic l-amino acid decar☐ylase-containing cells in the rat diencephalon: Comparison between formaldehyde-induced histofluorescence and immunofluorescence

Abstract

The morphology, number and distribution of catecholaminergic neurons, as visualized either with the aluminum-catalysed formaldehyde method for catecholamines or with the immunohistochemical method for the catecholamine-synthesizing enzymes tyrosine hydroxylase and aromatic l-amino acid decar☐ylase, respectively, were analysed within the rat dorsal hypothalamus, ventral thalamus and adjoining regions (A11 and A13 cell groups). Both polyclonal rabbit and monoclonal mouse tyrosine hydroxylase antibodies were used in elution-restaining and double-staining experiments, respectively. Some of the animals also received spinal injections of the fluorescent tracer True Blue in order to retrogradely label cells projecting to the spinal cord. With respect to the number and distribution of catecholaminergic neurons in the A11 and medial A13 cell groups, including the spinal-projecting subpopulation, the results obtained with the two methods were very similar, indicating that within these regions of the CNS the two methods in principle visualize identical cell populations. However, the catecholaminergic cells were distinctly larger and their processes appeared more extensive with the immunohistochemical method. Animals processed for immunohistochemistry exhibited a lower total number of retrogradely labelled cells in the A11 area than those analysed with aldehyde-induced fluorescence despite the fact that both methods revealed similar numbers of retrogradely labelled tyrosine hydroxylase-positive and catecholamine-containing cells, respectively. The reason for these discrepancies, which are probably of methodological nature, are discussed. While this study shows that the results obtained with the two methods within the A11 and medial A13 cell group are very similar and thus strengthens the earlier proposed concept of the organization of the diencephalospinal dopaminergic system, it also documents that in intermingling and nearby CNS regions there are cell bodies which cannot be demonstrated with the aldehyde fluorescence method, but which still contain tyrosine hydroxylase and/or aromatic l-amino acid decar☐ylase-like immunoreactivity. One explanation is low levels of enzyme and/or dopamine combined with a comparatively low sensitivity of the histochemical method. Thus, neurons containing both enzymes are probably dopaminergic, even if catecholamine fluorescence cannot be demonstrated. Neurons containing tyrosine hydroxylase, but lacking both aldehyde induced fluorescence and aromatic l-amino acid decar☐ylase, may also still be dopaminergic. In contrast, aromatic l-amino acid decar☐ylase-positive neurons apparently lacking tyrosine hydroxylase may decar☐ylate another amino acid than l-dopa. However, a final decision must await further studies employing more sensitive histochemical techniques and more “powerful” antisera.