Post-translational processing of the neurotensin/neuromedin N precursor in the central nervous system of the rat—II. Immunohistochemical localization of maturation products

The neurotensin/neuromedin N precursor molecule possesses four lysine-arginine dibasic residues which represent potential sites of cleavage by proteolytic maturation enzymes. As shown in the preceding paper, all of these dibasic residues are cleaved to a variable extent in rat brain. The aim of the present study was to localize immunohistochemically the resulting maturation products using site-specific antibodies directed against neurotensin, as well as against the exposed KLPLVL (K6L) and EKEEVI (E6I) sequences of the precursor. In a first set of experiments, each antigen was singly labelled in serial adjacent sections through the rat brain using a peroxidase-antiperoxidase technique. In a second series of experiments, neurotensin and either E61 or K6L antigens were double labelled in pairs using indirect immunofluorescence and visualized by confocal microscopy. In both types of preparations, immunoreactivity for all three antigens was detected in nerve cell bodies and axon terminals. In the absence of colchicine pretreatment, labelled nerve cell bodies were sparse in both neurotensin- and E6I-immunostained material and virtually undetectable in K6L-immunoreacted sections. By contrast, terminal immunostaining was intense and comparable in distribution for both neurotensin and E6I in most regions examined. K6L axonal labelling showed the same topographic pattern as that of E6I and neurotensin but was consistently weaker, except in the globus pallidus, where both E6I- and K6L-immunoreactive arbors were more widespread than those of neurotensin. These results suggest that the cleavage of the dibasic sites adjacent to the E6I and K6L sequences is more extensive in certain brain regions than in others. Colchicine pretreatment markedly increased the number of neurotensin- and, to a lesser extent. E6I-immunoreactive perikarya throughout the rat brain. However, it only marginally augmented the number of K6L-immunoreactive cell bodies, which remained sparse throughout. These results suggest that the maturation cleavages exposing the E6I and K6L sequences occur further distal to the cell body than the one giving rise to neurotensin. Both E6I- and K6L-immunoreactive perikarya were essentially confined to areas displaying neurotensin immunoreactivity. Furthermore, E6I and K6L antigens were shown in double labeling experiments to be present in the same cells as neurotensin, indicating that even if it is quantitatively different among brain regions, the basic pattern of neurotensin/neuromedin N precursor processing remains qualitatively similar throughout the brain.