An immunohistochemical technique for simultaneously visualizing two different antigens has been used to investigate the presence of the acidic dipeptide, N-acetyl-aspartyl-glutamate (NAAG), in cholinergic, noradrenergic-adrenergic, and serotonergic neurons within CNS. The brain slices were processed sequentially with purified antisera against NAAG and then monoclonal antibody against choline acetyltransferase (ChAT), a marker for cholinergic neurons, or antiserum against dopamine-β-hydroxylase (DBH), a marker of noradrenergic-adrenergic neurons, or antiserum against serotonin (5HT). Both antigens were revealed by the peroxidase reaction but with different chromogens, which are easily distinguishable.
An intense double staining of NAAG-like immunoreactivity (NAAG-LI) and ChAT was observed in the motoneurons of the spinal cord as well as in the several motor components of cranial nerve nuclei including facial, ambiguus, and trigeminal nuclei. A partial colocalization of NAAG-LI and ChAT was evident in the perikarya of the basal forebrain cholinergic system, whereas cholinergic neurons of the medial septum exhibited only sporadic staining for NAAG-LI. A complete coexistence of NAAG-LI and DBH was observed in the locus coeruleus. Most of the other noradrenergic and adrenergic cell groups of the medulla region exhibited substantial co-localization with the exception of the A2 cell group, which was virtually devoid of NAAG-LI.
In the dorsal raphe, only a low percentage of serotonergic neurons stained for NAAG-LI. The co-existence of NAAG-LI and serotonin was more evident in the neurons of the median raphe, although the majority of cells failed to show double staining. In the raphe pallidus and raphe magnus, the co-existence with NAAG-LI remained partial but involved a large percentage of serotonergic neurons. These results suggest a more widespread neuronal function of NAAG in addition to its proposed role as putative neurotransmitter in a subgroup of glutamatergic neurons.