Identification of tryptamine and tryptamine‐serotonin neurons in the rat dorsal raphe nuclei using specific antibodies

Previous studies have shown that tryptamine (T) is closely associated with the serotonergic system but have failed to determine whether neurons containing only T exist in the raphe nuclei (RN) and/or if T colocalizes with serotonin (5‐HT). The use of rabbit‐conjugated T antisera (TAS) and rat‐conjugated 5‐HT antisera (5‐HTAS) in a double‐labelling technique has made the study of the colocalization of T and 5‐HT in neurons of the rat dorsal RN (DR) possible. Slices taken from the same zone of the DR were treated following four different procedures of double‐immunolabelling: DAB coloration for the rabbit TAS and DAB‐nickel coloration for the rat 5‐HTAS either first or second; or DAB coloration for the 5‐HTAS and DAB‐nickel coloration for the TAS first or second. Control sections were treated according to a single immunoperoxidase staining in the same zone. The immunolabelled neurons were computed using the Biocom 200 program at the same magnification. The results of this double‐immunolabelling show that three different cell types exist in the rat DR: (1) T‐only‐containing neurons, (2) 5‐HT‐only‐containing neurons displaying either homogeneous DAB or homogeneous DAB‐nickel, and (3) neurons where T colocalizes with 5‐HT (T‐positive/5‐HT‐positive neurons) displaying heterogeneous DAB‐nickel coloration. The results were identical whatever the procedure performed or the order of the DAB‐nickel revelation. In contrast, the single immunolabelling method failed to stain T‐only‐containing neurons (or 5‐HT‐only‐containing neurons); T‐immunoreactive cells (or 5‐HT‐immunoreactive cells) correspond in fact to neurons containing either T (or 5‐HT) or T and 5‐HT. © 1993 Wiley‐Liss, Inc.