160 Braw Research. i~3:. ~85) li~i, ~ i¢~a BRE 20806 Evidence for synenkephalin-like immunoreactivity in pontobuibar monoaminergic neurons of the cat YVES CHARNAY1, LUCIENNE LEGER 2, JEAN ROSSIER3, MICHEL JOUVET2 and PAUL MARIE DUBOIS t ICNRS-ERA 981, Laboratoire d'Histologie-Embryologie, FacultO de MOdecine Lyon-Sud, B.P. 12, F-69600 Oullins; 2DOpartement de MOdecine ExpOrimentale, INSERM U.52 et U. 171, FacultO de MOdecine, 8, avenue Rockefeller, ['-69373 Lyon Cddex: and 3CNRS, Laboratoire de Physiologie nerveuse, F-91190 Gif-sur- Yvette (France) (Accepted December 18th, 1984) Key words: synenkephalin - - enkephalins - - monoamines - - coexistence - - central nervous system Using indirect immunofluorescence, evidence that enkephalin- and synenkephalin-like-immunoreactivities are colocalized within numerous monoaminergic neurons of the cat pontobulbar formation is presented. The colocalization concerns most catecholaminergic cell bodies in the locus coeruleus region and numerous serotoninergic cell bodies in the raphe nuclei. Synenkephalin is the 1-70 N-ter- minal region of the bovine adrenal medulla proenkephalin. Therefore, the proenkephalin (or a related) system seems to represent the biosynthetic pathway for the enkephalins immunodetected within these monoaminergic neurons. Using an indirect immunofluorescence technique we have previously shown that most of the noradren- ergic cell bodies in the cat locus coeruleus complex contain an enkephalin-like immunoreactivity (ELI)4,15. More recently, the presence of ELI in se- rotoninergic neurons in nuclei raphe pallidus, obscu- rus, magnus and dorsalis has been observed in the same species (refs. 8 and 13 and L6ger et al., in prep- aration), Thus as reported in the endocrine and pe- ripheral nervous systems (see Lundberg et al. 21) the coexistence of enkephalins and monoamines in one cell is also strongly~uggested in the brain4,8,13, ~5. We report here evidence indicating that synenkephalin- like immunoreactivity (SLI) is also colocalized with enkephalins in monoaminergic neurons of the locus coeruleus complex and raphe nuclei. Synenkephalin18 is the 1-70 N-terminal region of bovine proenkepha- lin (also called proenkephalin A). Proenkephalin A includes one sequence synenkephalin, four copies of Met-, one copy of Leu-enkephalin, one copy of hep- ta-Met-enkephalin-Arg6-Phe 7 and one copy of octa- Met-enkephalin-Arg6-Gly7-Leu8 (ref. 10). Synenke- phalin does not contain an enkephalin sequence and is supposed to have a role in the processing of proen- kephalin. Previous studies have suggested that, in en- kephalinergic neurons, synenkephalin was a better marker than either Met- or Leu-enkephalin: Indeed, the sequence of synenkephalin is found only in proenkephalin and not in prodynorphin or proopio- melanocortin (POMC), This is not the case for Met- enkephalin that is also found in the fl-endorphin part of POMC and for Leu-enkephalin that is also found in prodynorphin. Therefore the use of antisera against Leu- or Met-enkephalin could not dissociate if these peptides arise from POMC, prodynorphin or proenkephalin A. On the other hand, the presence of synenkephalin immunoreactivity in a cell is a good in- dication that this cell contains proenkephalin-derived peptides. Preliminary results of the present work have been presented elsewhere3,16. Sixteen young adult cats of the European race re- ceived stereotaxically a unilateral injection of colchi- cine (10 ~1 of colchicine in 1 ktl of Ringer-lactate) in the vicinity of the locus coeruleus or raphe nuclei 4.15. After a survival time of 24-48 h the animals were an- esthetized with 5% sodium pentobarbital and per- fused transcardially with 2 liter of ice-cold McLean fixative mixture lz (4% paraformaldehyde in 0.04 M Correspondence and reprints requests: Y. Charnay. Present address: CNRS-ERA 981, Laboratoire d'Histologie-Embryologie, Facult6 de M6decine Lyon-Sud, B.P. 12, F-69600 Oullins, France. 0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B .V. (Biomedical Division)