Neuroscience 128 (2004) 843–859 AN IMMUNOCYTOCHEMICAL MAPPING OF METHIONINE- ENKEPHALIN-ARG6-GLY7-LEU8 IN THE HUMAN BRAINSTEM R. COVEÑAS,a* F. MARTÍN,a P. SALINAS,b E. RIVADA,b et al., 1987; Murakami et al., 1987). This peptide has been b a c V. SMITH, L. A. AGUILAR, Z. DÍAZ-CABIALE, shown to act as a neurotransmitter or neuromodulator and c d J. A. NARVÁEZ AND G. TRAMU has been implicated in pain, cardiovascular, and motor aInstitute of Neurosciences of Castilla y León, Laboratory of Neuro- mechanisms (Akill et al., 1984; Basbaum and Fields, 1984; anatomy of the Peptidergic Systems, Facultad de Medicina, Campus Pasternak, 1987; Douglas and Kitchan, 1992). Its distribu- Unamuno, c/ Alfonso X El Sabio s/n, 37007 Salamanca, Spain tion has been studied using immunocytochemical and ra- b University of Málaga, School of Medicine, Department of Anatomy, dioimmunoassay techniques in the rat, cat and monkey 29080 Málaga, Spain central nervous systems (Pittius et al., 1984; Zamir et al., c University of Málaga, School of Medicine, Department of Physiology, 1985; Fallon and Leslie, 1986; Ibuki et al., 1989; Murakami 29080 Málaga, Spain d et al., 1987, 1989; Tanaka et al., 1993; Belda et al., 2003). University of Bordeaux I, Laboratory of Functional Neurocytochemis- Previous reports using a combination of radioimmunoas- try, C.N.R.S., 33405 Talence, France say, gel filtration, and high-performance liquid chromatog- raphy have only demonstrated the presence of Met8 in Abstract—Using an indirect immunoperoxidase technique, eight nuclei of the human brainstem (e.g. periaqueductal we studied the distribution of immunoreactive fibers and cell gray, substantia nigra) and in a general form in the dorsal bodies containing methionine-enkephalin-Arg6-Gly7-Leu8 in and ventral medulla oblongata (Pittius et al., 1984; Iadarola the adult human brainstem. Immunoreactive cell bodies were found in the reticular formation of the medulla oblongata (in et al., 1991). Thus, at the present very few data are avail- 8 which we observed the highest density of immunoreactive able on the distribution of Met in the human brainstem cell bodies) and the pons, the solitary nucleus, the hypoglos- (Pittius et al., 1984; Iadarola et al., 1991), and in this sal nucleus, the medial and spinal vestibular nuclei, the lat- sense, to date the distribution of Met8-immunoreactive eral cuneate nucleus, the nucleus prepositus, the central (Met8-ir) fibers and cell bodies in the human CNS has not gray of the pons and mesencephalon, the central and peri- been studied. central nuclei of the inferior colliculus, the superior collicu- In light of the above and in order to know a more lus, ventral to the superior olive and in the midline region of 8 the pons and mesencephalon. The highest density of immu- complete distribution of Met in the human brainstem, here noreactive fibers containing methionine-enkephalin-Arg6- we were prompted to study the distribution of cell bodies Gly7-Leu8 was found in the spinal trigeminal nucleus, the and fibers containing Met8 in the whole human brainstem central gray and the reticular formation of the medulla oblon- using an immunocytochemical technique. gata, pons and mesencephalon, the solitary nucleus, the spinal vestibular nucleus, the dorsal accessory olivary nu- cleus, the raphe obscurus, the substantia nigra and in the EXPERIMENTAL PROCEDURES interpeduncular nucleus. The widespread distribution of im- munoreactive structures containing methionine-enkephalin- Brainstem material was obtained from five adult human brains Arg6-Gly7-Leu8 in the human brainstem indicates that this (School of Medicine, University of Málaga, Spain) with no previous neuropeptide might be involved in several physiological history of neurological or psychiatric disease (two men, 81 (M-1) mechanisms, acting as a neurotransmitter and/or and 82 (M-2) years; three women, 69 (W-1), 84 (W-2) and 87 neuromodulator. © 2004 IBRO. Published by Elsevier Ltd. All (W-3) years), who died from renal failure, anemia, or myocardial rights reserved. infarct. The experimental design, protocols, and procedures of this work were performed under the guidelines of the ethics and legal Key words: man, medulla oblongata, mesencephalon, pons, recommendations of Spanish and European law, as well as in pro-enkephalin. accordance with the Declaration of Helsinki. This work has been approved by the research commission of the University of Málaga (Spain). All procedures were carried out with the adequate under- Methionine-enkephalin-Arg6-Gly7-Leu8 (Met8) is a peptide standing and written consent of the subjects. derived from the precursor pro-enkephalin. It is known that Autopsies were carried out within 24–48 h after death. The Met8 is exclusively produced from pro-enkephalin and not brains were removed and perfused ex situ via the carotid and vertebral arteries. Both arteries were connected to a pressure- by the other two precursors (pro-opiomelanocortin and driven pump and the brains were perfused at normal mean pro-dynorphin) of opioid peptides. Thus, the presence of arterial pressure with 1000 ml of 0.15 M phosphate-buffered 8 Met is a good marker for the pro-enkephalin system (Abe saline (PBS; pH 7.2), followed by 3000 ml of 4% paraformal- dehyde in the same buffer. After postfixation (30 days in the *Corresponding author. Tel: ϩ34-923-294400x1856; fax: ϩ34-923-294549. latter fixative solution) at 4 °C, the brains were kept in PBS at E-mail address: [email protected] (R. Coveñas). 4 °C and cryoprotected, at the same temperature, by immersion Abbreviations: ir, immunoreactive; Met8, methionine-enkephalin-Arg6- in increasing sucrose baths (10–30%) until they sank. The Gly7-Leu8; PBS, phosphate-buffered saline. brainstems were dissected out and, using a cryostat, 60 ␮m- 0306-4522/04$30.00ϩ0.00 © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2004.07.009 843 844 R. Coveñas et al. / Neuroscience 128 (2004) 843–859 Fig. 1. Distribution of Met8-ir fibers and cell bodies in frontal planes of the human medulla oblongata according to the atlas of the human brain of Haines (1987). Cell bodies containing the neuropeptide are represented by closed circles (high density), and closed squares (low density). Ir fibers are represented by dotted lines (single fibers or low density), continuous lines (moderate density) and crossed lines (high density). See list of abbreviations for nomenclature. thick frontal sections of the mesencephalon, pons and medulla absorbed with synthetic Met8 (1.11ϫ10Ϫ4 M) and after the immuno- oblongata were cut and collected in PBS, kept at 4 °C, and histochemical protocol no immunostaining appeared (Fig. 17A–C). processed for immunostaining. In general, six out of seven Also, sections were incubated omitting the synthetic Met8 antibody sections were used for immunocytochemistry, and the remain- and no labeling was detected. In addition, no significant reduction in ing section was stained for the Nissl technique. immunoreactivity was observed upon incubating the first antibody with an excess (10Ϫ7 M) of synthetic methionine-enkephalin, leucine- Immunocytochemistry enkephalin, methionine-enkephalin-Arg6-Phe7, ␣-, ␤-, and ␥-melano- cyte-stimulating hormone, dynorphin A, or ␣-neo-endorphin. In order to avoid possible interference by endogenous peroxidase, Mapping free-floating sections were treated with distilled water containing NH3 (20%), H2O2 (30%) and NaOH (1%) for 20 min (Guntern et al., 1989). Mapping was carried out according to Haines’ (1987) atlas of the Then, the sections were washed for 20 min in PBS and preincubated human brain, and the same atlas was used for the terminology of for 30 min in PBS containing 1% normal horse serum and 0.3% the brainstem nuclei. In addition, the atlas of Paxinos et al. (1990) Triton X-100. The sections were then washed for 30 min in the latter was consulted. solution containing casein (0.5%) and were incubated overnight at The number of immunostained cell bodies appearing in each 4 °C in PBS containing Triton X-100 (0.3%) and normal horse serum section from each person was counted: a high density of Met8-ir (1%), as well as anti-Met8 antiserum (diluted 1/30,000). The sections perikarya was considered when we found more than 20 profiles/ were then washed in PBS (30 min) and incubated for 60 min at room region/section; a moderate density when we found 10–20 profiles/ temperature with biotinylated antirabbit immuno-␥ globulin (made in region/section, and a low density when we found fewer than 10 horse) (Vector, USA) diluted 1/200 in PBS. After a 30 min wash with profiles/region/section. The density of the immunopositive fibers PBS, the sections were incubated for 1 h with avidin–biotin–peroxi- was graded under microscopic observation into four categories: dase complex (Vectastain; diluted 1/100). Finally, after washing the high, moderate, low and single. This was accomplished by viewing sections in PBS (30 min) and Tris–HCl buffer (pH 7.6; 10 min), the the sections under bright-field illumination at a constant magnifi- tissue-bound peroxidase was developed by H2O2 using 3,3= diami- cation with reference to photographs of defined series of densities nobenzidine as chromogen. The sections were rinsed with PBS and (see Marcos et al., 1998). coverslipped with PBS/Glycerol (1/1). Finally, photomicrographs were obtained with an 8-bit CoolSNAP digital camera attached to a Zeiss Axiophot micro- Specificity of the antiserum scope. To improve the visualization of the results, only the brightness and contrast of the images were adjusted, without Polyclonal anti-Met8 antibody was raised in rabbits against immuno- any further manipulation of the photographs. Adobe Photo- gens prepared by coupling the whole synthetic Met8 peptide graph 6.0 was used to view the images and alter their bright- (Bachem, Switzerland) to a carrier protein (human serum albumin) ness and contrast. with glutaraldehyde, as previously reported (Marcos et al., 1999; Belda et al., 2003).