Neuroscience 128 (2004) 843–859

AN IMMUNOCYTOCHEMICAL MAPPING OF METHIONINE- ENKEPHALIN-ARG6-GLY7-LEU8 IN THE HUMAN

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, ) and in a general form in the dorsal bodies containing methionine-enkephalin-Arg6-Gly7-Leu8 in and ventral (Pittius et al., 1984; Iadarola the adult human brainstem. Immunoreactive cell bodies were found in the 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 , the , the hypoglos- (Pittius et al., 1984; Iadarola et al., 1991), and in this sal nucleus, the medial and spinal , 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 , 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 . 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. 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 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). Rabbit antiserum was preabsorbed with the RESULTS carrier protein and the couplin agent in order to prevent non-specific immunoreactivity due to the anti-carrier antibodies. This preabsorp- General considerations tion was carried out before the immunocytochemical applications. 8 To determine the specificity of the immunostaining, several his- As shown in Figs. 1–6, Met structures were found to be tologic controls were carried out. The primary antibody was pre- widely distributed throughout the human brainstem. Our R. Coveñas et al. / Neuroscience 128 (2004) 843–859 845 results indicate that Met8 is present in the human brain- observed in the human brainstems studied. Thus, the stem, at least until 48 h after death. In general, both the distribution and density of Met8-ir fibers and cell bodies distribution of the ir structures (fibers and cell bodies), in the brainstem of the youngest person studied (69 and their density, were fairly similar in the five brain- years) were in general similar to those observed in the stems studied. Thus, we did not find great differences in older persons (81, 82, 84 and 87 years). However, we the human brainstems, which were perfused ex situ from observed some minor differences among the speci- 24 h to 48 h after death, nor in the density of the mens. Thus, ir cell bodies located in the medial vestib- immunoreactivity nor in the distribution of the ir struc- ular nucleus, the and in the nucleus tures. In general, no sex or age differences were found prepositus were respectively only found in the brains in the distribution and density of the Met8-ir structures M-1 (81 years; perfused 24 h after death), W-2 (84

Abbreviations used in the figures

4 MAO medial accessory olivary nucleus AbdNr abducens nerve MCP middle cerebellar peduncle AbdNu abducens nucleus MesNu mesencephalic nucleus AccNu accessory nucleus MesTr mesencephalic tract ACSp anterior corticospinal tract ML medial ALS anterolateral system MLF medial longitudinal fasciculus AMV anterior medullary velum MVN medial vestibular nucleus ArNu arcuate nucleus NigSt nigrostriatal fibers CA NuAm Cbl cerebellum NuCu cuneate nucleus CC crus cerebri NuGr gracile nucleus CeGy central gray NuPp Nucleus prepositus CoBul corticobulbar fibers OCbF olivocerebellar fibers CocNr cochlear nerve OPon occipitopontine fibers CortNig corticonigral fibers PalNig pallidonigral fibers CSNu chief sensory nucleus (caudal end) PBNu pontobulbar nucleus CSp corticospinal fibers PCbF pontocerebellar fibers CTT central tegmental tract PO principal (inferior) olivary nucleus DAO dorsal accessory olivary nucleus PonNu pontine nuclei DCNu dorsal cochlear nucleus PPon parietopontine fibers DLF dorsal longitudinal fasciculus Py pyramid DMNu dorsal motor nucleus of the vagus RB restiform body DNu dentate nucleus RetF reticular formation DSCT dorsal spinocerebellar tract RetSp reticulospinal tract DTTr dorsal trigeminothalamic tract RuSp EAF external arcuate fibers SC Fac, G facial nerve, internal genu SCP superior cerebellar peduncle FacNr facial nerve SCP, Dec superior cerebellar peduncle, decussation FacNu facial nucleus SM stria medullares of fourth ventricle FCu cuneate fasciculus SN substantia nigra FGr gracile fasciculus SO superior olive FPon frontopontine fibers SolNu solitary nucleus g gelatinosa part of the spinal trigeminal nucleus SolTr GIF glossopharyngeal fibers (intramedullary) SpTec spinotectal tract GINr glossopharyngeal nerve SpTh HyNu hypoglossal nucleus SpTNu spinal trigeminal nucleus IAF internal arcuate fibers SpTT spinal trigeminal tract IC, Br inferior colliculus, brachium SpVN spinal (inferior) vestibular nucleus IC, CNu inferior colliculus, central nucleus SSNu superior salivatory nucleus IC, Com inferior colliculus, commissure SVN superior vestibular nucleus IC, LZ inferior colliculus, lateral zone TecSp ICP inferior cerebellar peduncle TPon temporopontine fibers IC, PNu inferior colliculus, pericentral nucleus TrapB trapezoid body IPFo TrapNu trapezoid nucleus IPNu interpeduncular nucleus TriMoNu trigeminal motor nucleus (caudal part) ISNu inferior salivatory nucleus TriNr trigeminal nerve JRB juxtarestiform body TroNr trochlear nerve LCNu lateral cuneate nucleus TroNu LL VCNu ventral cochlear nucleus LL, Nu lateral lemniscus, nucleus VesSp vestibulospinal tract LoCer locus ceruleus VSCT ventral spinocerebellar tract LRNu lateral reticular nucleus VTTr ventral trigeminothalamic tract m magnocellular part of the spinal trigeminal nucleus 846 R. Coveñas et al. / Neuroscience 128 (2004) 843–859

Fig. 2. 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), closed triangles (moderate density) or 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. years; perfused 37 h after death) and W-1 (69 years; dorsal parts. In addition, the clusters of cell bodies con- perfused 40 h after death). taining Met8 were almost always observed in the dorsal In all the cases studied, in the three brainstem regions part of the human brainstem. The highest density of ir cell (medulla oblongata, pons and mesencephalon), the high- bodies was found in the reticular formation of the medulla est density of ir structures was generally observed in their oblongata.

Fig. 3. Distribution of Met8-ir fibers and cell bodies in frontal planes of the human pons, according to the atlas of the human brain of Haines (1987). Cell bodies containing the neuropeptide are represented by closed circles (high density), closed triangles (moderate density) or 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. R. Coveñas et al. / Neuroscience 128 (2004) 843–859 847

Fig. 4. Distribution of Met8-ir fibers and cell bodies in frontal planes of the human pons, according to the atlas of the human brain of Haines (1987). Cell bodies containing the neuropeptide are represented by closed circles (high density), closed triangles (moderate 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.

Distribution of Met8-ir structures in the human olivary nucleus (Figs. 2A, B, 11B), the nucleus preposi- medulla oblongata tus (Fig. 2B) and in the dorsal and ventral cochlear nuclei (Figs. 2B, 11C–F). A low density of ir fibers was Met8-ir cell bodies. A high density of ir cell bodies found in the rostral midline (Fig. 2B), the medial longi- was found along the reticular formation, except in its tudinal fasciculus (Figs. 1B, 2B), the tectospinal tract caudalmost part (Figs. 1B, 2A, B, 7A, B). A moderate (Figs. 1B, 2B), the lateral vestibular nucleus, the solitary density was visualized in the caudal part of the medial tract (Figs. 2A, B, 3A, 9A, B), the vestibular nucleus (Figs. 2A, 7E, F) and in the rostral (Figs. 1B, 2A, B), the anterolateral system (Figs. 1B, 2A, parts of the spinal (inferior) vestibular nucleus (Figs. 2B, B, 10F, 11A) and in the spinal trigeminal tract (Figs. 1B, 8A, B) and the medial longitudinal fasciculus (Figs. 2B, 2A, B). Finally, single fibers were found in the restiform 8C, D), while a low density of cell bodies containing Met8 body (Figs. 1B, 2A, B) and the . was observed in the solitary nucleus (caudal part) (Fig. Moreover, the gelatinosa part of the spinal trigeminal 1B), the rostral part of the hypoglossal nucleus (Fig. 2A), nucleus (pars caudalis; Figs. 1A, 12C, D) showed a high the nucleus prepositus (Fig. 2B) and the rostral part of density of ir fibers, but a moderate density was found in the lateral cuneate nucleus (Fig. 2A). pars interpolaris and pars oralis (Figs. 1B, 2A, B, 12A, B). Met8-ir fibers. A high density of ir fibers containing In addition, a moderate density of Met8-ir fibers was found Met8 was observed in the central gray (Fig. 1A, B), the in the magnocellular part of the spinal trigeminal nucleus dorsal motor nucleus of the vagus (Figs. 1A, B, 2A, 9D, (pars caudalis; Fig. 1A). In the spinal vestibular nucleus F), the solitary nucleus (Figs. 1B, 2A, B, 3A, 9A, B), the (Figs. 2A, B, 8A, B), we observed a low density of ir fibers. reticular formation (Figs. 1B, 2A, B, 7B, 9A, C), the However, in its ventral region, we found a high density of ir raphe obscurus (Fig. 10A, B) and in the dorsal acces- fibers at caudal level (Fig. 2A) and a moderate density at sory olivary nucleus (Figs. 2A, B, 8E, F). A moderate rostral level (Fig. 2B). density was found in the gracile nucleus (Fig. 1B, 10C, In the hypoglossal nucleus (Figs. 1A, B, 2A, 9D, E), D), the lateral cuneate nucleus (Figs. 1B, 2A), the nu- at caudal level a moderate density of fibers containing cleus ambiguus (Figs. 1B, 2A, B), the lateral reticular Met8 was visualized (Fig. 1A, B) and a low density at nucleus (Figs. 1B, 2A, 10F, 11A), the medial accessory rostral level (Fig. 2A). In the cuneate nucleus (Fig. 1A, B, 848 R. Coveñas et al. / Neuroscience 128 (2004) 843–859

Fig. 5. Distribution of Met8-ir fibers and cell bodies in frontal plane of the human mesencephalon, according to the atlas of the human brain of Haines (1987). Cell bodies containing the neuropeptide are represented by 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.

Fig. 6. Distribution of Met8-ir fibers and cell bodies in frontal plane of the human mesencephalon, according to the atlas of the human brain of Haines (1987). Cell bodies containing the neuropeptide are represented by 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. R. Coveñas et al. / Neuroscience 128 (2004) 843–859 849

Fig. 7. Met8-ir cell bodies and fibers in the human medulla oblongata and pons. (A) A low magnification of the RetF of the medulla oblongata and the ML. Thick dashed lines indicate the midline. This photograph was taken from brain M-1 (see Experimental Procedures). D, dorsal; M, medial. Scale barϭ500 ␮m. (B) High-power image of the area delimited in Fig. 7A. Cluster of ir perikarya and Met8-ir fibers located in the RetF of the medulla oblongata. Scale barϭ100 ␮m. (C) Low-power image showing the CeGy and the RetF of the pons (RetF). Photograph taken from brain W-1. D, dorsal; M, medial. Scale barϭ500 ␮m. (D) High-power image of the area delimited in Fig. 7C. Ir cell bodies and fibers in the RetF of the pons. Scale barϭ100 ␮m. (E) Low-power image of the region around the SolNu and SolTr. For a nomenclature of the nuclei, see list of abbreviations. The area delimited into the MVN is shown at higher magnification in Fig. 7F. Photograph taken from brain M-1. D, dorsal; M, medial. Scale barϭ500 ␮m. (F) Cluster of ir cell bodies in the MVN. Scale barϭ100 ␮m.

10C, E) a low density was found at caudal level (Fig. 1A) whereas a moderate density was found in the midline (at and a moderate density at rostral level (Fig. 1B). Finally, caudal level; Figs. 3A, 13C–F) and rostrally in the retic- the medial vestibular nucleus (Figs. 2A, B, 7E, F) ular formation located close to the midline (Fig. 4). A low showed a moderate density, but its anteroventrolateral density was visualized in the caudal midline (Fig. 3A, B), part showed a low density of ir fibers (Fig. 2B). the central gray (Figs. 3 B, 14A), and in the rostral part of the medial longitudinal fasciculus (Fig. 3A). Distribution of Met8-ir structures in the human pons Met8-ir cell bodies. A high density of ir cell bodies Met8-ir fibers. A high density of ir fibers was observed was caudally located ventral to the superior olive (Figs. in the nucleus prepositus (Fig. 3A), the reticular formation 3A, 12E, F), in the reticular formation (laterally; Figs. 3B, (Figs. 3A, B, 4, 7C, D, 13A, B) and the central gray (Figs. 3B, 7C, D, 13A, B) and in the central gray (rostral; Fig. 4), 4, 14A). A low density was seen in the abducens nucleus 850 R. Coveñas et al. / Neuroscience 128 (2004) 843–859

Fig. 8. Met8-ir cell bodies and fibers in the human medulla oblongata. (A) A low-power magnification of the dorso-lateral region of the medulla oblongata. For a nomenclature of the nuclei, see list of abbreviations. The area delimited into the SpVN is shown at higher magnification in Fig. 8B. Photograph taken from brain M-1. D, dorsal; M, medial. Scale barϭ500 ␮m. (B) Cluster of ir cell bodies located in the SpVN. Scale barϭ100 ␮m. (C) A low-power magnification of the midline region of the medulla oblongata. Thick dashed lines indicate the midline. For a nomenclature of the nuclei, see list of abbreviations. Photograph taken from brain W-2. D, dorsal. Scale barϭ500 ␮m. (D) High-power magnification of the region delimited in Fig. 8 C. Note the ir cell bodies located in the MLF. Scale barϭ100 ␮m. (E) A low-power magnification of the ventral region of the medulla oblongata. For a nomenclature of the nuclei, see list of abbreviations. The area delimited is shown at higher magnification in Fig. 8F. Photograph taken from brain W- 3. D, dorsal; M, medial. Scale barϭ500 ␮m. (F) Ir fibers in the dorsal accessory olivary nucleus. Scale barϭ100 ␮m.

(Fig. 3A), the facial nucleus (Fig. 3A), the spinal trigeminal nuclei of the inferior colliculus (Figs. 5, 14B–E), the tract (Fig. 3A), the medial longitudinal fasciculus (Figs. 3A, B, central gray (Figs. 5, 6, 9A, 14F), the midline (Figs. 5, 6) 4), the tectospinal tract (Fig. 3A), the central tegmental tract and the superior colliculus (Figs. 6, 15D). (Figs. 3A, B, 4), the anterolateral system (Figs. 3A, B, 4), the medial lemniscus (Figs. 3A, B, 4) and the superior cerebellar Met8-ir fibers. A high density of ir fibers was ob- peduncle (Fig. 4). served in the central gray (Figs. 5, 6, 14F, 15A–C), the

8 reticular formation (Figs. 5, 6), the substantia nigra (Fig. 6) Distribution of Met -ir structures in the human and the interpeduncular nucleus (Figs. 6, 16A, B) and in mesencephalon the region placed between the last two nuclei (Fig. 16A, C), Met8-ir cell bodies. A low density of cell bodies con- whereas a moderate density was found in the superior taining Met8 was observed in the central and pericentral colliculus (Figs. 6, 15D) and in the pericentral nucleus of R. Coveñas et al. / Neuroscience 128 (2004) 843–859 851

Fig. 9. Met8-ir fibers in the human medulla oblongata. (A) A low magnification of the dorsal region of the medulla oblongata. For a nomenclature of the nuclei, see list of abbreviations. Photograph taken from brain W-1. D, dorsal; L, lateral. Scale barϭ500 ␮m. (B) High-power image of the area delimited in Fig. 9A including the SolNu and the SolTr. Note the ir fibers (arrows) located in the solitary tract. Scale bar: 100 ␮m. (C) High-power magnification of the region delimited in the corner of Fig. 9A showing ir fibers placed in the RetF. Scale barϭ100 ␮m. (D) A low magnification of the dorso-medial region of the medulla oblongata. For a nomenclature of the nuclei, see list of abbreviations. Photograph taken from brain M-2. D, dorsal. Scale barϭ500 ␮m. (E) High-power image of the region delimited in the ventral rectangle of Fig. 9D. Note the ir fibers containing Met8 in the HyNu. Scale barϭ50 ␮m. (F) High-power magnification of the region delimited in the DMNu of Fig. 9D. Ir fibers located in the DMNu. Scale barϭ50 ␮m. the inferior colliculus (Figs. 5, 14B–D). A low density of DISCUSSION Met8-ir fibers was located in the central nucleus of the Comparison of the distribution of Met8 inferior colliculus (Figs. 5, 14E), the medial longitudinal in the human brainstem fasciculus (Figs. 5, 6), the lateral lemniscus (Fig. 5), the anterolateral system (Figs. 5, 6), the central tegmental This is the first detailed study showing the distribution of tract (Figs. 5, 6), the superior cerebellar peduncle (Fig. 5), Met8-ir fibers and cell bodies in the human brainstem the medial lemniscus (Figs. 5, 6) and the brachium of the without neurological disease. inferior colliculus (Fig. 6). Finally, single fibers were ob- In comparison with two previous studies carried out served in the and in the red nucleus on several nuclei (eight in total) of the human brainstem, (not shown in Figs. 1–6). in which a combination of radioimmunoassay, gel filtra- 852 R. Coveñas et al. / Neuroscience 128 (2004) 843–859

Fig. 10. Met8-ir fibers in the human medulla oblongata.. (A) Ir fibers in the RaNu. Photograph taken from brain M-1. Dashed lines indicate the midline. D, dorsal. Scale barϭ100 ␮m. (B) A higher magnification of the region delimited in Fig. 10A. Scale barϭ50 ␮m. (C) A low magnification of the DMNu. Regions delimited in the NuGr and NuCu are respectively observed at higher magnification in Figs. 10D and 10E. For a nomenclature of the nuclei, see list of abbreviations. Photograph taken from brain W-3. D, dorsal. Scale barϭ500 ␮m. (D) Ir fibers located in the gracile nucleus. Scale barϭ50 ␮m. (E) Presence of Met8-ir fibers in the NuCu. Scale barϭ50 ␮m. (F) A low magnification of the ventro-lateral part of the medulla oblongata. The region delimited can be observed at higher magnification in Fig. 11A. For a nomenclature of the nuclei, see list of abbreviations. Photograph taken from brain W-2. D, dorsal. L, lateral. Scale barϭ500 ␮m. tion and high-performance liquid chromatography tech- structures containing Met8. By contrast, Pittius et al., niques were applied in order to detect the presence of (1984) reported the presence of Met8 in the locus cer- Met8 (Pittius et al., 1984; Iadarola et al., 1991), our uleus, the and in the area pos- results are in general in agreement with these studies. trema of the human brainstem, in which we failed to find Thus, using these techniques and immunocytochemistry Met8-ir structures using an immunocytochemical the presence of Met8 was detected in the central gray of technique. the , the inferior and superior colliculi, the sub- These differences could be due to the sensitivity of stantia nigra and in the red nucleus of the human brain- the techniques used (radioimmunoassay, gel filtration, stem. Pittius et al. (1984) found the highest concentra- immunocytochemistry, etc.). Moreover, Pittius et al. tions of Met8 in the human , sub- (1984) show a higher concentration in the dorsal than in stantia nigra and in other non-brainstem nuclei. In the the ventral medulla oblongata. This is in agreement with former two nuclei, we also observed a high density of ir our results, since we also observed a higher density of ir R. Coveñas et al. / Neuroscience 128 (2004) 843–859 853

Fig. 11. Met8-ir fibers in the human medulla oblongata. (A) Ir fibers (arrows) located in the ALS and in the LRNu. Scale barϭ100 ␮m. (B) Fibers containing Met8 in the MAO. Photograph taken from brain W-3. D, dorsal; M, medial. Scale barϭ100 ␮m. (C) Ir fibers located in the DCNu. Photograph taken from brain W-3. D, dorsal; L, lateral. Scale barϭ200 ␮m. (D) High-power image of the region delimited in Fig. 11C. Scale barϭ50 ␮m. (E) A low magnification of the ventro-lateral region of the medulla oblongata. For a nomenclature of the nuclei, see list of abbreviations. Photograph taken from brain M-1. D, dorsal; L, lateral. Scale barϭ500 ␮m. (F) A high magnification of the region delimited in Fig. 11E (VCNu). Note the ir fibers located in the ventral cochlear nucleus. Scale barϭ50 ␮m. structures containing Met8 in the dorsal than in the on the Met8-ir fibers (Ibuki et al., 1989). However, some ventral medulla oblongata. In sum, our results show a differences are apparent. Thus, in the human, but not in very more widespread distribution of Met8 in the human the monkey, Met8-ir fibers have been found in the hypo- brainstem than previously reported (Pittius et al., 1984; glossal nucleus, the nucleus ambiguus, the medial and Iadarola et al., 1991), since this is the first study in which lateral vestibular nuclei and in the abducens nucleus. By 8 the presence of Met in a large number of human brain- contrast, in the monkey but not in the human, Met8-ir stem nuclei and tracts has been demonstrated. fibers have been observed in the locus ceruleus and in the trapezoid body. In comparison with the distribution of Comparison of the distribution of Met8 in the primate Met8-ir cell bodies in the monkey brainstem (Ibuki et al., brainstem 1989), our results for the human brainstem showed Our results are also in general in agreement with those differences. In this sense, whereas in both human and described in the monkey brainstem for the distribution monkey cell bodies containing Met8 are found in the 854 R. Coveñas et al. / Neuroscience 128 (2004) 843–859

Fig. 12. Met8-ir fibers and cell bodies in the human medulla oblongata and pons. (A) A low magnification of the lateral region of the medulla oblongata. The area delimited (SpTNu) can be observed at higher magnification in Fig. 12B. For a nomenclature of the nuclei, see list of abbreviations. Photograph taken from brain M-2. D, dorsal; M, medial. Scale barϭ500 ␮m. (B) Ir fibers (arrows) in the rostral part of the spinal trigeminal nucleus. Scale barϭ50 ␮m. (C) A low magnification of the SpNu. Photograph taken from brain M-2. D, dorsal; g, gelatinosa part; M, medial. Scale barϭ500 ␮m. (D) High-power image of the region delimited in Fig. 11C. Scale barϭ100 ␮m. (E) Ir cell bodies located ventral to the SO. Photograph taken from brain W-3. D, dorsal; M, medial. Scale barϭ200 ␮m. (F) High-power magnification of the region delimited in Fig. 12E. Note the ir cell bodies. Scale barϭ100 ␮m. reticular formation of the medulla oblongata and pons, tion of the midbrain, the interpeduncular nucleus and in the solitary nucleus, the nucleus prepositus and in the the trapezoid body. It therefore appears that the distri- central gray of the pons and midbrain, in other brainstem bution of cell bodies containing Met8 in the monkey nuclei the presence of Met8-ir cell bodies is found in only brainstem shows significant differences in comparison one of the two species. Thus, in humans but not in the with the distribution of perikarya containing the octapep- monkey, Met8-ir perikarya are present in the hypoglos- tide in the human brainstem. The differences observed sal nucleus, the lateral cuneate nucleus, the medial in both species concerning the distribution of cell bodies longitudinal fasciculus and in the inferior and superior containing Met8 could be due to technical consider- colliculi. However, in the monkey, but not in humans, ations; for example the antisera used, the administration Met8-ir cell bodies are located in the central gray of the of colchicine, and/or species differences. It should be medulla oblongata, the dorsal motor nucleus of the va- noted that such a discrepancy could be due to the gus, the spinal trigeminal nucleus, the reticular forma- administration of colchicine, since in the monkey the R. Coveñas et al. / Neuroscience 128 (2004) 843–859 855

Fig. 13. Met8-ir cell bodies in the human pons. (A) A low magnification of the dorsal region of the pons. The area delimited can be observed at higher magnification in Fig. 13B. Photograph taken from brain M-1. D, dorsal; M, medial. Scale barϭ200 ␮m. (B) Cluster of ir cell bodies located in the RetF. Scale barϭ100 ␮m. (C) Ir cell bodies located in the midline. Photograph taken from brain W-2. D, dorsal; M, medial. Scale barϭ500 ␮m. (D) High-power magnification of the region delimited in Fig. 13C. Scale barϭ100 ␮m. (E) A low magnification of the midline of the pons. The area delimited can be observed at higher magnification in Fig. 13F. Dashed lines indicate the midline. Photograph taken from brain M-2. D, dorsal. Scale barϭ500 ␮m. (F) Cluster of ir cell bodies located in the midline. Scale barϭ100 ␮m. drug was administered into the ventricle in order to baum 1985; Belda et al., 2003), our results in the human accumulate the neuropeptide in cell bodies of the brain- brainstem point to a more widespread distribution of stem (Ibuki et al., 1989). Finally, in agreement with the Met8-ir fibers. On comparing the distribution of Met8-ir above, this paper shows for the first time the presence of perikarya in the brainstem, it seems that in the human 8 Met -ir structures in the primate hypoglossal nucleus, brainstem the distribution of Met8-ir cell bodies is slightly the medial, spinal and lateral vestibular nuclei, the ab- more widespread than in the cat (Belda et al., 2003). ducens nucleus and in the facial nucleus. Thus, for example, in humans, but not in the cat, Met8-ir

8 perikarya were found in the hypoglossal nucleus, the Comparison of the distribution of Met in the lateral cuneate nucleus, the medial and spinal vestibular mammalian non-primate brainstem nuclei, the reticular formation, the central gray of the In comparison with the distribution of ir fibers containing pons, and in the pericentral and central nuclei of the Met8 found in the brainstem of the cat (Cruz and Bas- inferior colliculus. In both species, the methodology and 856 R. Coveñas et al. / Neuroscience 128 (2004) 843–859

Fig. 14. Met8-ir cell bodies and fibers in the human pons and mesencephalon. (A) Ir fibers and cell bodies in the pons central gray (CeGy). D, dorsal; M, medial. Photograph taken from brain W-2. Scale barϭ100 ␮m. (B) A low magnification of the inferior colliculus. For a nomenclature of the nuclei, see list of abbreviations. Photograph taken from brain W-1. D, dorsal; M, medial. Scale barϭ500 ␮m. (C) High-power image of the region delimited in Fig. 14B. The areas delimited in the dorsal and ventral rectangles are respectively observed at higher magnification in Figs. 14D and 14E. Scale barϭ200 ␮m. (D) Ir cell bodies (arrows) and fibers in the pericentral nucleus of the inferior colliculus. Scale barϭ50 ␮m. (E) Cell bodies (arrows) in the central nucleus of the inferior colliculus. Scale barϭ50 ␮m. (F) A low magnification of the mesencephalic central gray. The area delimited is observed at higher magnification in Fig. 15A. Photograph taken from brain W-3. D, dorsal; M, medial. Scale barϭ500 ␮m. the primary antiserum used was the same, except that in Finally, on comparing the distribution of Met8-ir the feline colchicine was administered into the fourth structures in the human and rat brainstems (Fallon and ventricle (Belda et al., 2003). Despite the administration Leslie 1986; Harlan et al., 1987; Murakami et al., 1987; of the drug in the cat, as mentioned, Met8-ir cell bodies Palkovits 1988), it seems that in general the distribution are slightly more widespread distributed in the human of Met8-ir fibers is quite similar. However, the distribution brainstem than in the same region of the cat. This sug- of Met8-ir perikarya is more widespread in the rat than in gests that the discrepancies found in both species re- humans. This difference could also be due to technical garding the distribution of Met8-ir perikarya would be considerations, since in the rat colchicine was adminis- due to species differences and not to methodological tered in the ventricle (Fallon and Leslie 1986; Murakami considerations. et al., 1987), and in this rodent not only immunocyto- R. Coveñas et al. / Neuroscience 128 (2004) 843–859 857

Fig. 15. Met8-ir cell bodies and fibers in the human mesencephalon. (A) Cell body (arrow) and fibers containing Met8 in the central gray. Scale barϭ100 ␮m. (B) Low-power image of the mesencephalic central gray. Photograph taken from brain W-. D, dorsal; M, medial. Scale barϭ200 ␮m. (C) High-power image of the region delimited in Fig. 15B. Note the ir fibers. Scale barϭ50 ␮m. (D) Ir cell body (arrow) and fibers in the superior colliculus. Photograph taken from brain M-1. D, dorsal; L, lateral. Scale barϭ50 ␮m. chemical techniques were applied (Fallon and Leslie stance, there is substantial evidence that opioid pep- 1986; Murakami et al., 1987; Palkovits 1988) but also an tides in brainstem nuclei, including the solitary nucleus, in situ hybridization (Harlan et al., 1987) technique was are involved in respiratory, cardiovascular, and gastric applied in the brainstem. By contrast, here in the human regulation (Palkovits and Eskay, 1987; Cheng et al., brainstem only immunocytochemical techniques were 1996; Yuan and Foss, 1999), and also on the modulation carried out. of taste responses (Cheng-Shu et al., 2003). In this

8 sense, it has been described that the respiratory distur- Possible Met -containing pathways in human bances observed in Rett syndrome are associated with Regarding the results found here, we have no data indi- a decreased expression of Met8 in the parabraquial, cating whether the Met8-ir perikarya observed in the hu- hypoglossal, dorsal vagal and solitary nuclei (Saito et man brainstem are local and/or projecting neurons. How- al., 2001). ever, it can be suggested, for example, that the dorsal Furthermore, an analgesic role for Met8 has been motor nucleus of the vagus, the spinal trigeminal nucleus, described in the spinal trigeminal nucleus (Langemark et the dorsal accessory olivary nucleus, the reticular forma- al., 1995), and also in patients suffering infantile tion of the midbrain, the substantia nigra and the interpe- spasms, Met8 was almost completely vanished in this duncular nucleus would receive Met8-ir afferences, since nucleus (Hayashi et al., 2000). Finally, the presence of in these nuclei a high density of ir fibers containing Met8, Met8 in the substantia nigra suggests its possible in- but no cell bodies containing the neuropeptide, were volvement on motor function. Moreover, it has been observed. demonstrated a loss of Met8 in this area in Parkinson’s

8 and Huntington’s diseases (Iadarola et al., 1991; de Possible physiological functions of Met in the Ceballos and López-Lozano, 1999). Thus, the data pre- human brainstem sented in this paper support the involvement of Met8 in The widespread distribution of Met8-ir structures in the these physiological functions in humans human brainstem suggests that this peptide might be We hope that our study on the distribution of fibers and involved in several physiological actions, acting as a cell bodies containing Met8 in the human brainstem will neurotransmitter and/or neuromodulator. Some of these serve to compare the distribution of Met8-ir structures physiological actions are well documented. For in- found in normal human brainstems with those observed in 858 R. Coveñas et al. / Neuroscience 128 (2004) 843–859

8 8 Fig. 16. Met8-ir fibers in the human mesencephalon. (A) Low-power Fig. 17. Specificity of the Met antiserum. (A) Met -ir fibers (arrows) in magnification of the ventro-medial mesencephalon. Observe the pres- the dorsal medulla oblongata in a section to which the immunocyto- ence of ir fibers in the IPNu. Photograph taken from brain M-2. D, chemistry method was applied. The delimited area is observed at dorsal; M, medial. Scale barϭ200 ␮m. (B) High-power magnification of higher magnification in Fig. 17C. D, dorsal; M, medial. Scale ϭ ␮ the area delimited in the left rectangle of Fig. 16A. Scale barϭ50 ␮m. bar 200 m. (B) No immunoreactivity appeared in the same region in (C) High-power magnification of the area delimited in the right rectan- a section adjacent to that shown in Fig. 17A when preabsorption of the 8 8 gle of Fig. 16A. Scale barϭ50 ␮m. first antiserum (anti-Met ) with synthetic Met was carried out prior to applying the immunocytochemical technique. Arrowheads indicate a blood vessel. Scale barϭ200 ␮m. (C) Ir fibers in the midline region. brainstems showing a given pathology (e.g. Parkinson or Scale barϭ100 ␮m. Alzheimer disease), as well as for studying the physiolog- ical actions of Met8 in the human brainstem. In this sense, phrenic patients in comparison with those found in the it has been demonstrated that the concentration of Met8 same brainstem region of normal patients (Iadarola et al., was significantly elevated in the substantia nigra of schizo- 1991). R. Coveñas et al. / Neuroscience 128 (2004) 843–859 859

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(Accepted 12 July 2004)