7
Peptides, Vol. 14, pp. 1263-1269, 1993 0196-9781/93 $6.00 + .00 Printed in the USA. Copyright © 1993PergamonPress Ltd. Alpha-Neo-Endorphin-Like Immunoreactivity in the Cat Brain Stem P. MARCOS, *1 R. COVElqAS,* J. A. NARVAEZ,I" G. TRAMU,J; J. A. AGUIRREt AND S. GONZALEZ-BARONt *Departamento de Biolog{a Celular y Patolog{a, Facultad de Medicina, Universidad de Salamanca, Avda. Campo Charro s/n, 37007 Salamanca, Spain, -?Departamento de Fisiolog{a, Facultad de Medicina, Universidad de Mdlaga, Mdlaga, Spain, and ¢Laboratoire de Neurocytochimie Fonctionnelle, C.N.R.S., U.R.A. 339, Universit£ de Bordeaux L France Received 30 March 1993 MARCOS, P., R. COVENAS, J. A. NARVAEZ, G. TRAMU, J. A. AGUIRRE AND S. GONZ,g, LEZ-BARt~N. Alpha-neo- endorphin-like immunoreactivity in thecat brain stem. PEPTIDES 14(6) 1263-1269, 1993.--This paper examines the distribution of fibers and cell bodies containing alpha-neo-endorphin in the cat brain stem by using an indirect immunoperoxidase technique. A high or moderate density of immunoreactive cell bodies was found in the superior central nucleus, nucleus incertus, dorsal tegmental nucleus, nucleus of the trapezoid body, and in the laminar spinal trigeminal nucleus, whereas a low density of such perikarya was observed in the inferior colliculus, nucleus praepositus hypoglossi, dorsal nucleus of the raphe, nucleus of the brachium of the inferior colliculus, and in the nucleus of the solitary tract. The highest density of immunoreactive fibers was found in the substantia nigra, dorsal motor nucleus of the vagus, nucleus coeruleus, lateral tegmental field, marginal nucleus of the brachium conjunctivum, and in the inferior and medial vestibular nuclei. These results indicate that alpha-neo-endorphin is widely distributed in the cat brain stem and suggest that the peptide could play an important role in several physiological functions, e.g., those involved in respiratory, cardiovascular, auditory, and motor mechanisms. Alpha-neo-endorphin Immunocytochemistry Brain stem Cat PRODYNORPHIN (proenkephalin B) (...-Lys-Arg-Tyr ~75- • . . -Pro-Lys184-Arg-Ser-Ser-Glu - . . . -Arg-Tyr 2°9- . . . - Gin225-Lys-Arg-Tyr228-... -Va1256) is the precursor of a series of opiate peptides such as alpha-neo-endorphin (...-Tyr~TL . . . - L y s 184 . . . ) , beta-endorphin (...-Tyrl75-...-Pro 183 • . .), dynorphin A ( .... Tyr 2°9- . . .-Gln 22s . . .) and dynor- phin B (... -Tyr228- . . . -Va1256)(see 12). Alpha-neo-endorphin has been extracted from the hypothalamus and fully sequenced. It is known that the sequence of this peptide is conserved in all species thus far examined (16). A large number of immunocy- tochemical and radioimmunoassay studies have been carried out on the distribution of prodynorphin and the different dy- norphins in the central nervous system of mammals (3,11,14,15,17,19-21), but few studies have examined the dis- tribution of alpha-neo-endorphin (3,15,17,21). These studies have mainly been carried out in rats and humans. However, no comprehensive studies have been done in other animal models (e.g., cat). Over the last 3 years, we have described in the cat brain stem the anatomical distribution of cell bodies and fibers containing somatostatin-28(1-12), neurotensin, cholecystokinin octapep- tide, and neuropeptide Y (5,8-10). However, no data are avail- able on the distribution of immunoreactive structures containing alpha-neo-endorphin in the cat central nervous system. Thus, the aim of this paper was to determine the distribution ofalpha- neo-endorphin in the brain stem of the cat using an immuno- peroxidase technique and to compare our findings with the dis- tribution of the neuropeptides previously described in the feline brain stem ( 1,4,5,8-10,18). METHOD Seven male, adult cats (2-3 kg body weight) were used in this study• Under deep ketamine anesthesia (40-50 mg/kg), two animals received unilateral intraventricular (fourth ven- tricle) injections ofcolchicine (300 ~g in 5 #1 of distilled water). Two days after administration of the drug, the treated and un- treated animals were anesthesized and perfused via the as- cending aorta with 500 ml of 0.9% NaCI and 3 1 of 4% para- formaldehyde in 0.15 M phosphate buffer 7.2. The brains were removed, the brain stems were dissected out, postfixed over- night in the latter solution, and placed in increasing sucrose baths (10-30%) until they sank. Using a cryostat, 80-tzm frontal sections were cut and processed for immunostaining as pre- viously described (5,6,8-10). The immunological properties of the alpha-neo-endorphin antiserum have been reported previously (6,7). Moreover, the Requests for reprints should be addressed to P. Marcos. 1263

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Peptides, Vol. 14, pp. 1263-1269, 1993 0196-9781/93 $6.00 + .00 Printed in the USA. Copyright © 1993 Pergamon Press Ltd.

Alpha-Neo-Endorphin-Like Immunoreactivity in the Cat Brain Stem

P. M A R C O S , *1 R. COVElqAS,* J. A. N A R V A E Z , I " G. T R A M U , J ; J. A. A G U I R R E t A N D S. G O N Z A L E Z - B A R O N t

*Departamento de Biolog{a Celular y Patolog{a, Facultad de Medicina, Universidad de Salamanca, Avda. Campo Charro s/n, 37007 Salamanca, Spain, -?Departamento de Fisiolog{a, Facultad de Medicina,

Universidad de Mdlaga, Mdlaga, Spain, and ¢Laboratoire de Neurocytochimie Fonctionnelle, C.N.R.S., U.R.A. 339, Universit£ de Bordeaux L France

Rece ived 30 M a r c h 1993

MARCOS, P., R. COVENAS, J. A. NARVAEZ, G. TRAMU, J. A. AGUIRRE AND S. GONZ,g, LEZ-BARt~N. Alpha-neo- endorphin-like immunoreactivity in the cat brain stem. PEPTIDES 14(6) 1263-1269, 1993.--This paper examines the distribution of fibers and cell bodies containing alpha-neo-endorphin in the cat brain stem by using an indirect immunoperoxidase technique. A high or moderate density of immunoreactive cell bodies was found in the superior central nucleus, nucleus incertus, dorsal tegmental nucleus, nucleus of the trapezoid body, and in the laminar spinal trigeminal nucleus, whereas a low density of such perikarya was observed in the inferior colliculus, nucleus praepositus hypoglossi, dorsal nucleus of the raphe, nucleus of the brachium of the inferior colliculus, and in the nucleus of the solitary tract. The highest density of immunoreactive fibers was found in the substantia nigra, dorsal motor nucleus of the vagus, nucleus coeruleus, lateral tegmental field, marginal nucleus of the brachium conjunctivum, and in the inferior and medial vestibular nuclei. These results indicate that alpha-neo-endorphin is widely distributed in the cat brain stem and suggest that the peptide could play an important role in several physiological functions, e.g., those involved in respiratory, cardiovascular, auditory, and motor mechanisms.

Alpha-neo-endorphin Immunocytochemistry Brain stem Cat

PRODYNORPHIN (proenkephalin B) ( . . . - L y s - A r g - T y r ~75- • . . -Pro-Lys184-Arg-Ser-Ser-Glu - . . . -Arg-Tyr 2°9- . . . -

Gin225-Lys-Arg-Tyr228-... -Va1256) is the precursor of a series of opiate peptides such as alpha-neo-endorphin ( . . . - T y r ~ T L . . . - L y s 184 . . . ) , beta-endorphin ( . . . - T y r l 7 5 - . . . - P r o 183

• . .), dynorphin A ( . . . . Tyr 2°9- . . . -Gln 22s . . .) and dynor- phin B ( . . . -Tyr 228- . . . -Va1256) (see 12). Alpha-neo-endorphin has been extracted from the hypothalamus and fully sequenced. It is known that the sequence of this peptide is conserved in all species thus far examined (16). A large number of immunocy- tochemical and radioimmunoassay studies have been carried out on the distribution of prodynorphin and the different dy- norphins in the central nervous system of mammals (3,11,14,15,17,19-21), but few studies have examined the dis- tribution of alpha-neo-endorphin (3,15,17,21). These studies have mainly been carried out in rats and humans. However, no comprehensive studies have been done in other animal models (e.g., cat).

Over the last 3 years, we have described in the cat brain stem the anatomical distribution of cell bodies and fibers containing somatostatin-28(1-12), neurotensin, cholecystokinin octapep- tide, and neuropeptide Y (5,8-10). However, no data are avail- able on the distribution of immunoreactive structures containing

alpha-neo-endorphin in the cat central nervous system. Thus, the aim of this paper was to determine the distribution ofalpha- neo-endorphin in the brain stem of the cat using an immuno- peroxidase technique and to compare our findings with the dis- tribution of the neuropeptides previously described in the feline brain stem ( 1,4,5,8-10,18).

METHOD

Seven male, adult cats (2-3 kg body weight) were used in this study• Under deep ketamine anesthesia (40-50 mg/kg), two animals received unilateral intraventricular (fourth ven- tricle) injections ofcolchicine (300 ~g in 5 #1 of distilled water). Two days after administrat ion of the drug, the treated and un- treated animals were anesthesized and perfused via the as- cending aorta with 500 ml of 0.9% NaCI and 3 1 of 4% para- formaldehyde in 0.15 M phosphate buffer 7.2. The brains were removed, the brain stems were dissected out, postfixed over- night in the latter solution, and placed in increasing sucrose baths (10-30%) until they sank. Using a cryostat, 80-tzm frontal sections were cut and processed for immunos ta in ing as pre- viously described (5,6,8-10).

The immunological properties of the alpha-neo-endorphin antiserum have been reported previously (6,7). Moreover, the

Requests for reprints should be addressed to P. Marcos.

1263

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.7

FIG. 1. Distribution of alpha-NEO-E-IR cell bodies and fibers in frontal planes of the brain stem of the cat corresponding to the anteroposterior stereotaxic plane levels from A 1.6 to P 16.0 of the Berman stereotaxic atlas (2). lmmunoreactive fibers are represented by continuous lines, whereas cell bodies are represented by closed circles, triangles, and squares, their shape being related to the density of perikarya (O, high density: > l0 cell bodies; A, middle density: 5-10 cell bodies; I , lower density: < 5 cell bodies). The anterior (A) or posterior (P) level, in mm with respect to the zero stereotaxic point of each section, is indicated at the lower right. 5P: principal sensory trigeminal nucleus; 5SL: laminar spinal trigeminal nucleus; 5SM: alaminar spinal trigeminal nucleus, magnocellular division; 5SP: alaminar spinal trigeminal nucleus, parvocellular division; 5ST: spinal trigeminal tract; 7M: facial nucleus; AQ: aqueduct; BC: brachium conjunctivum; BCM: marginal nucleus of the brachium conjunctivum; BIN: nucleus of the brachium of the inferior colliculus; CAE: nucleus coeruleus; CE: central canal; CS: superior central nucleus; CUC: cuneate nucleus, caudal division; CUR: cuneate nucleus, rostral division; DMV: dorsal motor nucleus of the vagus; DRM: dorsal nucleus of the raphe, medial division; FIG: gigantocellular tegmental field; FTL: lateral tegmental field; GRR: gracile nucleus, rostral division; ICC: central nucleus of the inferior colliculus; ICP: pericentral

1264

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ALPHA-NEO-ENDORPHIN IN THE CAT BRAIN STEM 1265

specificity of the immunostaining was controlled by: a) the preabsorption of the antiserum with synthetic alpha-neo-en- dorphin (100 ~g/ml diluted antiserum); b) omitting the alpha- neo-endorphin antiserum in the first incubation bath (in both cases, no residual immunoreactivity was found); c) no significant reduction in the immunolabeling was found when alpha-neo- endorphin antiserum was preabsorbed with an excess (10 -7 M) of synthetic dynorphin A(1-8), dynorphin A( 1-17), dynorphin B, beta-neo-endorphin, beta-endorphin, methionine- enkephalin, or leucine-enkephalin; d) possible interference by endogenous peroxidases was ruled out by staining some sections beginning with the diaminobenzidine step. No reaction was visualized. Mapping was carried out according to the stereotaxic atlas of Berman (2), and the same atlas was used for the terminology of the brain stem nuclei. Finally, the term alpha-neo-endorphin- like immunoreactive (alpha-NEO-E-IR) was used to describe the staining results in our material.

RESULTS

Figure 1 shows the distribution ofalpha-NEO-E-IR cell bodies and fibers in the cat brain stem. The densest clusters of alpha- NEO-E-IR neurons were found in the superior central nucleus, nucleus of the trapezoid body, laminar spinal trigeminal nucleus, nucleus incertus, and in the pericentral division of the dorsal tegmental nucleus, whereas the highest density of alpha-NEO- E-IR fibers was observed in the substantia nigra, dorsal motor nucleus of the vagus, inferior and medial vestibular nuclei, nu- cleus coeruleus, marginal nucleus of the brachium conjunctivum, lateral tegmental field, and in the region located below the retro- rubral nucleus.

At rostral level (A 1.6) [Fig. I(A)], a low density of both alpha-NEO-E-IR cell bodies and fibers was visualized in the nu- cleus of the brachium of the inferior colliculus. In addition, a moderate density of immunoreactive fibers was found in the outer division of the posterior interpeduncular nucleus and in the paramedian interpeduncular nucleus, and a high density was observed extending laterally from the latter nucleus to the region located below the retrorubral nucleus [Fig. 2(A)].

More caudally, a low density of cell bodies containing alpha- NEO-E was observed in the central and pericentral nuclei of the inferior colliculus [Fig. I(B)], paracentral division of the teg- mental reticular nucleus [Figs. I(B), 2(B)], below the medial longitudinal bundles [Fig. I(C)], and in the median division of the dorsal nucleus of the raphe [Fig. I(B)], whereas a high density of immunoreactive cell bodies was found in the superior central nucleus [Figs. I(B,C), 2(C-E), 3(A)], nucleus of the trapezoid body [Figs. 2(C-E), 3(C,D)], and between the medial longitudinal bundles [Fig. I(C)]; a low/moderate density was seen in the nu- cleus incertus [Figs. I(B,C), 3(A)] and a moderate density in the pericentral division of the dorsal tegmental nucleus [Figs. I(C),

3(A,B)]. Scanty alpha-NEO-E-IR fibers were observed in the pericentral nucleus of the inferior colliculus [Fig. l(B)], pericen- tral division of the dorsal tegmental nucleus [Fig. I(B,C)], medial, dorsolateral and lateral divisions of the pontine gray [Fig. l(B)], accessory dorsal tegmental nucleus [Fig. l(C)], nucleus of the trapezoid body [Fig. I(C-E)], Kflliker-Fuse nucleus [Fig. l(C)], and in the gigantocellular tegmental field [Fig. 1(C)]. In addition, immunoreactive fibers, at a moderate density, were visualized in the following nuclei: median division of the dorsal nucleus of the raphe [Fig. I(B)], nucleus coeruleus [Fig. I(B,C)], nucleus incertus [Fig. I(B,C)], central division of the tegmental reticular nucleus [Fig. 1 (B)], nucleus sagulum [Fig. 1 (B)], and in the mar- ginal nucleus of the brachium conjunctivum [Fig. l(C)].

At P 5.2 [Fig. l(D)], cell bodies containing alpha-NEO-E were visualized close to the midline and the fourth ventricle [Fig. 2(F)]. A moderate density of alpha-NEO-E-IR fibers was visualized in the principal sensory trigeminal nucleus, magno- cellular division of the alaminar spinal trigeminal nucleus, and in the lateral tegmental field [Fig. I(D-H)].

A low density of alpha-NEO-E-IR perikarya was found in the nucleus praepositus hypoglossi [Fig. I(E,F)]. The latter nu- cleus [Fig. I(E,F)], the medial division of the facial nucleus [Fig. l(E)], and the infratrigeminal nucleus [Fig. l(E-G)] showed a low density of immunoreactive fibers. A moderate density of fibers containing alpha-NEO-E-IR was found in the inferior ves- tibular nucleus [Fig. I(E,F)], and a low/moderate density in the medial vestibular nucleus [Fig. I(E,F)] and in the parvocellular division of the alaminar spinal trigeminal nucleus [Fig. I(E-G)].

Finally, a low density of alpha-NEO-E-IR cell bodies was visualized in the medial nucleus of the solitary tract [Fig. I(G,H)] and a moderate density in the laminar spinal trigeminal nucleus (Figs. I(H), 3(E,F)]. A low number ofalpha-NEO-E-IR processes was found in the nucleus intercalatus [Fig. I(G)], rostral and caudal divisions of the cuneate nucleus [Fig. I(G,H)], laminar spinal trigeminal nucleus [Fig. I(H)], and in the internal and external divisions of the lateral reticular nucleus [Fig. l(H)]. In the medial nucleus of the solitary tract (Figs. I(G,H), 2(G)], a low/moderate density ofalpha-NEO-E-IR fibers was visualized, whereas a high density of immunoreactive fibers was found in the dorsal motor nucleus of the vagus [Fig. I(G)].

DISCUSSION

We have described for the first time the distribution ofalpha- NEO-E-IR cell bodies and fibers in the brain stem of the cat. Thus, the midbrain and pons showed more alpha-NEO-E-IR cell bodies than the medulla oblongata, whereas in the three brain stem regions a broad distribution of immunoreactive fibers was found. Most alpha-NEO-E-IR cell bodies were seen only in the brains of the colchicine-treated animals. In those not treated with the drug only very few perikarya in the inferior colliculus

nucleus of the inferior colliculus; IFF: infratrigeminal nucleus; INC: nucleus incertus; INT: nucleus intercalatus; 10: inferior olive; IOD: dorsal accessory nucleus of the inferior olive; IOM: medial accessory inferior olive; IPO: posterior interpeduncular nucleus, outer division; IPP: paramedian interpeduncular nucleus; KF: KiSlliker-Fuse nucleus; LRI: lateral reticular nucleus, internal division; LRX: lateral reticular nucleus, external division; MLB: medial longitudinal bundle; P: pyramidal tract; PGD: pontine gray, dorsolateral division; PGL: pontine gray, lateral division; PGM: pontine gray, medial division; PH: nucleus praepositus hypogiossi; RR: retrorubral nucleus; S: solitary tract; SAG: nucleus sagulum; SM: medial nucleus of the solitary tract; T: nucleus of the trapezoid body; TAD: accessory dorsal tegmental nucleus; TB: trapezoid body; TDP: dorsal tegmental nucleus, pericentral division; TRC: tegmental reticular nucleus, central division; TRP: tegmental reticular nucleus, paracentral division; V4: fourth ventricle; VIN: lateral vestibular nucleus; VMN: medial vestibular nucleus.

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0 [-n > .m

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ALPHA-NEO-ENDORPHIN IN THE CAT BRAIN STEM 1267

showed immunoreactivity. There were no significant differences in fiber distribution and staining intensities between the normal and colchicine-treated animals.

In general, our results are in agreement with the studies carried out by Maysinger et al. (15) and Cone et al. (3), since these authors found alpha-NEO-E in the rat midbrain, pons, and me- dulla oblongata, and in the human pons and medulla oblongata. Thus, in humans Maysinger et al. (15) observed more alpha- NEO-E in the pons than in the medulla. However, they observed alpha-NEO-E in the human periaqueductal gray, in which we did not find alpha-NEO-E-IR structures in the cat. Moreover, in rats Cone et al. (3) found more alpha-NEO-E in the midbrain than in the medulla-pons.

In comparison with previous studies on the distribution of alpha-NEO-E-IR fibers and cell bodies in the rat brain stem (20,21), it seems that in general the distribution observed in the brain stem of the cat is more widespread. Thus, we visualized brain stem nuclei of the cat containing alpha-NEO-E-IR peri- karya, which were not found in rats (20,21): e.g., the inferior colliculus, superior central nucleus, paracentral division of the tegmental reticular nucleus, dorsal nucleus of the raphe, nucleus incertus, nucleus of the trapezoid body, nucleus praepositus hy- poglossi, nucleus of the solitary tract, and the laminar spinal trigeminal nucleus. In the cat, we also observed alpha-NEO-E- IR fibers in the interpeduncular nucleus, inferior colliculus, nu- cleus coeruleus, nucleus of the trapezoid body, inferior and me- dial vestibular nuclei, dorsal nucleus of the raphe, Krl l iker- Fuse nucleus, lateral tegmental field, dorsal motor nucleus of the vagus, and in the nucleus praepositus hypoglossi, whereas in the rat alpha-NEO-E-IR fibers were not found in these brain stem regions (20,21 ). However, in the rat, immunoreactive fibers have been observed in the solitary tract (20,21), but not in the cat.

In sum, alpha-NEO-E-IR structures seem to be more widely distributed in the brain stem of the cat compared with the rat (20,21). This discrepancy could be due to technical considera- tions (antisera, fixative and the immunocytochemical technique used, injections ofcolchicine) and/or species differences. To an- swer this question, additional experiments should be carried out in the rat using the same protocol that we used in the feline.

Previously, it was not known whether the alpha-NEO-E-IR perikarya found in the cat brain stem are local or projecting neurons; additionally, the origin of the described immunoreac- tive fibers remains unknown. However, according to the mor- phological data observed in the feline, it appears that the sub- stantia nigra, marginal nucleus of the brachium conjunctivum, medial and inferior vestibular nuclei, and the dorsal motor nu- cleus of the vagus could receive alpha-NEO-E-IR afferents, since in all these nuclei a high or moderate density ofimmunoreactive fibers was visualized but no alpha-NEO-E-IR perikarya.

This observation is consistent with the data reported for the rat (13) in which, for example, a pathway containing alpha- NEO-E has been described to course from the striatum to the substantia nigra. The immunoreactive perikarya observed in the superior central nucleus and in the nucleus of the trapezoid body could be projecting neurons, since in both regions a high

density of alpha-NEO-E-IR cell bodies was found, but no, or a very low density of, immunoreactive fibers. The neurons visu- alized in the inferior colliculus could be interneurons, since a low density of both immunoreactive cell bodies and fibers was observed. Alternatively, alpha-NEO-E-IR perikarya might be projecting neurons and the fibers containing alpha-NEO-E might originate in other nuclei of the cat CNS. In sum, the cell bodies that give rise to the alpha-NEO-E-IR fibers found in the cat brain stem and whether the perikarya containing alpha-NEO-E are interneurons or projecting neurons remain unknown.

The distribution of fibers and cell bodies containing methi- onine-enkephalin, calcitonin gene-related peptide, substance P, neurotensin, somatostatin-28(1-12), cholecystokinin octapep- tide, and neuropeptide Y has been described previously in the cat brain stem (1,4,5,8-10,18). In general, except for the cho- lecystokinin octapeptide, the above-mentioned neuropeptides were broadly distributed and showed more immunoreactive structures than the alpha-NEO-E in the cat brain stem. Fur- thermore, the anatomical relationship between alpha-NEO-E- IR fibers and substance P, neurotensin, somatostatin, and neu- ropeptide Y immunoreactive fibers is greater than between aipha-NEO-E-IR fibers and methionine-enkephalin, cholecys- tokinin octapeptide, and calcitonin gene-related peptide positive processes.

Thus, at least four of the neuropeptides--alpha-NEO-E, sub- stance P, neurotensin, somatostatin, and neuropeptide Y--have been observed in fibers in the cat brain stem nuclei: the nucleus of the solitary tract, interpeduncular nucleus, laminar spinal tri- geminal nucleus, lateral tegmental field, dorsal motor nucleus of the vagus, marginal nucleus of the brachium conjunctivum, Krlliker-Fuse nucleus, nucleus coeruleus, dorsal tegmental nu- cleus, inferior colliculus, and the nucleus of the trapezoid body. These data suggest possible interactions among some of the five above-cited neuropeptides in the brain stem of the cat, and an elaborate modulation of functions in which these brain stem nuclei are involved. These data also suggest a possible colocal- ization of such neuroactive substances in the cat brain stem. Moreover, in the nucleus of the solitary tract, medial vestibular nucleus, interpeduncular nucleus, substantia nigra, laminar spi- nal trigeminal nucleus, and in the lateral tegmental field, me- thionine-enkephalin and alpha-NEO-E-IR fibers have been observed in the feline, whereas alpha-NEO-E-IR fibers and calcitonin gene-related peptide immunoreactive processes were visualized in the nucleus of the solitary tract, lateral tegmental field, dorsal motor nucleus of the vagus, and in the laminar spinal trigeminal nucleus. Finally, a weak relationship between the distribution of alpha-NEO-E-IR fibers and cholecystokinin octapeptide positive fibers was observed, since in the cat brain stem both types of fibers were found in the nucleus of the solitary tract, interpeduncular nucleus, dorsal motor nucleus of the vagus, marginal nucleus of the brachium conjunctivum, Krlliker-Fuse nucleus, and in the nucleus coeruleus.

On comparing the distribution of cell bodies containing alpha- NEO-E, substance P, neurotensin, or neuropeptide Y, the co- existence of the two former neuropeptides can be suggested in neurons of the superior central nucleus, nucleus incertus, and

FIG. 2. Alpha-neo-endorphin immunoreactive cell bodies and fibers in the cat brain stem. (A) A 1.6. Immunoreactive fibers (arrows) located below the retrorubral nucleus. BP: Brachium pontis (×25). (B) P 1.5. Immunoreactive cell bodies (arrows) in the paracentral division of the tegmental reticular nucleus (X 100). (C) P 1.5. Cluster of immunoreactive neurons in the superior central nucleus (CS). MLB: medial longitudinal bundle (x25). (D) P 1.5. High power image of the area delimited in (C) (× 100). (E) P 3. I. Cell bodies containing alpha-neo-endorphin in the superior central nucleus (X 100). (F) P 5.2. Immunoreactive cell bodies (arrows) located close to the midline and the fourth ventricle (x 100). (G) P 16.0. Immunoreactive fibers (arrows) in the medial nucleus of the solitary tract. CE: central canal; GRR: gracile nucleus, rostral division (x 100).

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1268 M A R C O S ET AL.

FIG. 3. Alpha-neo-endorphin immunoreactivity in the cat brain stem. (A) P 3.1. Clusters of immunoreactive cell bodies in the superior central nucleus (CS), nucleus incertus (INC), and in the pericentral division of the dorsal tegmental nucleus (TDP). MLB: medial longitudinal bundle (x25). (B) P 3.1. High power image of the area delimited in (A) (X 100). (C) P 3. I. Immunoreactive cell bodies in the nucleus of the trapezoid body (T). P: pyramidal tract; TB: trapezoid body (x25). (D) P 3.1. A high magnification of the region delimited in (C) (Xl00). (E) P 16.0. Immunoreactive neurons (arrows) in the laminar spinal trigeminal nucleus (x25). (F) P 16.0. High power image of the region delimited in (E) (Xl00).

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ALPHA-NEO-ENDORPHIN IN THE CAT BRAIN STEM 1269

the laminar spinal trigeminal nucleus. Alpha-Neo-endorphin and neurotensin would also coexist in cell bodies of the nucleus of the trapezoid body and the inferior colliculus, and alpha-NEO- E and neuropeptide Y in the perikarya of the nucleus of the trapezoid body and the laminar spinal trigeminal nucleus.

The widespread distribution of alpha-NEO-E-IR structures in the cat brain stem indicates that the peptide could be involved in several physiological functions. In this sense, the presence of alpha-NEO-E-IR fibers in the Krl l iker-Fuse nucleus, nucleus of the solitary tract, dorsal motor nucleus of the vagus, and in the marginal nucleus of the brachium conjunct ivum suggests

the involvement of this peptide in respiratory and cardiovascular mechanisms, whereas the presence ofalpha-NEO-E-IR structures in the inferior colliculus suggests that alpha-NEO-E might be involved in auditory mechanisms. Finally, alpha-NEO-E could be involved in motor mechanisms, since alpha-NEO-E-IR structures were observed in the interpeduncular and vestibular nuclei.

ACKNOWLEDGEMENT

The author express their gratitude to Mr. Nicholas Skinner for kindly revising the English version of the manuscript.

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