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Distribution of neurotensin-like immunoreactive cell bodies and fibers in the brainstem of the adult male cat
Peptides,Vol. 12, pp. 1201-1209. ¢ PergamonPress plc, 1991.Printedin the U.S.A.
0196-9781/91 $3.00 + .00
Distribution of Neurotensin-Like Immunoreactive Cell Bodies and Fibers in the Brainstem of the Adult Male Cat M. DE LEON,* R. COVElqAS,* J. A. NARV/~EZ,I" G. T R A M U , ~ J. A. AGUIRRE§ AND S. GONZ,~LLEZ-BARONt
*Departamento de Biologfa Celular y Patologfa, Facuhad de Medicina Avda. Campo Charro s/n, 37007 Salamanca, Spain ~Departamento de Fisiologfa, Facultad de Medicina, Mdlaga, Spain SLaboratoire de Neurocytochimie Fonctionelle, CNRS, URA 339, Universitd de Bordeaux L France §Department of Histology and Neurobiology, Karolinska lnstitutet, Stockholm, Sweden Received 25 April 1991 DE LEON, M., R. COVElqAS, J. A. NARVAEZ, G. TRAMU, J. A. AGUIRREAND S. GONZALEZ-BARON.Distribution of neurotensin-like immunoreactive cell bodies and fibers in the brainstem of the adult male cat. PEPTIDES 12(6) 1201-1209, 1991.--We studied the distributionof cell bodies and fibers containingneurotensinin the brainstem of the cat using an indirect immunoperoxidasetechnique. A high or moderate density of immunoreactiveperikarya was found in the interpeduncularnucleus, inferior colliculus, nucleus of the brachiumof the inferior colliculusand in the lateral tegmentalfield. Moreover, a high density of neurotensin-immunoreactivefibers was observed in the periaqueductal gray, locus coeruleus and in the marginal nucleus of the brachium conjunctivum. The interpeduncularnucleus, nucleus of the solitary tract and the dorsal motor nucleus of the vagus contained a moderate density of immunoreactivefibers. Neurotensin
Immunocytochemistry Brainstem
Cat
SINCE neurotensin (NT) was isolated from bovine hypothalamus by Carraway and Leeman (6), immunocytochemical and radioimmunoassay studies have been carded out on the distribution of this peptide in the CNS of rats (16,30), the guinea pig (29), monkeys (18) and humans (20). These studies showed that NT is widely distributed in the mammalian brain, this finding suggesting a potential role of NT as a neurotransmitter responsible for a wide range of physiological effects. Thus, within the CNS, this peptide elicits many biological activities affecting hemodynamic, glucoregulatory, nociceptive, neuroendocrine and thermoregulatory systems (4, 6, 19, 22). Moreover, NT induces muscle relaxation, a decrease in locomotor activity and a reduction in food consumption (15,16). Despite the data on the distribution of NT in the mammalian CNS, the location of this peptide in the CNS of the cat has not been examined in depth, except in the diencephalon, in which the findings of an immunocytochemical study have recently been published (11). Accordingly, only partial data can be obtained from the literature; e.g., the presence of NT in the forebraln, the substantia nigra, and spinal cord has been described following the use of radioimmunoassay techniques (14), whereas by using immunocytochemical procedures, NT has been visualized in the nucleus of the solitary tract and in the spinal trigeminal tract (2), as well as in the ventrolateral medulla (7). Accordingly, there are no detailed data on the distribution of NT-containing fibers and cell bodies in the cat brainstem. Therefore, in
the present work, we attempted to study this distribution using an immunoperoxidase technique and then compared our findings with the distribution of several other neuropeptides previously described in the cat brainstem (1, 10, 12). METHOD Ten adult male cats (2-4 kg body weight) were used in the study. Under deep ketamine anesthesia (40-50 mg/kg), four were pretreated with colchicine (300 g.g in 5 ILl of distilled water). The drug was injected unilaterally into the fourth ventricle. Two days after the administration, both the treated and untreated animals were anesthetized with ketamine and perfused through the ascending aorta with 500 ml of 0.9% saline followed by 3 1 of 4% paraformaldehyde in 0.15 M phosphate buffer (pH 7.2). The brains were removed, and the brainstem was dissected out and posffixed in the same solution for twelve hours. Using a vibratome, 60 ~,m frontal sections were cut and processed for immunostaining, as has been previously described (11). The immunological characteristics of the NT antiserum used here have been reported previously (27). The specificity of the immunostaining was controlled by preabsorption of the primary antiserum with synthetic NT (100 o,g per ml diluted antiserum) and by omitting the NT antiserum in the fu'st incubation bath. No residual immunoreactivity was found in either case. In addition, no significant reduction in immunoreactivity was observed
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NEUROTENSIN IN THE CAT BRAINSTEM
upon incubating NT antiserum absorbed with an excess of substance P, angiotensin II, 13-endorphin, somatostatin and vasoactive intestinal peptide. Possible interference by endogenous peroxidase was ruled out by staining some sections beginning with the diaminobenzidine step. No reaction was visualized. Mapping was carded out according to the stereotaxic atlas of Berman (3). The same atlas was used for the terminology of the brainstem nuclei. Finally, the term "neurotensin-like immunoreactive" (NT-ir) is used to describe the staining results in our material. RESULTS Fibers and cell bodies containing NT are widely distributed throughout the cat brainstem (Fig. 1). Immunoreactivity was found in the midbrain, for example, in the periaqueductal gray, interpeduncular nucleus, inferior colliculus and dorsal raphe nucleus; in the pons, in the locus coeruleus, dorsal tegmental nucleus, marginal nucleus of the brachium conjunctivum and KiSllikerFuse area, whereas in the medulla oblongata NT-ir fibers or cell bodies were observed in the dorsal motor nucleus of the vagus, area postrema, nucleus of the solitary tract and inferior olive. In general, the medulla oblongata showed a lower degree of immunoreactivity as compared with the levels observed in the pons and midbrain. In this sense, the densest clusters of NT-ir cell bodies were found in the interpeduncular nucleus, inferior colliculus, nucleus of the brachium of the inferior colliculus and in the lateral tegmental field, whereas the densest network of NT-ir fibers was visualized in the periaqueductal gray, interpeduncular nucleus, locus coeruleus and marginal nucleus of the brachium conjunctivum. In rostral brainstem sections (A 1.6) (Fig. 1A), NT-ir fibers were observed in dorsal, medial and ventral regions of the section. Low numbers of immunoreactive processes were found in the intermediate division of the superior coUiculus and in the nucleus of the brachium of the inferior colliculus, extending dorsally across the central tegmental field a low density of NT-ir fibers and ventrally towards the interpeduncular nucleus. Other immunoreactive fibers were visualized at a low density extending from the ventral region of the periaqueductal gray to the central tegmental field. In this, in some cases, NT-ir fibers were seen to surround nonlabeled perikarya. The periaqueductal gray had a high density of fibers containing NT, mainly in the region located close to the aqueduct (Fig. 2A). Moreover, in the midline region a moderate density of immunoreactive fibers was ob-
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served in the central linear nucleus, in the outer division of the posterior interpeduncular nucleus, and in the paramedian interpeduncular nucleus. Finally, few immunoreactive fibers were visualized between the central linear nucleus and the interpeduncular nucleus. At this level (A 1.6), a moderate and high density of NT-ir cell bodies was found, respectively, in the nucleus of the brachium of the inferior colliculus and laterally in the interpeduncular nucleus (Fig. 2B, C); these were fusiform and medium in size in the former nucleus and small and round in the latter. At P 0.9 (Fig. 1B), a low number of immunoreactive fibers was observed in the pericentral nucleus and central nucleus of the inferior colliculus, whereas single NT-ir fibers were visualized in the commissure of the inferior colliculi, poutine gray and pyramidal tract. In the periaqueductal gray and in the locus coeruleus (Fig. 2D), a rich network of immunoreactive fibers was found. In addition, a low density of these fibers was observed in the nucleus incertus, the median division of the dorsal nucleus of the raphe (Fig. 2E, G), paralemniscal tegmental field and cuneiform nucleus, and a moderate density in some regions of the central tegmental field. Finally, in the midline region a moderate density was found in the area located between the medial longitudinal bundle and the superior central nucleus, and a low density in this latter nucleus and in the region extending from the superior central nucleus to the medial division of the pontine gray. At P 0.9, NT-ir perikarya were found in the section dorsally, ventrally and also located near to the aqueduct. A high density of fusiform and medium-sized immunoreactive cell bodies was observed dorsally in the pericentral nucleus of the inferior colliculus and in the dorsal-most region of the central nucleus of the inferior colliculus (Figs. 2H, 3A, B, C). Ventrally, scanty small and round cell bodies containing NT were located in the dorsolateral and lateral divisions of the pontine gray (Fig. 3F). Small and round immunoreactive cell bodies were also located in regions situated near the aqueduct, such as the locus coeruleus, periaqueductal gray and the median division of the dorsal nucleus of the raphe (Fig. 2F). In the three former regions, there was a low density of cell bodies containing NT. Finally, a few round and small cell bodies were found in the ventral nucleus of the lateral lemniscus. More caudally, at P 3.1 (Fig. 1C), a high density of NT-ir fibers was found in the marginal nucleus of the brachium conjunctivum (Fig. 3D, E) and in the locus coeruleus; a low density was observed in the following regions: Ktilliker-Fuse area
FACING PAGE FIG. 1. Distribution of NT-ir cell bodies and fibers in frontal planes of the bralnstem of the cat corresponding to the anteroposterior stereotaxic plane levels from A 1.6 to P 16.0 of the Berman stereotaxic atlas (3). Immunoreactive 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: >10 cell bodies; &, middle density: 5-10 cell bodies; l , lower density: <5 cell bodies). The anteriority (A) or posteriority (P), in mm with respect to the zero stereotaxic point of each section, is indicated at the lower right. 5SL: laminar spinal trigeminal nucleus; 5SP: alaminar spinal trigeminal nucleus; 5ST: spinal trigeminal tract; AQ: aqueduct; BC: brachium conjunctivum; BCM: marginal nucleus of the brachium conjunctivum; BIN: nucleus of the brachium of the inferior colliculus; BP: brachium pontis; CAE: locus coeruleus; CE: central canal; CI: inferior central nucleus; CNF: cuneiform nucleus; CS: superior central nucleus; c u e : cuneate nucleus, caudal division; DMV: dorsal motor nucleus of the vagus; DRM: dorsal nucleus of the raphe, medial division; FTC: central tegmental field; FTG: gigantocellular tegmental field; FTL: lateral tegmental field; FTM: magnocellular tegmental field; FTP: paralemniscal tegmental field; GRR: gracile nucleus, rostral division; ICC: central nucleus of the inferior colliculus; ICO: commissure of the inferior colliculi; ICP: pericentral nucleus of the inferior colliculus; INC: nucleus incertus; IO: inferior olive; IPI: posterior interpeduncular nucleus, inner division; IPO: posterior interpeduncular nucleus, outer division; IPP: paramedian interpoduncular nucleus; KF: K611iker-Fuse area; LC: central linear nucleus; LLD: dorsal nucleus of the lateral lemniscus; LLV: ventral nucleus of the lateral lemniscus; LRI: lateral reticular nucleus, internal division; LRX: lateral reticular nucleus, external division; MLB: medial longitudinal bundle; P: pyramidal tract; PAG: periaqueductal gray; PDG: pontine gray, dorsal division; PGL: pontine gray, lateral division; PGM: pontine gray, medial division; PPR: postpyramidal nucleus of the raphe; S: solitary tract; SAG: nucleus sagulum; SCI: superior colliculus, intermedial division; 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; TRC: tegmental reticular nucleus, central division; TRP: tegmental reticular nucleus, paracentral division; VMN: medial vestibular nucleus.
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(Fig. 3D), accessory dorsal tegmental nucleus, dorsal tegmental nucleus, nucleus incertus, superior central nucleus, gigantocellular tegmental field, paralemniscal tegmental field and nucleus of the trapezoid body, whereas few fibers were found in the trapezoid body. At this level, two clusters of immunoreactive cell bodies located ventrally and dorsally were seen. In both locations, the density of perikarya was low. Dorsally, small and round immunoreactive neurons were observed in the locus coemleus, accessory dorsal tegmental nucleus, marginal nucleus of the brachium conjunctivum and extending below the two former nuclei. Finally, in the nucleus of the trapezoid body, as well as between this nucleus and the pyramidal tract, small and round immunoreactive perikarya were visualized. At P 5.2 (Fig. 1D), no immunoreactive cell bodies were found. However, a low density of fibers was observed in the gigantocellular tegmental field, lateral tegmental field, inferior central nucleus and nucleus of the trapezoid body. At this, posteriorly, the immunoreactive fibers found dorsolaterally and in the midline at previous levels had disappeared, except in the inferior central nucleus. At P 9.2 (Fig. 1E), immunoreactive fibers were mainly located in the ventrolateral region, although they were also observed dorsally in the medial vestibular nucleus. Thus, in the midline, only the ventral-most regions, the postpyramidal nucleus of the raphe (Fig. 3G) and the inferior central nucleus, showed immunoreactivity. Fibers containing NT also extended to the magnocellular tegmental field, inferior olive, gigantocellular tegmental field, lateral tegmental field, the parvocellular division of the alaminar spinal trigeminal nucleus and the medial vestibular nucleus. In all the regions mentioned, a low or very low density of immunoreactive fibers was visualized. Finally, a low number of fusiform and small NT-ir perikarya was observed in the ventrolateral medulla. More caudally, at P 14.7 (Fig. IF), a moderate density of the immunoreactive processes was found in the dorsal motor nucleus of the vagus and in the nucleus of the solitary tract; in the latter nucleus (Fig. 3H), a scarce number of small and fusiform or triangular cell bodies was also observed. Furthermore, fibers containing NT were visualized in a region situated ventrolaterally to the solitary tract, as well as extending from the dorsal motor nucleus of the vagus to the lateral tegmental field and the parvocellular division of the alaminar spinal trigeminal nucleus. Finally, at P 16.0 (Fig. 1G), a low density of NT-ir fibers was found, extending from the region situated close to the central canal (Fig. 31) to the lateral tegmental field and the laminar spinal trigeminal nucleus. Two clusters of immunoreactive perikarya were found: the first, located in the nucleus of the solitary tract and in the region located below this nucleus, showed a low density, whereas the second, observed in the lateral tegmental field (Fig. 3J), had a moderate density. In the nucleus of the solitary tract the cell bodies were small and fusiform or triangular; in the lateral tegmental field they were medium in size and fusiform.
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DISCUSSION
In the present study, we described in detail for the first time the distribution of NT-ir fibers and cell bodies in the cat brainstem. Our results are in agreement with previous data found by Batten and Lo (2) and by Ciriello et al. (7) in the brainstem of the cat since, like these authors, we also found immunoreactive cell bodies and fibers in the nucleus of the solitary tract and in the ventrolateral medulla. However, we did not visualize NT immunoreactivity in the substantia nigra of the cat, in contrast to the findings of Goedert and Emson (14), who observed substantial amounts of NT in this region of the same species. In general, the findings observed in this work are also in agreement with those found concerning the distribution of NT in the brainstem of the rat (16,30). For example, in both the rat and the cat NT-ir fibers have been observed in the periaqueductal gray, superior colliculus, locus coeruleus, dorsal nucleus of the raphe, superior central nucleus, nucleus parabrachialis medialis, tegmental fields, K611iker-Fuse area, nucleus of the solitary tract, dorsal motor nucleus of the vagus, nucleus of the trapezoid body, spinal trigeminal nucleus, cuneiform nucleus and area postrema. However, some minor differences merit comment. In the interpeduncular nucleus, nucleus of the brachium of the inferior colliculus, medial vestibular nucleus and in the inferior colliculus we visualized NT-ir fibers in the cat, but no immunoreactive fibers have been found in the rat in these nuclei. By contrast, in the rat NT-ir fibers were observed in the nucleus ambiguus, ventral nucleus of the lateral lemniscus, lateral reticular nucleus, cuneate nucleus and dorsal cochlear nucleus in which, in the cat, no immunoreactive fibers were observed. On comparing the distribution of NT-ir fibers in the brainstem of the cat and humans (20), it can be stated that, in general, the location of such fibers is very similar since NT-ir processes have been visualized in the following brainstem regions of both the cat and humans: periaqueductal gray, locus coeruleus, superior central nucleus, cuneiform nucleus, nucleus parabrachialis medialis, nucleus of the solitary tract, dorsal motor nucleus of the vagus, spinal trigeminal nucleus, medial vestibular nucleus, inferior olive, inferior colliculus, interpeduncular nucleus and superior colliculus. Moreover, our results are also in agreement with the study carried out by Cooper et al. (9) in the human brain, since these authors found NT in the periaqueductal gray, locus coeruleus, superior colliculus and inferior colliculus. However, in the nucleus ambiguus, lateral reticular nucleus, nucleus praepositus hypoglossii and retrofacial nucleus NT-ir fibers have been observed in humans but not in the cat. In the latter species we visualized immunoreactive processes in the dorsal tegmental nucleus, in which Mai et al. (20) did not observe them in humails.
In comparison with previous studies on the distribution of NT-ir cell bodies in the rat brainstem (16,30), it seems that, in general, the distribution of NT-ir perikarya in the brainstem of the cat is quite similar. In this sense, in both species cell bodies containing NT were observed in the periaqueductal gray, locus
FACING PAGE FIG. 2. NT-ir cell bodies and fibers in the cat brainstem. (A) NT-ir fibers (arrows) in the periaqueductalgray (PAG). AQ: aqueduct ( x 24). (B) NT-ir cell bodies (arrows) in the interpeduncularnucleus (IP) ( x 24). (C) High power image of a cell body containingNT located in the region delimitedby dashed lines in B (x 97). (D) Immunoreactivefibers in the locus coeruleus (CAE). Note an immunoreactivecell body (arrow) (× 24). (E) NT-ir fibers in the dorsal nucleus of the raphe (DRM) and in the nucleus incertus (arrow) (INC). MLB: medial longitudinalbundle. (F) Two immunoreacfive cell bodies (arrows), one located in the dorsal nucleus of the raphe and the other close to the medial longitudinalbundle ( x 24). (G) High power image of the delimited area shown in (E) in which NT-ir fibers can be observed in the dorsal nucleus of the raphe (x 97). (H) NT-ir perikarya (arrows) located in the pericentralnucleus of the inferior colliculus(ICP) and in the central nucleus of the inferior colliculus(ICC) ( × 24).
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coeruleus, dorsal nucleus of the raphe, superior central nucleus, nucleus parabrachialis medialis and nucleus of the solitary tract. In addition, the following nuclei showed NT-ir cell bodies in the cat but not in the rat: nucleus of the brachium of the inferior colliculus, interpeduncular nucleus, inferior colliculus, ventral nucleus of the lateral lemniscus and accessory dorsal tegmental nucleus. On the contrary, in the superior central nucleus, the spinal trigeminal nucleus, nucleus ambiguus, nucleus parabrachialis lateralis and dorsal cochlear nucleus cell bodies containing NT have been found in the rat but not in the cat. In comparison with the human brainstem, a widespread distribution of immunoreactive cell bodies appears in the brainstem of the cat, since in man only the periaqueductal gray, superior colliculus and spinal trigeminal nucleus showed NT-ir cell bodies. In the cat, cell bodies containing NT were found in the former but not in either of the latter two; moreover, in the feline such perikarya were visualized in the above-mentioned nuclei. In sum, it can be said that, in general, the distribution of immunoreactive fibers in the rat, the cat and humans is quite similar, although some minor differences can be observed among the three species. Moreover, it seems that the distribution of NT-ir cell bodies in the mammalian brainstem is, in general, similar in the rat and cat, although some differences also appear, and wider in both species in comparison with humans. Such differences are probably due to the technical conditions, such as the injections of colchicine, rather than species differences, and would thus be responsible for the differences found in the distribution of cell bodies containing NT, since in both the rat and cat colchicine was administered intraventricularly. This study also demonstrates NT-ir structures in several brainstem nuclei in which no immunoreactivity for NT has been detected previously. Thus NT-ir fibers have been observed for the first time in mammals in the dorsal tegmental nucleus, and NT-ir cell bodies in the interpeduncular nucleus, inferior colliculus and ventral nucleus of the lateral lenmiscus. Intraventricularly administered colchicine increased the number of immunoreactive perikarya in the cat brainstem, since in control animals--not treated with the drug--cell bodies containing NT were only observed in the inferior colliculus, nucleus of the solitary tract and in the nucleus of the trapezoid body. However, in animals treated with colchicine, NT-ir cell bodies were also observed in the periaqueductal gray, accessory dorsal tegmental nucleus, locus coeruleus, dorsal nucleus of the raphe, nucleus of the brachium conjunctivum and in the interpeduncular nucleus. Moreover, the number and extent of immunoreactive perikarya were increased in the inferior colliculus after treatment with colchicine. The distribution of fibers and cell bodies containing methionine-enkephalin, neuropeptide Y or cholecystokinln octapeptide has been reported previously (8, 10, 12). A clear anatomical relationship between NT-ir fibers and neuropeptide Y-immunoreactive fibers can be suggested, since both neuropeptides have been observed in the following nuclei of the cat brainstem: periaqueductal gray, pericentral nucleus of the inferior coUiculus, locus coeruleus, nucleus incertus, dorsal nucleus of the raphe,
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superior central nucleus, marginal nucleus of the brachium conjunctivum, K611iker-Fuse area, dorsal tegmental nucleus, inferior central nucleus, postpyramidal nucleus of the raphe, medial vestibular nucleus, nucleus of the solitary tract, laminar spinal lrigeminal nucleus and dorsal motor nucleus of the vagus. However, the relationship between NT-ir fibers and cholecystokinin octapeptide-immunoreactive fibers is less marked in the cat brainstem, since both types of fibers were only observed in the periaqueductal gray, nucleus incertus, marginal nucleus of the brachium conjunctivum, dorsal tegmental nucleus, nucleus of the solitary tract, dorsal motor nucleus of the vagus, interpeduncular nucleus and cuneiform nucleus. Moreover, a weak relationship between the distribution of NT-ir fibers and methionine-enkephalin positive processes was observed. In this sense, in the cat both types of fibers were found in the periaqueductal gray, postpyramidal nucleus of the raphe, medial vestibular nucleus, interpeduncular nucleus and laminar spinal trigeminal nucleus. The possible coexistence of two or more neuropeptides in some of the above-mentioned nuclei of the cat brainstem should be investigated in the future. In addition, in the pericentral nucleus of the inferior colliculus, locus coeruleus, interpeduncular nucleus and nucleus of the solitary tract, in which we observed NT-ir cell bodies, fibers or perikarya containing neuropeptide Y, methionine-enkephalin or cholecystokinin octapeptide have also been reported (8, 10, 12). Finally, on comparing the distribution of perikarya containing NT, neuropeptide Y, methionine-enkephalin or cholecystokinin octapeptide, the coexistence of the two former neuropeptides can be suggested in the neurons of the locus coeruleus, interpeduncular nucleus and the pericentral nucleus of the inferior colliculus. NT and cholecystokinin octapeptide would also coexist in the cell bodies of the nucleus of the solitary tract, and NT and methionine-enkephalinin the perikarya of the interpeduncular nucleus. However, future work should be carried out to corroborate this hypothesis. In this sense, previous data have reported the coexistence of NT and cholecystokinin in neurons located in the cat anterodorsal thalamic nucleus (28). Moreover, the presence of NT, neuropeptide Y, cholecystokinin octapeptide and methionine-enkephalin in certain regions of the brainstem of the cat, such as periaqueductal gray and interpeduncular nucleus, as well as of the three former neuropeptides and angiotensin II (1) in the marginal nucleus of the brachium conjunctivum, dorsal tegmental nucleus, nucleus of the solitary tract and dorsal motor nucleus of the vagus, suggests a possible interaction among such substances and an elaborate modulation of functions in which these brainstem nuclei are involved. The origin of the NT-ir fibers, as well as whether the cell bodies containing NT are local or projecting neurons in the cat brainstem, is unknown. Knowledge of the fiber connections of the neurotensinergic neuronal system in the mammalian CNS is somewhat incomplete. In the rat, neurotensinergic pathways have been described coursing from the nucleus of the solitary tract to the parabrachial area (25) and from the central nucleus of the amygdala to the parabrachial area (21). In addition, in the rat, NT-ir cell bodies have been visualized in the paraventricular
FACING PAGE FIG. 3. NT-ir fibers and perikarya in the cat brainstem. (A) Immunoreactivecell bodies in the central nucleus of the inferior colliculus (× 21). (B) High power image of the NT-ir cell body shown in the square in (A) ( × 83). (C) An immunoreactiveneuron in the central nucleus of the inferior colliculus ( × 83). (D) Immunoreactivefibers in the brachium conjunctivum(arrows) (BC), marginal nucleus of the brachium conjunctivum(BCM), and K611iker-Fusearea (arrow) (KF) ( × 21). (E) A high magnificationof the delimitedregion in (D) ( × 83). (F) Cluster of immunoreactiveperikarya in the pontine gray ( × 83). ((3) NT-ir fibers in the postpyramidalnucleus of the raphe ( × 21). (H) An immunoreactivecell body (arrow) located in the nucleus of the solitary tract. S: solitary tract (× 21). (I) NT-ir fibers (arrows) located close to the central canal (CE) (× 21). (J) Immunoreactivecell bodies (arrows) in the lateral tegmentalfield ( × 21).
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nucleus of the hypothalamus, amygdala, periaqueductal gray and nucleus of the solitary tract, all these regions projecting to the locus coeruleus (23,24). These data suggest the possible sources of the neurotensinergic innervation of the rat locus coeruleus. According to the morphological data observed by us in the cat, the parabrachial area could receive neurotensinergic afferents, since a high density of immunoreactive fibers and a low density of NT-ir perikarya were observed. An origin of such fibers in the nucleus of the solitary tract, as occurs in the rat, is quite improbable, since a low density of cell bodies containing NT was visualized in the cat. The possible neurotensinergic pathway in this species coursing from the central nucleus of the amygdala to the parabrachial area should be investigated. Currently, it is not known whether NT-ir cell bodies are present or not in this nucleus of the amygdala. It seems that the locus coeruleus might also receive neurotensinergic afferents, since a high and a low density of cell bodies and fibers containing NT, respectively, were found in the cat. Projections from the paraventricular nucleus of the hypothalamus, nucleus of the solitary tract and/or the periaqueductal gray are quite improbable in the feline because no (11) or very scarce NT-ir cell bodies were seen in such regions. In addition to the locus coeruleus and the parabrachial area, it seems that the periaqueductal gray, nucleus of the solitary tract and dorsal nucleus of the vagus could also receive neurotensinergic afferents according to the presence in such nuclei of a high or moderate density of fibers containing NT and a scarce number of immunoreactive cell bodies or an absence of them. In the rat, neurotensinergic pathways from the ventral mesencephalon to the nucleus accumbens, prefrontal cortex and amygdala (17,26),
as well as from the parabrachial complex to the central nucleus of the amygdala (5), have been demonstrated. In the cat, the presence of these pathways is quite unlikely, since in the feline ventral mesencephalon we have not observed NT-ir cell bodies, whereas in the parabrachial complex a high density of NT-ir fibers and a low density of NT-ir perikarya were found, suggesting that the parabrachial complex receives neurotensinergic afferences. Finally, the cell bodies observed in the inferior colliculus and in the nucleus of the brachium of the inferior colliculus could be projecting neurons, since numerous immunoreactive perikarya were observed and the density of NT-containing fibers was low. In sum, the cell bodies which originate the NT-ir fibers observed in the cat brainstem, as well as whether the cell bodies that contain NT are interneurons or projecting neurons, remain unknown. Future works are required to elucidate these aspects. The widespread distribution of NT-ir structures in the cat brainstem suggests that the peptide could be involved in several physiological roles. In the rat, several data suggest that NT is involved in antinociception (22) and in cardiovascular mechanisms (13). Our findings are in agreement with these data, since we visualized NT immunoreactivity in the nucleus of the solitary tract, dorsal motor nucleus of the vagus, periaqueductal gray and spinal trigeminal nucleus. The involvement of NT in other mechanisms should also be investigated, since the presence of NT-ir perikarya and fibers in the inferior colliculus indicates that NT might be involved in auditory mechanisms. ACKNOWLEDGEMENT This work was supported by the University of Salamanca (Spain).
REFERENCES 1. Aguirre, J. A.; Covefias, R.; Croix, D.; Alonso, J. R.; Narv~iez, J. A.; Tramu, G.; Gonz,flez-Bar6n, S. Immunocytochemical study of angiotensin-II fibres and cell bodies in the brainstem respiratory areas of the cat. Brain Res. 489:311-317; 1989. 2. Batten, T. F. C.; Lo, V. K. F. Immunohistochemical localization of peptides and amines in the brain stem of the cat. J. Physiol. (Lond.) 388:103P; 1986. 3. Berman. A. L. The brain stem of the cat. A cytoarchitectonic atlas with stereotaxic coordinates. Madison, WI: The University of Wisconsin Press; 1969. 4. Bissette, G.; Nemeroff, C. B.; Loosen, P. T.; Prange, A. J.; Lipton, M. A. Hypothermia and cold tolerance induced by the intracisternal administration of the hypothalamic peptide neurotensin. Nature 262:607-609; 1976. 5. Block, C. H.; Hoffman, G.; Kapp, B. S. Peptide-containing pathways from the parabrachial complex to the central nucleus of the amygdala. Peptides 10:465-471; 1989. 6. Carraway, R.; Leeman, S. F. The isolation of a new hypotensive peptide, neurotensin, from bovine hypothalami. J. Biol. Chem. 248:6854-6861; 1973. 7. Ciriello, J.; Caverson, M. M.; Calaresu, F. R.; Krukoff, T. L. Neuropeptide and serotonin immunoreactive neurons in the cat ventrolateral medulla. Brain Res. 440:53-66; 1988. 8. Conrath-Verrier, M.; Dietl, M.; Arluison, M.; Cesselin, F.; Bourgoin, S.; Hamon, M. Localization of met-enkephalin-like immunoreactivity within pain-related nuclei of cervical spinal cord, brainstem and midbrain in the cat. Brain Res. Bull. 11:587--604; 1983. 9. Cooper, P.; Fernstrom, M. H.; Rorstad, O. P.; Leeman, S. E.; Martin, J. B. The regional distribution of somatostatin, substance P and neurotensin in human brain. Brain Res. 218:219-232; 1981. 10. Covefias, R.; Aguirre, J. A.; de Le6n, M.; Alonso, J. R.; Narv~iez, J. A.; Ar6valo, R.; Gonz~tlez-Bar6n, S. Distribution of neuropeptide Y-like immunoreactive cell bodies and fibers in the brain stem of the cat. Brain Res. Bull. 25:675-683; 1990. 11. De Le6n, M.; Covefias, R.; Narv~iez, J. A.; Tramu, G.; Aguirre, J.
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13. 14. 15. 16. 17. 18. 19. 20. 21.
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A.; Gonz~ilez-Bar6n, S. Neurotensin-like immunoreactivity in the diencephalon of the adult male cat. Peptides 12:257-264; 1991. De Le6n, M.; Covefias, R.; Narv~iez, J. A.; Tramu, G.; Aguirre, J. A.; Gonz~ilez-Bar6n, S. Distribution of cholecystokinin octapeptide in the cat brainstem: An immunocytochemical study. Arch. Ital. Biol., in press; 1991. Di Paola, E. D.; Richelson, E. Cardiovascular effects of neurotensin and some analogues on rats. Eur. J. Pharmacol. 175:279-283; 1990. Goedert, M.; Emson, P. C. The regional distribution of neurotensin-like immunoreactivity in central and peripheral tissues of the cat. Brain Res. 272:291-297; 1983. Hawkins, M. F. Central nervous system neurotensin and feeding. Physiol. Behav. 36:1-8; 1986. Jennes, L.; Stumpf, W. E.; Kalivas, P. W. Neurotensin: Topographical distribution in rat brain by immunohistochemistry. J. Comp. Neurol. 210:211-224; 1982. Kalivas, P. W.; Miller, J. S. Neurotensin neurons in the ventral tegmental area project to the medial nucleus accumbens. Brain Res. 300:157-160; 1984. Kataoka, K.; Mizuno, N.; Frohman, L. A. Regional distribution of immunoreactive neurotensin in monkey brain. Brain Res. Bull. 4:57-60; 1979. Maeda, K.; Frohman, L. A. Dissociation of systemic and central effects of neurotensin on the secretion of growth hormone, prolactin, and thyrotropin. Endocrinology 103:1903-1909; 1978. Mai, J. K.; Triepel, J.; Metz, J. Neurotensin in the human brain. Neuroscience 22:499-524; 1987. Moga, M. M.; Gray, T. S. Evidence for corticotropin-releasing factor, neurotensin, and somatostatin in the neural pathway from the central nucleus of the amygdala to the parabrachial nucleus. J. Comp. Neurol. 241:275-284; 1985. Osbahr, A. J.; Nemeroff, C. B.; Luttinger, D.; Mason, G. A.; Prange, A. J.. Neurotensin-induced antinociception in mice: Antagonism by thyrotropin-releasing hormone. J. Pharmacol. Exp. Ther.
NEUROTENSIN IN THE CAT BRAINSTEM
217:645-651; 1981. 23. Palkovits, M. Neuropeptides in the brain. In: Martini, L.; Ganong, W. F., eds. Frontiers in neuroendocrinology, vol. 10. New York: Raven; 1988:1-44. 24. Palkovits, M.; Brownstein, M. J. Locus coeruleus. In: Fedoroff, S.; Hartz, L., eds. Advances in cellular neurobiology. New York: Academic Press; 1983:81-103. 25. Riche, D.; De Pommery, J.; Menetrey, D. Neuropeptides and catecholamines in efferent projections of the nuclei of the solitary tract in the rat. J. Comp. Neurol. 293:399-424; 1990. 26. Seroogy, K. B.; Mehta, A.; Fallon, J. H. Neurotensin and cholecystokinin coexistence within neurons of the ventral mesencephalon: Projections to forebrain. Exp. Brain Res. 68:277-289; 1987.
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27. Studler, J. M.; Kitabgi, P.; Tramu, G.; Herve, D.; Glowinski, J.; Tassin, J. P. Extensive colocalization of neurotensin with dopamine in rat meso-cortico-frontal dopaminergic neurons. Neuropeptides 11: 95-100; 1988. 28. Sugimoto, T.; Itoh, K.; Yasui, Y.; Kaneko, T.; Mizuno, N. Coexistence of neuropeptides in projection neurons of the thalamus in the cat. Brain Res. 347:381-384; 1985. 29. Triepel, J.; Weindl, A.; Heiurich, D.; Forsmann, W. G.; Metz, J. Distribution of neurotensin-immunoreactive perikarya in the brainstem of the guinea-pig. Histochemistry 13:229-312; 1984. 30. Uhl, G. R.; Goodman, R. R.; Snyder, S. H. Neurotensin-containing cell bodies, fibers and nerve terminals in the brain stem of the rat: Immunohistochemical mapping. Brain Res. 167:77-91; 1979.
0196-9781/91 $3.00 + .00
Distribution of Neurotensin-Like Immunoreactive Cell Bodies and Fibers in the Brainstem of the Adult Male Cat M. DE LEON,* R. COVElqAS,* J. A. NARV/~EZ,I" G. T R A M U , ~ J. A. AGUIRRE§ AND S. GONZ,~LLEZ-BARONt
*Departamento de Biologfa Celular y Patologfa, Facuhad de Medicina Avda. Campo Charro s/n, 37007 Salamanca, Spain ~Departamento de Fisiologfa, Facultad de Medicina, Mdlaga, Spain SLaboratoire de Neurocytochimie Fonctionelle, CNRS, URA 339, Universitd de Bordeaux L France §Department of Histology and Neurobiology, Karolinska lnstitutet, Stockholm, Sweden Received 25 April 1991 DE LEON, M., R. COVElqAS, J. A. NARVAEZ, G. TRAMU, J. A. AGUIRREAND S. GONZALEZ-BARON.Distribution of neurotensin-like immunoreactive cell bodies and fibers in the brainstem of the adult male cat. PEPTIDES 12(6) 1201-1209, 1991.--We studied the distributionof cell bodies and fibers containingneurotensinin the brainstem of the cat using an indirect immunoperoxidasetechnique. A high or moderate density of immunoreactiveperikarya was found in the interpeduncularnucleus, inferior colliculus, nucleus of the brachiumof the inferior colliculusand in the lateral tegmentalfield. Moreover, a high density of neurotensin-immunoreactivefibers was observed in the periaqueductal gray, locus coeruleus and in the marginal nucleus of the brachium conjunctivum. The interpeduncularnucleus, nucleus of the solitary tract and the dorsal motor nucleus of the vagus contained a moderate density of immunoreactivefibers. Neurotensin
Immunocytochemistry Brainstem
Cat
SINCE neurotensin (NT) was isolated from bovine hypothalamus by Carraway and Leeman (6), immunocytochemical and radioimmunoassay studies have been carded out on the distribution of this peptide in the CNS of rats (16,30), the guinea pig (29), monkeys (18) and humans (20). These studies showed that NT is widely distributed in the mammalian brain, this finding suggesting a potential role of NT as a neurotransmitter responsible for a wide range of physiological effects. Thus, within the CNS, this peptide elicits many biological activities affecting hemodynamic, glucoregulatory, nociceptive, neuroendocrine and thermoregulatory systems (4, 6, 19, 22). Moreover, NT induces muscle relaxation, a decrease in locomotor activity and a reduction in food consumption (15,16). Despite the data on the distribution of NT in the mammalian CNS, the location of this peptide in the CNS of the cat has not been examined in depth, except in the diencephalon, in which the findings of an immunocytochemical study have recently been published (11). Accordingly, only partial data can be obtained from the literature; e.g., the presence of NT in the forebraln, the substantia nigra, and spinal cord has been described following the use of radioimmunoassay techniques (14), whereas by using immunocytochemical procedures, NT has been visualized in the nucleus of the solitary tract and in the spinal trigeminal tract (2), as well as in the ventrolateral medulla (7). Accordingly, there are no detailed data on the distribution of NT-containing fibers and cell bodies in the cat brainstem. Therefore, in
the present work, we attempted to study this distribution using an immunoperoxidase technique and then compared our findings with the distribution of several other neuropeptides previously described in the cat brainstem (1, 10, 12). METHOD Ten adult male cats (2-4 kg body weight) were used in the study. Under deep ketamine anesthesia (40-50 mg/kg), four were pretreated with colchicine (300 g.g in 5 ILl of distilled water). The drug was injected unilaterally into the fourth ventricle. Two days after the administration, both the treated and untreated animals were anesthetized with ketamine and perfused through the ascending aorta with 500 ml of 0.9% saline followed by 3 1 of 4% paraformaldehyde in 0.15 M phosphate buffer (pH 7.2). The brains were removed, and the brainstem was dissected out and posffixed in the same solution for twelve hours. Using a vibratome, 60 ~,m frontal sections were cut and processed for immunostaining, as has been previously described (11). The immunological characteristics of the NT antiserum used here have been reported previously (27). The specificity of the immunostaining was controlled by preabsorption of the primary antiserum with synthetic NT (100 o,g per ml diluted antiserum) and by omitting the NT antiserum in the fu'st incubation bath. No residual immunoreactivity was found in either case. In addition, no significant reduction in immunoreactivity was observed
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DE LEON ET AL
NEUROTENSIN IN THE CAT BRAINSTEM
upon incubating NT antiserum absorbed with an excess of substance P, angiotensin II, 13-endorphin, somatostatin and vasoactive intestinal peptide. Possible interference by endogenous peroxidase was ruled out by staining some sections beginning with the diaminobenzidine step. No reaction was visualized. Mapping was carded out according to the stereotaxic atlas of Berman (3). The same atlas was used for the terminology of the brainstem nuclei. Finally, the term "neurotensin-like immunoreactive" (NT-ir) is used to describe the staining results in our material. RESULTS Fibers and cell bodies containing NT are widely distributed throughout the cat brainstem (Fig. 1). Immunoreactivity was found in the midbrain, for example, in the periaqueductal gray, interpeduncular nucleus, inferior colliculus and dorsal raphe nucleus; in the pons, in the locus coeruleus, dorsal tegmental nucleus, marginal nucleus of the brachium conjunctivum and KiSllikerFuse area, whereas in the medulla oblongata NT-ir fibers or cell bodies were observed in the dorsal motor nucleus of the vagus, area postrema, nucleus of the solitary tract and inferior olive. In general, the medulla oblongata showed a lower degree of immunoreactivity as compared with the levels observed in the pons and midbrain. In this sense, the densest clusters of NT-ir cell bodies were found in the interpeduncular nucleus, inferior colliculus, nucleus of the brachium of the inferior colliculus and in the lateral tegmental field, whereas the densest network of NT-ir fibers was visualized in the periaqueductal gray, interpeduncular nucleus, locus coeruleus and marginal nucleus of the brachium conjunctivum. In rostral brainstem sections (A 1.6) (Fig. 1A), NT-ir fibers were observed in dorsal, medial and ventral regions of the section. Low numbers of immunoreactive processes were found in the intermediate division of the superior coUiculus and in the nucleus of the brachium of the inferior colliculus, extending dorsally across the central tegmental field a low density of NT-ir fibers and ventrally towards the interpeduncular nucleus. Other immunoreactive fibers were visualized at a low density extending from the ventral region of the periaqueductal gray to the central tegmental field. In this, in some cases, NT-ir fibers were seen to surround nonlabeled perikarya. The periaqueductal gray had a high density of fibers containing NT, mainly in the region located close to the aqueduct (Fig. 2A). Moreover, in the midline region a moderate density of immunoreactive fibers was ob-
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served in the central linear nucleus, in the outer division of the posterior interpeduncular nucleus, and in the paramedian interpeduncular nucleus. Finally, few immunoreactive fibers were visualized between the central linear nucleus and the interpeduncular nucleus. At this level (A 1.6), a moderate and high density of NT-ir cell bodies was found, respectively, in the nucleus of the brachium of the inferior colliculus and laterally in the interpeduncular nucleus (Fig. 2B, C); these were fusiform and medium in size in the former nucleus and small and round in the latter. At P 0.9 (Fig. 1B), a low number of immunoreactive fibers was observed in the pericentral nucleus and central nucleus of the inferior colliculus, whereas single NT-ir fibers were visualized in the commissure of the inferior colliculi, poutine gray and pyramidal tract. In the periaqueductal gray and in the locus coeruleus (Fig. 2D), a rich network of immunoreactive fibers was found. In addition, a low density of these fibers was observed in the nucleus incertus, the median division of the dorsal nucleus of the raphe (Fig. 2E, G), paralemniscal tegmental field and cuneiform nucleus, and a moderate density in some regions of the central tegmental field. Finally, in the midline region a moderate density was found in the area located between the medial longitudinal bundle and the superior central nucleus, and a low density in this latter nucleus and in the region extending from the superior central nucleus to the medial division of the pontine gray. At P 0.9, NT-ir perikarya were found in the section dorsally, ventrally and also located near to the aqueduct. A high density of fusiform and medium-sized immunoreactive cell bodies was observed dorsally in the pericentral nucleus of the inferior colliculus and in the dorsal-most region of the central nucleus of the inferior colliculus (Figs. 2H, 3A, B, C). Ventrally, scanty small and round cell bodies containing NT were located in the dorsolateral and lateral divisions of the pontine gray (Fig. 3F). Small and round immunoreactive cell bodies were also located in regions situated near the aqueduct, such as the locus coeruleus, periaqueductal gray and the median division of the dorsal nucleus of the raphe (Fig. 2F). In the three former regions, there was a low density of cell bodies containing NT. Finally, a few round and small cell bodies were found in the ventral nucleus of the lateral lemniscus. More caudally, at P 3.1 (Fig. 1C), a high density of NT-ir fibers was found in the marginal nucleus of the brachium conjunctivum (Fig. 3D, E) and in the locus coeruleus; a low density was observed in the following regions: Ktilliker-Fuse area
FACING PAGE FIG. 1. Distribution of NT-ir cell bodies and fibers in frontal planes of the bralnstem of the cat corresponding to the anteroposterior stereotaxic plane levels from A 1.6 to P 16.0 of the Berman stereotaxic atlas (3). Immunoreactive 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: >10 cell bodies; &, middle density: 5-10 cell bodies; l , lower density: <5 cell bodies). The anteriority (A) or posteriority (P), in mm with respect to the zero stereotaxic point of each section, is indicated at the lower right. 5SL: laminar spinal trigeminal nucleus; 5SP: alaminar spinal trigeminal nucleus; 5ST: spinal trigeminal tract; AQ: aqueduct; BC: brachium conjunctivum; BCM: marginal nucleus of the brachium conjunctivum; BIN: nucleus of the brachium of the inferior colliculus; BP: brachium pontis; CAE: locus coeruleus; CE: central canal; CI: inferior central nucleus; CNF: cuneiform nucleus; CS: superior central nucleus; c u e : cuneate nucleus, caudal division; DMV: dorsal motor nucleus of the vagus; DRM: dorsal nucleus of the raphe, medial division; FTC: central tegmental field; FTG: gigantocellular tegmental field; FTL: lateral tegmental field; FTM: magnocellular tegmental field; FTP: paralemniscal tegmental field; GRR: gracile nucleus, rostral division; ICC: central nucleus of the inferior colliculus; ICO: commissure of the inferior colliculi; ICP: pericentral nucleus of the inferior colliculus; INC: nucleus incertus; IO: inferior olive; IPI: posterior interpeduncular nucleus, inner division; IPO: posterior interpeduncular nucleus, outer division; IPP: paramedian interpoduncular nucleus; KF: K611iker-Fuse area; LC: central linear nucleus; LLD: dorsal nucleus of the lateral lemniscus; LLV: ventral nucleus of the lateral lemniscus; LRI: lateral reticular nucleus, internal division; LRX: lateral reticular nucleus, external division; MLB: medial longitudinal bundle; P: pyramidal tract; PAG: periaqueductal gray; PDG: pontine gray, dorsal division; PGL: pontine gray, lateral division; PGM: pontine gray, medial division; PPR: postpyramidal nucleus of the raphe; S: solitary tract; SAG: nucleus sagulum; SCI: superior colliculus, intermedial division; 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; TRC: tegmental reticular nucleus, central division; TRP: tegmental reticular nucleus, paracentral division; VMN: medial vestibular nucleus.
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(Fig. 3D), accessory dorsal tegmental nucleus, dorsal tegmental nucleus, nucleus incertus, superior central nucleus, gigantocellular tegmental field, paralemniscal tegmental field and nucleus of the trapezoid body, whereas few fibers were found in the trapezoid body. At this level, two clusters of immunoreactive cell bodies located ventrally and dorsally were seen. In both locations, the density of perikarya was low. Dorsally, small and round immunoreactive neurons were observed in the locus coemleus, accessory dorsal tegmental nucleus, marginal nucleus of the brachium conjunctivum and extending below the two former nuclei. Finally, in the nucleus of the trapezoid body, as well as between this nucleus and the pyramidal tract, small and round immunoreactive perikarya were visualized. At P 5.2 (Fig. 1D), no immunoreactive cell bodies were found. However, a low density of fibers was observed in the gigantocellular tegmental field, lateral tegmental field, inferior central nucleus and nucleus of the trapezoid body. At this, posteriorly, the immunoreactive fibers found dorsolaterally and in the midline at previous levels had disappeared, except in the inferior central nucleus. At P 9.2 (Fig. 1E), immunoreactive fibers were mainly located in the ventrolateral region, although they were also observed dorsally in the medial vestibular nucleus. Thus, in the midline, only the ventral-most regions, the postpyramidal nucleus of the raphe (Fig. 3G) and the inferior central nucleus, showed immunoreactivity. Fibers containing NT also extended to the magnocellular tegmental field, inferior olive, gigantocellular tegmental field, lateral tegmental field, the parvocellular division of the alaminar spinal trigeminal nucleus and the medial vestibular nucleus. In all the regions mentioned, a low or very low density of immunoreactive fibers was visualized. Finally, a low number of fusiform and small NT-ir perikarya was observed in the ventrolateral medulla. More caudally, at P 14.7 (Fig. IF), a moderate density of the immunoreactive processes was found in the dorsal motor nucleus of the vagus and in the nucleus of the solitary tract; in the latter nucleus (Fig. 3H), a scarce number of small and fusiform or triangular cell bodies was also observed. Furthermore, fibers containing NT were visualized in a region situated ventrolaterally to the solitary tract, as well as extending from the dorsal motor nucleus of the vagus to the lateral tegmental field and the parvocellular division of the alaminar spinal trigeminal nucleus. Finally, at P 16.0 (Fig. 1G), a low density of NT-ir fibers was found, extending from the region situated close to the central canal (Fig. 31) to the lateral tegmental field and the laminar spinal trigeminal nucleus. Two clusters of immunoreactive perikarya were found: the first, located in the nucleus of the solitary tract and in the region located below this nucleus, showed a low density, whereas the second, observed in the lateral tegmental field (Fig. 3J), had a moderate density. In the nucleus of the solitary tract the cell bodies were small and fusiform or triangular; in the lateral tegmental field they were medium in size and fusiform.
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DISCUSSION
In the present study, we described in detail for the first time the distribution of NT-ir fibers and cell bodies in the cat brainstem. Our results are in agreement with previous data found by Batten and Lo (2) and by Ciriello et al. (7) in the brainstem of the cat since, like these authors, we also found immunoreactive cell bodies and fibers in the nucleus of the solitary tract and in the ventrolateral medulla. However, we did not visualize NT immunoreactivity in the substantia nigra of the cat, in contrast to the findings of Goedert and Emson (14), who observed substantial amounts of NT in this region of the same species. In general, the findings observed in this work are also in agreement with those found concerning the distribution of NT in the brainstem of the rat (16,30). For example, in both the rat and the cat NT-ir fibers have been observed in the periaqueductal gray, superior colliculus, locus coeruleus, dorsal nucleus of the raphe, superior central nucleus, nucleus parabrachialis medialis, tegmental fields, K611iker-Fuse area, nucleus of the solitary tract, dorsal motor nucleus of the vagus, nucleus of the trapezoid body, spinal trigeminal nucleus, cuneiform nucleus and area postrema. However, some minor differences merit comment. In the interpeduncular nucleus, nucleus of the brachium of the inferior colliculus, medial vestibular nucleus and in the inferior colliculus we visualized NT-ir fibers in the cat, but no immunoreactive fibers have been found in the rat in these nuclei. By contrast, in the rat NT-ir fibers were observed in the nucleus ambiguus, ventral nucleus of the lateral lemniscus, lateral reticular nucleus, cuneate nucleus and dorsal cochlear nucleus in which, in the cat, no immunoreactive fibers were observed. On comparing the distribution of NT-ir fibers in the brainstem of the cat and humans (20), it can be stated that, in general, the location of such fibers is very similar since NT-ir processes have been visualized in the following brainstem regions of both the cat and humans: periaqueductal gray, locus coeruleus, superior central nucleus, cuneiform nucleus, nucleus parabrachialis medialis, nucleus of the solitary tract, dorsal motor nucleus of the vagus, spinal trigeminal nucleus, medial vestibular nucleus, inferior olive, inferior colliculus, interpeduncular nucleus and superior colliculus. Moreover, our results are also in agreement with the study carried out by Cooper et al. (9) in the human brain, since these authors found NT in the periaqueductal gray, locus coeruleus, superior colliculus and inferior colliculus. However, in the nucleus ambiguus, lateral reticular nucleus, nucleus praepositus hypoglossii and retrofacial nucleus NT-ir fibers have been observed in humans but not in the cat. In the latter species we visualized immunoreactive processes in the dorsal tegmental nucleus, in which Mai et al. (20) did not observe them in humails.
In comparison with previous studies on the distribution of NT-ir cell bodies in the rat brainstem (16,30), it seems that, in general, the distribution of NT-ir perikarya in the brainstem of the cat is quite similar. In this sense, in both species cell bodies containing NT were observed in the periaqueductal gray, locus
FACING PAGE FIG. 2. NT-ir cell bodies and fibers in the cat brainstem. (A) NT-ir fibers (arrows) in the periaqueductalgray (PAG). AQ: aqueduct ( x 24). (B) NT-ir cell bodies (arrows) in the interpeduncularnucleus (IP) ( x 24). (C) High power image of a cell body containingNT located in the region delimitedby dashed lines in B (x 97). (D) Immunoreactivefibers in the locus coeruleus (CAE). Note an immunoreactivecell body (arrow) (× 24). (E) NT-ir fibers in the dorsal nucleus of the raphe (DRM) and in the nucleus incertus (arrow) (INC). MLB: medial longitudinalbundle. (F) Two immunoreacfive cell bodies (arrows), one located in the dorsal nucleus of the raphe and the other close to the medial longitudinalbundle ( x 24). (G) High power image of the delimited area shown in (E) in which NT-ir fibers can be observed in the dorsal nucleus of the raphe (x 97). (H) NT-ir perikarya (arrows) located in the pericentralnucleus of the inferior colliculus(ICP) and in the central nucleus of the inferior colliculus(ICC) ( × 24).
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F
NEUROTENSIN IN THE CAT BRAINSTEM
coeruleus, dorsal nucleus of the raphe, superior central nucleus, nucleus parabrachialis medialis and nucleus of the solitary tract. In addition, the following nuclei showed NT-ir cell bodies in the cat but not in the rat: nucleus of the brachium of the inferior colliculus, interpeduncular nucleus, inferior colliculus, ventral nucleus of the lateral lemniscus and accessory dorsal tegmental nucleus. On the contrary, in the superior central nucleus, the spinal trigeminal nucleus, nucleus ambiguus, nucleus parabrachialis lateralis and dorsal cochlear nucleus cell bodies containing NT have been found in the rat but not in the cat. In comparison with the human brainstem, a widespread distribution of immunoreactive cell bodies appears in the brainstem of the cat, since in man only the periaqueductal gray, superior colliculus and spinal trigeminal nucleus showed NT-ir cell bodies. In the cat, cell bodies containing NT were found in the former but not in either of the latter two; moreover, in the feline such perikarya were visualized in the above-mentioned nuclei. In sum, it can be said that, in general, the distribution of immunoreactive fibers in the rat, the cat and humans is quite similar, although some minor differences can be observed among the three species. Moreover, it seems that the distribution of NT-ir cell bodies in the mammalian brainstem is, in general, similar in the rat and cat, although some differences also appear, and wider in both species in comparison with humans. Such differences are probably due to the technical conditions, such as the injections of colchicine, rather than species differences, and would thus be responsible for the differences found in the distribution of cell bodies containing NT, since in both the rat and cat colchicine was administered intraventricularly. This study also demonstrates NT-ir structures in several brainstem nuclei in which no immunoreactivity for NT has been detected previously. Thus NT-ir fibers have been observed for the first time in mammals in the dorsal tegmental nucleus, and NT-ir cell bodies in the interpeduncular nucleus, inferior colliculus and ventral nucleus of the lateral lenmiscus. Intraventricularly administered colchicine increased the number of immunoreactive perikarya in the cat brainstem, since in control animals--not treated with the drug--cell bodies containing NT were only observed in the inferior colliculus, nucleus of the solitary tract and in the nucleus of the trapezoid body. However, in animals treated with colchicine, NT-ir cell bodies were also observed in the periaqueductal gray, accessory dorsal tegmental nucleus, locus coeruleus, dorsal nucleus of the raphe, nucleus of the brachium conjunctivum and in the interpeduncular nucleus. Moreover, the number and extent of immunoreactive perikarya were increased in the inferior colliculus after treatment with colchicine. The distribution of fibers and cell bodies containing methionine-enkephalin, neuropeptide Y or cholecystokinln octapeptide has been reported previously (8, 10, 12). A clear anatomical relationship between NT-ir fibers and neuropeptide Y-immunoreactive fibers can be suggested, since both neuropeptides have been observed in the following nuclei of the cat brainstem: periaqueductal gray, pericentral nucleus of the inferior coUiculus, locus coeruleus, nucleus incertus, dorsal nucleus of the raphe,
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superior central nucleus, marginal nucleus of the brachium conjunctivum, K611iker-Fuse area, dorsal tegmental nucleus, inferior central nucleus, postpyramidal nucleus of the raphe, medial vestibular nucleus, nucleus of the solitary tract, laminar spinal lrigeminal nucleus and dorsal motor nucleus of the vagus. However, the relationship between NT-ir fibers and cholecystokinin octapeptide-immunoreactive fibers is less marked in the cat brainstem, since both types of fibers were only observed in the periaqueductal gray, nucleus incertus, marginal nucleus of the brachium conjunctivum, dorsal tegmental nucleus, nucleus of the solitary tract, dorsal motor nucleus of the vagus, interpeduncular nucleus and cuneiform nucleus. Moreover, a weak relationship between the distribution of NT-ir fibers and methionine-enkephalin positive processes was observed. In this sense, in the cat both types of fibers were found in the periaqueductal gray, postpyramidal nucleus of the raphe, medial vestibular nucleus, interpeduncular nucleus and laminar spinal trigeminal nucleus. The possible coexistence of two or more neuropeptides in some of the above-mentioned nuclei of the cat brainstem should be investigated in the future. In addition, in the pericentral nucleus of the inferior colliculus, locus coeruleus, interpeduncular nucleus and nucleus of the solitary tract, in which we observed NT-ir cell bodies, fibers or perikarya containing neuropeptide Y, methionine-enkephalin or cholecystokinin octapeptide have also been reported (8, 10, 12). Finally, on comparing the distribution of perikarya containing NT, neuropeptide Y, methionine-enkephalin or cholecystokinin octapeptide, the coexistence of the two former neuropeptides can be suggested in the neurons of the locus coeruleus, interpeduncular nucleus and the pericentral nucleus of the inferior colliculus. NT and cholecystokinin octapeptide would also coexist in the cell bodies of the nucleus of the solitary tract, and NT and methionine-enkephalinin the perikarya of the interpeduncular nucleus. However, future work should be carried out to corroborate this hypothesis. In this sense, previous data have reported the coexistence of NT and cholecystokinin in neurons located in the cat anterodorsal thalamic nucleus (28). Moreover, the presence of NT, neuropeptide Y, cholecystokinin octapeptide and methionine-enkephalin in certain regions of the brainstem of the cat, such as periaqueductal gray and interpeduncular nucleus, as well as of the three former neuropeptides and angiotensin II (1) in the marginal nucleus of the brachium conjunctivum, dorsal tegmental nucleus, nucleus of the solitary tract and dorsal motor nucleus of the vagus, suggests a possible interaction among such substances and an elaborate modulation of functions in which these brainstem nuclei are involved. The origin of the NT-ir fibers, as well as whether the cell bodies containing NT are local or projecting neurons in the cat brainstem, is unknown. Knowledge of the fiber connections of the neurotensinergic neuronal system in the mammalian CNS is somewhat incomplete. In the rat, neurotensinergic pathways have been described coursing from the nucleus of the solitary tract to the parabrachial area (25) and from the central nucleus of the amygdala to the parabrachial area (21). In addition, in the rat, NT-ir cell bodies have been visualized in the paraventricular
FACING PAGE FIG. 3. NT-ir fibers and perikarya in the cat brainstem. (A) Immunoreactivecell bodies in the central nucleus of the inferior colliculus (× 21). (B) High power image of the NT-ir cell body shown in the square in (A) ( × 83). (C) An immunoreactiveneuron in the central nucleus of the inferior colliculus ( × 83). (D) Immunoreactivefibers in the brachium conjunctivum(arrows) (BC), marginal nucleus of the brachium conjunctivum(BCM), and K611iker-Fusearea (arrow) (KF) ( × 21). (E) A high magnificationof the delimitedregion in (D) ( × 83). (F) Cluster of immunoreactiveperikarya in the pontine gray ( × 83). ((3) NT-ir fibers in the postpyramidalnucleus of the raphe ( × 21). (H) An immunoreactivecell body (arrow) located in the nucleus of the solitary tract. S: solitary tract (× 21). (I) NT-ir fibers (arrows) located close to the central canal (CE) (× 21). (J) Immunoreactivecell bodies (arrows) in the lateral tegmentalfield ( × 21).
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nucleus of the hypothalamus, amygdala, periaqueductal gray and nucleus of the solitary tract, all these regions projecting to the locus coeruleus (23,24). These data suggest the possible sources of the neurotensinergic innervation of the rat locus coeruleus. According to the morphological data observed by us in the cat, the parabrachial area could receive neurotensinergic afferents, since a high density of immunoreactive fibers and a low density of NT-ir perikarya were observed. An origin of such fibers in the nucleus of the solitary tract, as occurs in the rat, is quite improbable, since a low density of cell bodies containing NT was visualized in the cat. The possible neurotensinergic pathway in this species coursing from the central nucleus of the amygdala to the parabrachial area should be investigated. Currently, it is not known whether NT-ir cell bodies are present or not in this nucleus of the amygdala. It seems that the locus coeruleus might also receive neurotensinergic afferents, since a high and a low density of cell bodies and fibers containing NT, respectively, were found in the cat. Projections from the paraventricular nucleus of the hypothalamus, nucleus of the solitary tract and/or the periaqueductal gray are quite improbable in the feline because no (11) or very scarce NT-ir cell bodies were seen in such regions. In addition to the locus coeruleus and the parabrachial area, it seems that the periaqueductal gray, nucleus of the solitary tract and dorsal nucleus of the vagus could also receive neurotensinergic afferents according to the presence in such nuclei of a high or moderate density of fibers containing NT and a scarce number of immunoreactive cell bodies or an absence of them. In the rat, neurotensinergic pathways from the ventral mesencephalon to the nucleus accumbens, prefrontal cortex and amygdala (17,26),
as well as from the parabrachial complex to the central nucleus of the amygdala (5), have been demonstrated. In the cat, the presence of these pathways is quite unlikely, since in the feline ventral mesencephalon we have not observed NT-ir cell bodies, whereas in the parabrachial complex a high density of NT-ir fibers and a low density of NT-ir perikarya were found, suggesting that the parabrachial complex receives neurotensinergic afferences. Finally, the cell bodies observed in the inferior colliculus and in the nucleus of the brachium of the inferior colliculus could be projecting neurons, since numerous immunoreactive perikarya were observed and the density of NT-containing fibers was low. In sum, the cell bodies which originate the NT-ir fibers observed in the cat brainstem, as well as whether the cell bodies that contain NT are interneurons or projecting neurons, remain unknown. Future works are required to elucidate these aspects. The widespread distribution of NT-ir structures in the cat brainstem suggests that the peptide could be involved in several physiological roles. In the rat, several data suggest that NT is involved in antinociception (22) and in cardiovascular mechanisms (13). Our findings are in agreement with these data, since we visualized NT immunoreactivity in the nucleus of the solitary tract, dorsal motor nucleus of the vagus, periaqueductal gray and spinal trigeminal nucleus. The involvement of NT in other mechanisms should also be investigated, since the presence of NT-ir perikarya and fibers in the inferior colliculus indicates that NT might be involved in auditory mechanisms. ACKNOWLEDGEMENT This work was supported by the University of Salamanca (Spain).
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