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Distribution of dopamine-immunoreactive fibers in the rat brainstem
Journal of Chemical Neuroanatomy 18 (2000) 1 – 9 www.elsevier.com/locate/jchemneu
Distribution of dopamine-immunoreactive fibers in the rat brainstem K. Kitahama a,*, I. Nagatsu b, M. Geffard c, T. Maeda d a
De´partement de Me´decine Expe´rimentale, INSERM U480, Faculte´ de Me´decine, Uni6ersite´ Claude Bernard, 8, a6enue Rockefeller, 69373 Lyon, Cedex 08, France b Department of Anatomy, School of Medicine, Fujita Health Uni6ersity, Toyoake, Japan c Institut de Biochimie Cellulaire et Neurochimie, Uni6ersite´ de Bordeaux II, Bordeaux, France d Department of Anatomy, Shiga Uni6ersity of Medical Science, Ohtsu, Japan Received 22 June 1999; received in revised form 12 October 1999; accepted 12 October 1999
Abstract We describe the distribution of axons immunoreactive for dopamine in pons and medulla oblongata of rat under normal conditions or after inhibition of monoamine oxidase or dopamine b-hydroxylase. In the pons of non-treated animal, fairly dense plexuses of dopamine-immunoreactive varicose fibers were found in the locus coeruleus, dorsal parabrachial and dorsal raphe nuclei, central gray and reticular formation dorsal to the superior olive. In the medulla oblongata, the immunoreactive fibers were abundant in the dorsal vagal complex, lateral paragigantocellular nucleus, midline raphe nuclei and spinal trigeminal nucleus. Monoamine oxidase inhibition made it possible to increase the intensity of immunoreactivity and consequently the number of labeled fibers in these areas, indicating that dopamine is perpetually oxidized by monoamine oxidase, and consequently in low concentration under normal conditions. Sparse dopamine-immunoreactive fibers were observed in the pontine gray, motor trigeminal nucleus, inferior olive and major axon bundles such as the dorsal and ventral tegmental bundles, where numerous noradrenergic fibers have been reported. In axons of these areas, intense dopamine-immunoreactivity was seen only after inhibition of dopamine-b-hydroxylase. It appears that dopamine is released and oxidized in response to autonomic changes such as hypoxia, hemorrhage, and cardiovascular variation in the caudal brainstem, as we have described elsewhere. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Catecholamines; Dopamine; Locus coeruleus; Medulla; Pons; Monoamine oxidase; Immunohistochemistry
Abbre6iations: 10, dorsal motor nucleus of the vagus; 12, hypoglossal nucleus; 7n, facial nerve; AQ, aqueduct; BIC, nucleus brachium inferior colliculus; CG, central gray; CLi, caudal linear raphe nucleus; COM, commissural nucleus, subnucleus of the NTS; cp, cerebral peduncle; CU, cuneate nucleus; DLL, dorsal nucleus lateral lemniscus; DpMe, deep mesencephalic nucleus; DRN, dorsal raphe nucleus; fr, fasciculus retroflexus; g7, genu facial nerve; Gi, gigantocellular reticular nucleus; GiA, gigantocellular reticular nucleus, pars alpha; IC, inferior colliculus; IF, interfascicular nucleus; IO, inferior olive; IP, interpeduncular nucleus; LC, locus coeruleus; ll, lateral lemniscus; LPB, lateral parabrachial nucleus; LPGi, lateral paragigantocellular nucleus; LRN, lateral reticular nucleus; LSO, superior olive; MdD, medullary reticular field, dorsal; MdV, medullary reticular nucleus, ventral; ml, medial lemniscus; mlf, medial longitudinal fasciculus; MnR, median raphe nucleus; Mo5, motor trigeminal nucleus; MPB, medial parabrachial nucleus; MVe, medial vestibular nucleus; NTS, nucleus of the solitary tract; PCRt, parvocellular reticular nucleus; Pn, pontine nucleus; PnC, pontine reticular nucleus, caudal; PnO, pontine reticular nucleus, oral; PnV, pontine reticular nucleus, ventral; Pr5, principal sensory trigeminal nucleus; PTA, pretectal area; py, pyramidal tract; pyx, pyramidal decussation; RLi, rostral linear raphe nucleus; RMg, raphe magnus nucleus; ROb, raphe obscurus nucleus; RPa, raphe pallidus nucleus; RPn, raphe pontis nucleus; RR, retrorubral field; SC, superior colliculus; scp, superior cerebellar peduncle; SNC, substantia nigra pars compacta; SNR, substantia nigra pars reticularis; Sp5C, spinal trigeminal nucleus, caudal; Sp5O, spinal trigeminal nucleus, rostral; st, solitary tract; SuVe, superior vestibular nucleus; VCA, ventral cochlear nucleus, anterior; VLL, ventral nucleus lateral lemniscus; VTA, ventral tegmental area; vtb, ventral tegmental bundle; xscp, decussation of the superior cerebellar peduncle. * Corresponding author. Tel.: + 33-4-78777197; fax: +33-4-78777172. E-mail address: [email protected] (K. Kitahama) 0891-0618/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 8 9 1 - 0 6 1 8 ( 9 9 ) 0 0 0 4 7 - 2
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1. Introduction Dopamine (DA) is synthesized from L-dihydroxyphenyl-alanine (L-DOPA), which is transformed from tyrosine by their first step synthesizing enzyme TH (Nagatsu et al., 1964). It is oxidized to 3,4-dihydroxyphenyl-acetic acid (DOPAC) by its degrading enzyme, monoamine oxidase (MAO), or hydroxylated to noradrenaline (NA) by the enzyme dopamine-b-hydroxylase (DBH). Cerebral DA content is thus strongly influenced by the activity of these enzymes. According to in vivo voltammetric technique studies from our laboratory, there is a significant decrease in oxidation current of DA in the locus coeruleus and ventrolateral medulla after MAO inhibition (Buda et al., 1983; Gillon et al., 1990). This indicates a perpetual oxidation of DA in these regions. We recently demonstrated that the metabolic rate of DA in the ventrolateral medulla is continuously related to the level of arterial blood pressure (Rentero et al., 1993), suggesting that DA in this region participates in regulation of the cardiovascular system. Although a high DA concentration has been reported in the lower brainstem (Versteeg et al., 1976; Saavedra et al., 1979), the distribution of DA fibers has not been described at this level in any animal species. This is mainly due to the difficulty in discriminating DA from the dense NA innervation visualized with the histofluorescence method (Dahlstro¨m and Fuxe, 1964; Levitt and Moore, 1979; Bjo¨rklund and Lindvall, 1984). It is also impossible to distinguish these two innervations by tyrosine hydroxylase (TH)-immunohistochemistry, which recognizes both the DA and NA components (Ho¨kfelt et al., 1984a,b). DA-immunohisto-chemistry and only makes it possible to trace specifically DA-components. In the present study, we have therefore used DA-immunohistochemistry to describe the distribution of DA axons in the pons and medulla oblongata of rat using a highly specific monoclonal DA-antiserum in rats after inhibition of MAO. In order to confirm that accumulated DA after MAO inhibition is a transmitter but not a precursor of NA, inhibition of DBH was also effected as a control study.
2. Experimental procedure Thirty male OFA rats weighing 180 – 220 g were used. They were divided into four groups: animals that were untreated (n=5); treated with an inhibitor of MAO (pargyline, 10 mg/kg, i.p.) (n =8) or saline (n = 4) 2 h before sacrifice; treated with an inhibitor of DBH (FLA63, 40 mg/kg, i.p.) (n =5) or saline (n =3) 45 min before sacrifice. Five rats were treated with both inhibitors 45 min before sacrifice. Under deep anesthesia, each rat was perfused through the ascending aorta with 100 ml of 0.01 M
phosphate-buffered saline (PBS) followed by 200 ml of fixative containing 2% glutaraldehyde, 1% sodium metabisulfite, and 0.25% picric acid in 0.1 M PB. The brain was removed and cut into several blocks, which were postfixed in the same fixative for 8 h, followed by a rinse for 3 days in PBS containing 20% sucrose and 1% sodium metabisulfite. Sections were cut with a cryostat (25 mm) in transverse plane. The sections were incubated in (1) monoclonal antiserum against DA diluted 1:30 000 in PBS containing 0.3% Triton X-100 at 4°C for 1 week; (2) biotinylated rabbit IgG (Vector Laboratory, 1:1000) at 4°C for 12 h; and avidin–biotin–peroxidase complex (Vector Laboratory, 1:1000) at room temperature for 2 h. Peroxidase activity was then revealed in 50 mM Tris–HCl buffer (pH 7.6) containing 0.0003% H2O2, 0.01% 3,3%-diaminobenzidine –4 HCl (DAB) and 1% nickel ammonium sulfate. The reaction was terminated by washes in a Tris saline rinse. Sections were floated onto Tris solution on slides coated with 0.1% chromogelatin. After draining excess water the mounted sections were dehydrated and coverslipped using Depex. Sections were traced on a camera lucida to accurately locate DA-ir structures. Details of the production, characterization, and specificity of the present DA antiserum have been described elsewhere (Chagnaud et al., 1987; Kitahama et al., 1990).
3. Results
3.1. DA-immunoreacti6ity in normal rat (untreated and saline-treated) No difference in intensity of DA-immunoreactivity was observed between untreated and saline-treated animals. Strong DA-immunoreactivity was detected in cell bodies and proximal dendrites of substantia nigra, pars compacta (SNC) (A9 cell group), ventral tegmental area of Tsai (VTA) (A10 cell group) (Fig. 1A), retrorubral field (RR) (A8 cell group) (Fig. 1B), and dorsal raphe nucleus (DRN) (Fig. 1C), in which numerous TH-ir neurons are known to be present. In the pons and medulla, neuronal cell bodies in the A1 to A7 noradrenergic cell groups displayed only weak immunostaining (Fig. 1D). DA-ir varicose fibers were readily distinguishable in the midbrain, and especially, its midline structures. Fig. 1C illustrates them in the DRN and neighboring central gray (CG). In the pons, they were seen in the locus coeruleus (LC) and its adjacent CG (Fig. 1D). Labeled axons were also seen in small numbers in the lateral and ventral reticular formation. In the medulla oblongata, there were very fine DA-ir fibers in the dorsal vagal complex, spinal trigeminal and
K. Kitahama et al. / Journal of Chemical Neuroanatomy 18 (2000) 1–9 3
Fig. 1. DA-ir cell bodies and axons in untreated animals (A – D) and in MAOI-treated rats (E, F): intensely stained cell bodies are aggregated in the VTA (A) and dispersed in the RR (B). DA-ir varicosities are seen in the DRN (C) and CG (D). DA-immunoreactivity is very weak in LC cell bodies (indicated by arrows). MAOI treatment enhanced intensity of DA-immunoreactivity and consequently increased the number of axons in the DRN (E) and CG (F). In cell bodies of the LC, DA-immunoreactivity becomes visible (F). Bars = 100 mm.
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3.2. DA-immunoreacti6ity after inhibition of MAO
are shown in Fig. 1E,F, to be compared with Fig. 1C,D, respectively. However, only a few or sparse DA-ir axons were visible in regions known to be densely innervated by noradrenergic axons, such as the inferior olive, pontine, facial, hypoglossal nuclei, and the adrenergic axon bundles.
After MAOI treatment, the DA-immunoreactivity was enhanced in cell bodies of the A1 – A7 noradrenergic cell groups (Fig. 1F, cf. Fig. 1D), but still less intense than that in midbrain DA-ir cells. The number of DA-ir fibers increased significantly in many discrete regions of the brainstem. Some examples
3.2.1. Midbrain At the level of the substantia nigra (Fig. 2A), a large number of strongly DA-immunostained cell bodies were closely packed in the SNC and VTA, and some were scattered in the CG, interfascicular, rostral linear and caudal linear raphe nuclei.
raphe nuclei, as well as in the A1/C1 ventrolateral adrenergic cell region. DA-ir axons were also observed running through the rostral gigantocellular reticular nucleus (GiA).
Fig. 2. Semi-schematic drawings of the distribution of DA-ir cell bodies and axons in the midbrain, pons and medulla oblongata of MAOI-treated rat (traced by a camera lucida). Cell bodies showing weak DA immunoreactivity after MAO inhibition are also presented.
K. Kitahama et al. / Journal of Chemical Neuroanatomy 18 (2000) 1–9
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Fig. 2. (Continued)
DA-ir axons were mainly seen in the midline structures (CG, rostral linear raphe and interfascicular nuclei). They extended dorsally to the pretectal region and laterally to the midbrain reticular formation (deep mesencephalic nucleus). In the caudal mesencephalon (Fig. 2B), a number of DA-ir fibers were concentrated in the DRN as well as in the CG, especially in the ventrolateral portion of the latter (Fig. 1E). Also noteworthy was the presence of numerous labeled axons in the nucleus brachium inferior colliculus (BIC) and a moderate number in the superior colliculus and retrorubral field.
3.2.2. Pons In the rostral pons (Fig. 2C), DA-ir axons were abundant in the dorsal and median raphe nuclei as well as in the entire extent of the CG. In the CG, they were concentrated along the edge of the aqueduct and within the ventrolateral portion. Dorsolateral to the superior cerebellar peduncle (scp), a substantial number of immunoreactive varicose fibers forming a dense plexus were oriented ventrally to the lateral part of the rostral pontine reticular formation (PnO), medial to the lateral lemniscus. DA-ir fibers were also numerous around the inferior colliculus, which itself contained only a few.
6 K. Kitahama et al. / Journal of Chemical Neuroanatomy 18 (2000) 1–9 Fig. 3. DA-ir axons in the medulla oblongata of a MAOI-treated rat. Intensely stained varicose fibers can be seen in the rostral (A) and caudal (B) portions of the NTS and 10. They are also visible in the GiA, LPGi, Rpa and Sp5. Bars = 100 mm.
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Laterally, numerous vertically oriented DA-ir axons could be seen along the edge of the lateral surface of the brain. In the mid-pons (Fig. 2D), DA-ir varicose axons were abundant within the LC and its adjacent CG (Fig. 1F). They extended laterally to the lateral parabrachial nucleus (LPB) to form a dense plexus. Numerous scattered DA-ir axons ran vertically through the lateral portion of the caudal pontine reticular formation (PnC), medial to the motor trigeminal nucleus (Mo5). Other DA-ir fibers coursed near the midline, notably in the dorsal and pontine raphe nuclei. In the caudal pons (Fig. 2E), DA-ir axons were still visible in the LC and CG. Some were traced in a stream running through the lateral part of the PnC and aggregated in the ventrolateral corner of the brain (area between the Mo5 and lateral superior olive (LSO)). Others invaded the principal sensory trigeminal nucleus (Pr5), laterally. At the level of the facial nerve (Fig. 2F), a number of DA-ir axons were present dorsomedial to the facial genu. Many were also clustered in the lateral PnC dorsal to the LSO, and ran horizontally throughout the ventral reticular nucleus (PnV), to reach the raphe pallidus nucleus (RPa). A few were visible in the ventral cochlear nucleus.
Fig. 4. Intense DA-immunoreactivity observed in noradrenergic cells and dendrites of the LC (A) after DBH inhibition effected as a control study. It is present in fibers of the Mo5 (B) after the same treatment. DA-immunoreactivity in axons passing through the dtb (C) was only visible after treatment of both DBHI and MAOI. DA as a precursor of NA is detectable only after DBH inhibition. This evidence indicates that under normal condition, DA is rapidly transformed to NA in DBH-containing axons, and that accumulated DA in fibers after MAO inhibition is not a precursor of NA. Bars =100 mm.
3.2.3. Medulla oblongata Rostrally (Fig. 2G,H), a small number of DA-ir axons were found in the spinal trigeminal nucleus (Sp5) (Fig. 2G) and, slightly below (Fig. 2H), they suddenly increased in number and spread to the entire nucleus. They also reached more medially into the parvocellular reticular formation (PCRt), but axons running through the adrenergic ventral tegmental bundle (vtb) were not stained. As shown in Fig. 3A,B, a dense plexus of DA-ir axons pervaded the dorsal motor nucleus of vagus (10) and nucleus of the solitary tract (NTS). Ventrally, labeled axons were seen in the gigantocellular reticular nucleus pars alpha (GiA) (Fig. 3C). Others ran between the lateral paragigantocellular nucleus (C1 adrenergic cell region) (Fig. 3D) and the raphe pallidus nucleus, which was particularly enriched in DA-ir axons (Fig. 3E). Caudally (Fig. 2I,J), DA-ir fibers were concentrated in the dorsal vagal complex (10 and NTS pars commissuralis (COM)), and more diffusely spread in the dorsal and ventral medullary reticular formation (MdD, MdV). A loose plexus was also visible in Sp5, particularly, in its external edge (Fig. 3F). 3.3. DA-immunoreacti6ity after inhibition of DBH acti6ity Forty-five minutes after treatment with DBH inhibitor (FLA-63), intense DA-immunoreactivity was observed in noradrenergic cells as shown in Fig. 4A. Immunostained axons were abundant in the reticular formation, pontine
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nucleus, Pr5, Mo5 (Fig. 4B), inferior olive and hypoglossal nucleus. In the other parts of the pons and medulla, a large number of labeled fibers had a distribution pattern similar to that of noradrenergic fibers. There was no DA-immunostaining of axons in the dorsal and ventral tegmental bundles after this treatment. It was only visible after treatment of both MAO and DBH inhibitors (Fig. 4C).
4. Discussion Owing to the use of a specific monoclonal DA antibody, the present report provides information on the distribution and chemical characteristics of DA-ir fibers in the rat lower brainstem. Comparison between untreated and DBH-inhibitor-treated rats indicated that DA is not present in detectable amounts in NA fibers under normal conditions. Following DBH inhibition marked increases in the number of labeled axons were observed in regions where sparse or a few DA-ir fibers were normally seen: inferior olive, pontine, motor trigeminal, facial and hypoglossal nuclei, all areas where high NA (over 10 pg/mg protein) but very low DA concentrations have been measured biochemically (Versteeg et al., 1976). Biochemical studies (Buda et al., 1983) have reported that blockade of DBH activity by FLA-63 induces a significant increase (208% over control) in DA in the LC region. This was confirmed immunohistochemically by a marked increase in the DA-immunoreactivity of cell bodies in this area. The DBH inhibitor-induced increases in DA immunoreactivity occurred within 45 min, indicating that DA is rapidly converted to NA in axons containing DBH (Ande´n et al., 1985). In contrast, MAO inhibition enhanced DA-immunoreactivity in presumed DA fibers and consequently increased the number of labeled axons in many lower brainstem regions. This indicated that DA is perpetually oxidized by MAO in these fibers. The present findings revealed a much more widespread distribution of DA-ir fibers in the pons and medulla than had been previously suspected. In the pons, DA-ir varicose fibers were concentrated in the dorsal tegmentum, in keeping with the biochemical results having demonstrated high amounts of DA in the LC region (16 pg/mg protein) compared to other pontine structures (1–3 pg/mg) (Versteeg et al., 1976). A substantial amount of DOPAC has been measured in the LC area (Buda et al., 1983), indicating utilization of DA in this region. DA-ir fibers were abundant in a noradrenergic dendritic area of central gray medial to the LC. We have recently reported that most DA varicosities in the LC region are of relatively large size and form asymmetric
synaptic contacts on noradrenergic dendrites (Maeda et al., 1995), suggesting strong DA influences on LC noradrenergic neurons. Such terminal fibers have been shown to arise exclusively from the A13 hypothalamic DA cell group (Luppi et al., 1995). We could distinguish numerous DA-ir fibers in other pontine structures such as the dorsal and median raphe nuclei, CG and parabrachial region. These could well originate from the VTA or medial aspect of the SNC which have been shown to send many axons to the midline, reticular formation, DRN, lateral CG, and LPB (Van Bockstaele et al., 1989). We have recently demonstrated that DA-ir axons in the DRN arise exclusively from the VTA and from the A11 dorsal hypothalamic DA cell group (Peyron et al., 1995). It should be underlined that a plexus composed of numerous DA-ir axons is present in the lateral part of the reticular formation at the pontine level and its ventrolateral part more caudally. Similar findings have been made in two amphibian species (Gonzalez and Smeets, 1991). These DA axons extend caudalward, as major descending DA pathways, to the ventral medulla or laterally to the Sp5. Some A11 DA cells also send their axons, probably via the Sp5, down to the spinal cord (Ho¨kfelt et al., 1979). In the dorsal vagal region, DA-ir fibers were abundant, as has been reported in teleost amphibians (Ekstro¨m et al., 1990). This result was in good agreement with earlier biochemical observations (Versteeg et al., 1976; Lambas-Senas et al., 1990). DA concentration is higher (2–10 pg/mg protein) in this region than in other parts of medulla (0–1 pg/mg) (Versteeg et al., 1976). These areas show high DA/NA ratios (20–30%). DA is also concentrated in the more caudally situated nucleus commissuralis (10 pg/mg), in which a very dense plexus of DA terminals is present. Since the release of DA in the NTS, measured by microdialysis, has been shown to be enhanced by hypoxia, it is likely that DA is involved in the central ventilatory response to severe hypoxia (Goiny et al., 1991). In the ventrolateral catecholaminergic cell region, we identified a large number of dispersed DA-ir axons. In this region, in vivo voltammetric studies have demonstrated an increase in DOPAC occurring immediately after hemorrhage and antagonized by clonidine treatment (Gillon et al., 1990). We also showed that the level of activity of catecholamine metabolism in this region changes in synchrony with arterial pressure (Rentero et al., 1993). This would suggest that DA in the caudal brainstem may be involved in the autonomic regulation of the cardiovascular system.
Acknowledgements This study was supported by INSERM U480.
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Distribution of dopamine-immunoreactive fibers in the rat brainstem K. Kitahama a,*, I. Nagatsu b, M. Geffard c, T. Maeda d a
De´partement de Me´decine Expe´rimentale, INSERM U480, Faculte´ de Me´decine, Uni6ersite´ Claude Bernard, 8, a6enue Rockefeller, 69373 Lyon, Cedex 08, France b Department of Anatomy, School of Medicine, Fujita Health Uni6ersity, Toyoake, Japan c Institut de Biochimie Cellulaire et Neurochimie, Uni6ersite´ de Bordeaux II, Bordeaux, France d Department of Anatomy, Shiga Uni6ersity of Medical Science, Ohtsu, Japan Received 22 June 1999; received in revised form 12 October 1999; accepted 12 October 1999
Abstract We describe the distribution of axons immunoreactive for dopamine in pons and medulla oblongata of rat under normal conditions or after inhibition of monoamine oxidase or dopamine b-hydroxylase. In the pons of non-treated animal, fairly dense plexuses of dopamine-immunoreactive varicose fibers were found in the locus coeruleus, dorsal parabrachial and dorsal raphe nuclei, central gray and reticular formation dorsal to the superior olive. In the medulla oblongata, the immunoreactive fibers were abundant in the dorsal vagal complex, lateral paragigantocellular nucleus, midline raphe nuclei and spinal trigeminal nucleus. Monoamine oxidase inhibition made it possible to increase the intensity of immunoreactivity and consequently the number of labeled fibers in these areas, indicating that dopamine is perpetually oxidized by monoamine oxidase, and consequently in low concentration under normal conditions. Sparse dopamine-immunoreactive fibers were observed in the pontine gray, motor trigeminal nucleus, inferior olive and major axon bundles such as the dorsal and ventral tegmental bundles, where numerous noradrenergic fibers have been reported. In axons of these areas, intense dopamine-immunoreactivity was seen only after inhibition of dopamine-b-hydroxylase. It appears that dopamine is released and oxidized in response to autonomic changes such as hypoxia, hemorrhage, and cardiovascular variation in the caudal brainstem, as we have described elsewhere. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Catecholamines; Dopamine; Locus coeruleus; Medulla; Pons; Monoamine oxidase; Immunohistochemistry
Abbre6iations: 10, dorsal motor nucleus of the vagus; 12, hypoglossal nucleus; 7n, facial nerve; AQ, aqueduct; BIC, nucleus brachium inferior colliculus; CG, central gray; CLi, caudal linear raphe nucleus; COM, commissural nucleus, subnucleus of the NTS; cp, cerebral peduncle; CU, cuneate nucleus; DLL, dorsal nucleus lateral lemniscus; DpMe, deep mesencephalic nucleus; DRN, dorsal raphe nucleus; fr, fasciculus retroflexus; g7, genu facial nerve; Gi, gigantocellular reticular nucleus; GiA, gigantocellular reticular nucleus, pars alpha; IC, inferior colliculus; IF, interfascicular nucleus; IO, inferior olive; IP, interpeduncular nucleus; LC, locus coeruleus; ll, lateral lemniscus; LPB, lateral parabrachial nucleus; LPGi, lateral paragigantocellular nucleus; LRN, lateral reticular nucleus; LSO, superior olive; MdD, medullary reticular field, dorsal; MdV, medullary reticular nucleus, ventral; ml, medial lemniscus; mlf, medial longitudinal fasciculus; MnR, median raphe nucleus; Mo5, motor trigeminal nucleus; MPB, medial parabrachial nucleus; MVe, medial vestibular nucleus; NTS, nucleus of the solitary tract; PCRt, parvocellular reticular nucleus; Pn, pontine nucleus; PnC, pontine reticular nucleus, caudal; PnO, pontine reticular nucleus, oral; PnV, pontine reticular nucleus, ventral; Pr5, principal sensory trigeminal nucleus; PTA, pretectal area; py, pyramidal tract; pyx, pyramidal decussation; RLi, rostral linear raphe nucleus; RMg, raphe magnus nucleus; ROb, raphe obscurus nucleus; RPa, raphe pallidus nucleus; RPn, raphe pontis nucleus; RR, retrorubral field; SC, superior colliculus; scp, superior cerebellar peduncle; SNC, substantia nigra pars compacta; SNR, substantia nigra pars reticularis; Sp5C, spinal trigeminal nucleus, caudal; Sp5O, spinal trigeminal nucleus, rostral; st, solitary tract; SuVe, superior vestibular nucleus; VCA, ventral cochlear nucleus, anterior; VLL, ventral nucleus lateral lemniscus; VTA, ventral tegmental area; vtb, ventral tegmental bundle; xscp, decussation of the superior cerebellar peduncle. * Corresponding author. Tel.: + 33-4-78777197; fax: +33-4-78777172. E-mail address: [email protected] (K. Kitahama) 0891-0618/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 8 9 1 - 0 6 1 8 ( 9 9 ) 0 0 0 4 7 - 2
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1. Introduction Dopamine (DA) is synthesized from L-dihydroxyphenyl-alanine (L-DOPA), which is transformed from tyrosine by their first step synthesizing enzyme TH (Nagatsu et al., 1964). It is oxidized to 3,4-dihydroxyphenyl-acetic acid (DOPAC) by its degrading enzyme, monoamine oxidase (MAO), or hydroxylated to noradrenaline (NA) by the enzyme dopamine-b-hydroxylase (DBH). Cerebral DA content is thus strongly influenced by the activity of these enzymes. According to in vivo voltammetric technique studies from our laboratory, there is a significant decrease in oxidation current of DA in the locus coeruleus and ventrolateral medulla after MAO inhibition (Buda et al., 1983; Gillon et al., 1990). This indicates a perpetual oxidation of DA in these regions. We recently demonstrated that the metabolic rate of DA in the ventrolateral medulla is continuously related to the level of arterial blood pressure (Rentero et al., 1993), suggesting that DA in this region participates in regulation of the cardiovascular system. Although a high DA concentration has been reported in the lower brainstem (Versteeg et al., 1976; Saavedra et al., 1979), the distribution of DA fibers has not been described at this level in any animal species. This is mainly due to the difficulty in discriminating DA from the dense NA innervation visualized with the histofluorescence method (Dahlstro¨m and Fuxe, 1964; Levitt and Moore, 1979; Bjo¨rklund and Lindvall, 1984). It is also impossible to distinguish these two innervations by tyrosine hydroxylase (TH)-immunohistochemistry, which recognizes both the DA and NA components (Ho¨kfelt et al., 1984a,b). DA-immunohisto-chemistry and only makes it possible to trace specifically DA-components. In the present study, we have therefore used DA-immunohistochemistry to describe the distribution of DA axons in the pons and medulla oblongata of rat using a highly specific monoclonal DA-antiserum in rats after inhibition of MAO. In order to confirm that accumulated DA after MAO inhibition is a transmitter but not a precursor of NA, inhibition of DBH was also effected as a control study.
2. Experimental procedure Thirty male OFA rats weighing 180 – 220 g were used. They were divided into four groups: animals that were untreated (n=5); treated with an inhibitor of MAO (pargyline, 10 mg/kg, i.p.) (n =8) or saline (n = 4) 2 h before sacrifice; treated with an inhibitor of DBH (FLA63, 40 mg/kg, i.p.) (n =5) or saline (n =3) 45 min before sacrifice. Five rats were treated with both inhibitors 45 min before sacrifice. Under deep anesthesia, each rat was perfused through the ascending aorta with 100 ml of 0.01 M
phosphate-buffered saline (PBS) followed by 200 ml of fixative containing 2% glutaraldehyde, 1% sodium metabisulfite, and 0.25% picric acid in 0.1 M PB. The brain was removed and cut into several blocks, which were postfixed in the same fixative for 8 h, followed by a rinse for 3 days in PBS containing 20% sucrose and 1% sodium metabisulfite. Sections were cut with a cryostat (25 mm) in transverse plane. The sections were incubated in (1) monoclonal antiserum against DA diluted 1:30 000 in PBS containing 0.3% Triton X-100 at 4°C for 1 week; (2) biotinylated rabbit IgG (Vector Laboratory, 1:1000) at 4°C for 12 h; and avidin–biotin–peroxidase complex (Vector Laboratory, 1:1000) at room temperature for 2 h. Peroxidase activity was then revealed in 50 mM Tris–HCl buffer (pH 7.6) containing 0.0003% H2O2, 0.01% 3,3%-diaminobenzidine –4 HCl (DAB) and 1% nickel ammonium sulfate. The reaction was terminated by washes in a Tris saline rinse. Sections were floated onto Tris solution on slides coated with 0.1% chromogelatin. After draining excess water the mounted sections were dehydrated and coverslipped using Depex. Sections were traced on a camera lucida to accurately locate DA-ir structures. Details of the production, characterization, and specificity of the present DA antiserum have been described elsewhere (Chagnaud et al., 1987; Kitahama et al., 1990).
3. Results
3.1. DA-immunoreacti6ity in normal rat (untreated and saline-treated) No difference in intensity of DA-immunoreactivity was observed between untreated and saline-treated animals. Strong DA-immunoreactivity was detected in cell bodies and proximal dendrites of substantia nigra, pars compacta (SNC) (A9 cell group), ventral tegmental area of Tsai (VTA) (A10 cell group) (Fig. 1A), retrorubral field (RR) (A8 cell group) (Fig. 1B), and dorsal raphe nucleus (DRN) (Fig. 1C), in which numerous TH-ir neurons are known to be present. In the pons and medulla, neuronal cell bodies in the A1 to A7 noradrenergic cell groups displayed only weak immunostaining (Fig. 1D). DA-ir varicose fibers were readily distinguishable in the midbrain, and especially, its midline structures. Fig. 1C illustrates them in the DRN and neighboring central gray (CG). In the pons, they were seen in the locus coeruleus (LC) and its adjacent CG (Fig. 1D). Labeled axons were also seen in small numbers in the lateral and ventral reticular formation. In the medulla oblongata, there were very fine DA-ir fibers in the dorsal vagal complex, spinal trigeminal and
K. Kitahama et al. / Journal of Chemical Neuroanatomy 18 (2000) 1–9 3
Fig. 1. DA-ir cell bodies and axons in untreated animals (A – D) and in MAOI-treated rats (E, F): intensely stained cell bodies are aggregated in the VTA (A) and dispersed in the RR (B). DA-ir varicosities are seen in the DRN (C) and CG (D). DA-immunoreactivity is very weak in LC cell bodies (indicated by arrows). MAOI treatment enhanced intensity of DA-immunoreactivity and consequently increased the number of axons in the DRN (E) and CG (F). In cell bodies of the LC, DA-immunoreactivity becomes visible (F). Bars = 100 mm.
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3.2. DA-immunoreacti6ity after inhibition of MAO
are shown in Fig. 1E,F, to be compared with Fig. 1C,D, respectively. However, only a few or sparse DA-ir axons were visible in regions known to be densely innervated by noradrenergic axons, such as the inferior olive, pontine, facial, hypoglossal nuclei, and the adrenergic axon bundles.
After MAOI treatment, the DA-immunoreactivity was enhanced in cell bodies of the A1 – A7 noradrenergic cell groups (Fig. 1F, cf. Fig. 1D), but still less intense than that in midbrain DA-ir cells. The number of DA-ir fibers increased significantly in many discrete regions of the brainstem. Some examples
3.2.1. Midbrain At the level of the substantia nigra (Fig. 2A), a large number of strongly DA-immunostained cell bodies were closely packed in the SNC and VTA, and some were scattered in the CG, interfascicular, rostral linear and caudal linear raphe nuclei.
raphe nuclei, as well as in the A1/C1 ventrolateral adrenergic cell region. DA-ir axons were also observed running through the rostral gigantocellular reticular nucleus (GiA).
Fig. 2. Semi-schematic drawings of the distribution of DA-ir cell bodies and axons in the midbrain, pons and medulla oblongata of MAOI-treated rat (traced by a camera lucida). Cell bodies showing weak DA immunoreactivity after MAO inhibition are also presented.
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Fig. 2. (Continued)
DA-ir axons were mainly seen in the midline structures (CG, rostral linear raphe and interfascicular nuclei). They extended dorsally to the pretectal region and laterally to the midbrain reticular formation (deep mesencephalic nucleus). In the caudal mesencephalon (Fig. 2B), a number of DA-ir fibers were concentrated in the DRN as well as in the CG, especially in the ventrolateral portion of the latter (Fig. 1E). Also noteworthy was the presence of numerous labeled axons in the nucleus brachium inferior colliculus (BIC) and a moderate number in the superior colliculus and retrorubral field.
3.2.2. Pons In the rostral pons (Fig. 2C), DA-ir axons were abundant in the dorsal and median raphe nuclei as well as in the entire extent of the CG. In the CG, they were concentrated along the edge of the aqueduct and within the ventrolateral portion. Dorsolateral to the superior cerebellar peduncle (scp), a substantial number of immunoreactive varicose fibers forming a dense plexus were oriented ventrally to the lateral part of the rostral pontine reticular formation (PnO), medial to the lateral lemniscus. DA-ir fibers were also numerous around the inferior colliculus, which itself contained only a few.
6 K. Kitahama et al. / Journal of Chemical Neuroanatomy 18 (2000) 1–9 Fig. 3. DA-ir axons in the medulla oblongata of a MAOI-treated rat. Intensely stained varicose fibers can be seen in the rostral (A) and caudal (B) portions of the NTS and 10. They are also visible in the GiA, LPGi, Rpa and Sp5. Bars = 100 mm.
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Laterally, numerous vertically oriented DA-ir axons could be seen along the edge of the lateral surface of the brain. In the mid-pons (Fig. 2D), DA-ir varicose axons were abundant within the LC and its adjacent CG (Fig. 1F). They extended laterally to the lateral parabrachial nucleus (LPB) to form a dense plexus. Numerous scattered DA-ir axons ran vertically through the lateral portion of the caudal pontine reticular formation (PnC), medial to the motor trigeminal nucleus (Mo5). Other DA-ir fibers coursed near the midline, notably in the dorsal and pontine raphe nuclei. In the caudal pons (Fig. 2E), DA-ir axons were still visible in the LC and CG. Some were traced in a stream running through the lateral part of the PnC and aggregated in the ventrolateral corner of the brain (area between the Mo5 and lateral superior olive (LSO)). Others invaded the principal sensory trigeminal nucleus (Pr5), laterally. At the level of the facial nerve (Fig. 2F), a number of DA-ir axons were present dorsomedial to the facial genu. Many were also clustered in the lateral PnC dorsal to the LSO, and ran horizontally throughout the ventral reticular nucleus (PnV), to reach the raphe pallidus nucleus (RPa). A few were visible in the ventral cochlear nucleus.
Fig. 4. Intense DA-immunoreactivity observed in noradrenergic cells and dendrites of the LC (A) after DBH inhibition effected as a control study. It is present in fibers of the Mo5 (B) after the same treatment. DA-immunoreactivity in axons passing through the dtb (C) was only visible after treatment of both DBHI and MAOI. DA as a precursor of NA is detectable only after DBH inhibition. This evidence indicates that under normal condition, DA is rapidly transformed to NA in DBH-containing axons, and that accumulated DA in fibers after MAO inhibition is not a precursor of NA. Bars =100 mm.
3.2.3. Medulla oblongata Rostrally (Fig. 2G,H), a small number of DA-ir axons were found in the spinal trigeminal nucleus (Sp5) (Fig. 2G) and, slightly below (Fig. 2H), they suddenly increased in number and spread to the entire nucleus. They also reached more medially into the parvocellular reticular formation (PCRt), but axons running through the adrenergic ventral tegmental bundle (vtb) were not stained. As shown in Fig. 3A,B, a dense plexus of DA-ir axons pervaded the dorsal motor nucleus of vagus (10) and nucleus of the solitary tract (NTS). Ventrally, labeled axons were seen in the gigantocellular reticular nucleus pars alpha (GiA) (Fig. 3C). Others ran between the lateral paragigantocellular nucleus (C1 adrenergic cell region) (Fig. 3D) and the raphe pallidus nucleus, which was particularly enriched in DA-ir axons (Fig. 3E). Caudally (Fig. 2I,J), DA-ir fibers were concentrated in the dorsal vagal complex (10 and NTS pars commissuralis (COM)), and more diffusely spread in the dorsal and ventral medullary reticular formation (MdD, MdV). A loose plexus was also visible in Sp5, particularly, in its external edge (Fig. 3F). 3.3. DA-immunoreacti6ity after inhibition of DBH acti6ity Forty-five minutes after treatment with DBH inhibitor (FLA-63), intense DA-immunoreactivity was observed in noradrenergic cells as shown in Fig. 4A. Immunostained axons were abundant in the reticular formation, pontine
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nucleus, Pr5, Mo5 (Fig. 4B), inferior olive and hypoglossal nucleus. In the other parts of the pons and medulla, a large number of labeled fibers had a distribution pattern similar to that of noradrenergic fibers. There was no DA-immunostaining of axons in the dorsal and ventral tegmental bundles after this treatment. It was only visible after treatment of both MAO and DBH inhibitors (Fig. 4C).
4. Discussion Owing to the use of a specific monoclonal DA antibody, the present report provides information on the distribution and chemical characteristics of DA-ir fibers in the rat lower brainstem. Comparison between untreated and DBH-inhibitor-treated rats indicated that DA is not present in detectable amounts in NA fibers under normal conditions. Following DBH inhibition marked increases in the number of labeled axons were observed in regions where sparse or a few DA-ir fibers were normally seen: inferior olive, pontine, motor trigeminal, facial and hypoglossal nuclei, all areas where high NA (over 10 pg/mg protein) but very low DA concentrations have been measured biochemically (Versteeg et al., 1976). Biochemical studies (Buda et al., 1983) have reported that blockade of DBH activity by FLA-63 induces a significant increase (208% over control) in DA in the LC region. This was confirmed immunohistochemically by a marked increase in the DA-immunoreactivity of cell bodies in this area. The DBH inhibitor-induced increases in DA immunoreactivity occurred within 45 min, indicating that DA is rapidly converted to NA in axons containing DBH (Ande´n et al., 1985). In contrast, MAO inhibition enhanced DA-immunoreactivity in presumed DA fibers and consequently increased the number of labeled axons in many lower brainstem regions. This indicated that DA is perpetually oxidized by MAO in these fibers. The present findings revealed a much more widespread distribution of DA-ir fibers in the pons and medulla than had been previously suspected. In the pons, DA-ir varicose fibers were concentrated in the dorsal tegmentum, in keeping with the biochemical results having demonstrated high amounts of DA in the LC region (16 pg/mg protein) compared to other pontine structures (1–3 pg/mg) (Versteeg et al., 1976). A substantial amount of DOPAC has been measured in the LC area (Buda et al., 1983), indicating utilization of DA in this region. DA-ir fibers were abundant in a noradrenergic dendritic area of central gray medial to the LC. We have recently reported that most DA varicosities in the LC region are of relatively large size and form asymmetric
synaptic contacts on noradrenergic dendrites (Maeda et al., 1995), suggesting strong DA influences on LC noradrenergic neurons. Such terminal fibers have been shown to arise exclusively from the A13 hypothalamic DA cell group (Luppi et al., 1995). We could distinguish numerous DA-ir fibers in other pontine structures such as the dorsal and median raphe nuclei, CG and parabrachial region. These could well originate from the VTA or medial aspect of the SNC which have been shown to send many axons to the midline, reticular formation, DRN, lateral CG, and LPB (Van Bockstaele et al., 1989). We have recently demonstrated that DA-ir axons in the DRN arise exclusively from the VTA and from the A11 dorsal hypothalamic DA cell group (Peyron et al., 1995). It should be underlined that a plexus composed of numerous DA-ir axons is present in the lateral part of the reticular formation at the pontine level and its ventrolateral part more caudally. Similar findings have been made in two amphibian species (Gonzalez and Smeets, 1991). These DA axons extend caudalward, as major descending DA pathways, to the ventral medulla or laterally to the Sp5. Some A11 DA cells also send their axons, probably via the Sp5, down to the spinal cord (Ho¨kfelt et al., 1979). In the dorsal vagal region, DA-ir fibers were abundant, as has been reported in teleost amphibians (Ekstro¨m et al., 1990). This result was in good agreement with earlier biochemical observations (Versteeg et al., 1976; Lambas-Senas et al., 1990). DA concentration is higher (2–10 pg/mg protein) in this region than in other parts of medulla (0–1 pg/mg) (Versteeg et al., 1976). These areas show high DA/NA ratios (20–30%). DA is also concentrated in the more caudally situated nucleus commissuralis (10 pg/mg), in which a very dense plexus of DA terminals is present. Since the release of DA in the NTS, measured by microdialysis, has been shown to be enhanced by hypoxia, it is likely that DA is involved in the central ventilatory response to severe hypoxia (Goiny et al., 1991). In the ventrolateral catecholaminergic cell region, we identified a large number of dispersed DA-ir axons. In this region, in vivo voltammetric studies have demonstrated an increase in DOPAC occurring immediately after hemorrhage and antagonized by clonidine treatment (Gillon et al., 1990). We also showed that the level of activity of catecholamine metabolism in this region changes in synchrony with arterial pressure (Rentero et al., 1993). This would suggest that DA in the caudal brainstem may be involved in the autonomic regulation of the cardiovascular system.
Acknowledgements This study was supported by INSERM U480.
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