Immunocytochemical localization and ontogenic development of ...

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Abstract Melanin-concentrating hormone (MCH) was first discovered in the pituitary of chum salmon because of its role in the regulation of skin pallor. Later ...
Cell Tissue Res (2003) 311:71–77 DOI 10.1007/s00441-002-0660-6

R E G U L A R A RT I C L E

Masafumi Amano · Akiyoshi Takahashi Yoshitaka Oka · Takeshi Yamanome Hiroshi Kawauchi · Kunio Yamamori

Immunocytochemical localization and ontogenic development of melanin-concentrating hormone in the brain of a pleuronectiform fish, the barfin flounder Received: 22 May 2002 / Accepted: 15 October 2002 / Published online: 14 November 2002 © Springer-Verlag 2002

Abstract Melanin-concentrating hormone (MCH) was first discovered in the pituitary of chum salmon because of its role in the regulation of skin pallor. Later, it was found that MCH could also play a role as a central neurotransmitter or neuromodulator in the brain. However, knowledge of the function of MCH in fish has been restricted to certain fish species. Therefore, in the present study, the immunocytochemical localization and ontogenic development of MCH in the brain of a pleuronectiform fish, the barfin flounder Verasper moseri, were examined to obtain a better understanding of this hormone. In adult barfin flounder, MCH-immunoreactive (ir) neuronal somata were most prevalent in the magnocellular neurons of the nucleus tuberis lateralis (NLT), which project to the pituitary. In the pituitary, MCH-ir fibers were distributed in the neurohypophysial tissues within the pars intermedia and, to a lesser extent, into the pars distalis. MCH-ir neuronal somata were also present in dorsally projecting parvocellular neurons, located more posteriorly in the area above the lateral ventricular recess (LVR). LVR-MCH neurons did not seem to project to the pituitary. In the brain, MCH-ir fibers were detected not only in the hypothalamus but also in areas such as the optic tectum and thalamus. MCH-ir neuronal somata and fibers were not detected on the day of hatching. MCH-ir neuronal somata and fibers were first detected in the hypothalamus and the pituitary, respectively, 7 days after hatching. Subsequently, MCH-ir neuronal somata were observed in the NLT M. Amano (✉) · A. Takahashi · H. Kawauchi · K. Yamamori School of Fisheries Sciences, Kitasato University, Sanriku, Ofunato, Iwate 022-0101, Japan e-mail: [email protected] Tel.: +81-192-441904, Fax: +81-192-441904 Y. Oka Misaki Marine Biological Station, Graduate School of Science, The University of Tokyo, Misaki, Kanagawa 238-0225, Japan T. Yamanome Iwate Fisheries Technology Center, Kamaishi, Iwate 026-0001, Japan

and in the area above the LVR 14 days after hatching. The distribution of MCH-ir neuronal somata and fibers showed a pattern similar to that in the adult fish 35–42 days after hatching. These results indicate that MCH neurons were located in the NLT and in the area above the LVR and that NLT-MCH neurons project to the pituitary. MCH neurons were first detected 7 days after hatching, suggesting that MCH plays some physiological role in the early development of barfin flounder. Keywords Melanin-concentrating hormone · Immunocytochemistry · Ontogeny · Barfin flounder · Verasper moseri (Teleostei) Abbreviations DF nucleus diffusus of the inferior lobe · HCo horizontal commissure · NAPv nucleus anterior periventricularis · NH habenula · NLTi nucleus lateralis tuberis pars inferior · NLTl nucleus lateralis tuberis pars lateralis · NLTp nucleus lateralis tuberis pars posterior · NPG nucleus preglomerulosus · NPGl nucleus preglomerulosus lateralis · NPOm nucleus preopticus pars magnocellularis · NRLd nucleus recessus lateralis pars dorsalis · NRLl nucleus recessus lateralis pars lateralis · OT optic tectum · Pc nucleus pretectalis centralis · PIT pituitary · SPV stratum periventriculare · SV saccus vasculosus

Introduction Melanin-concentrating hormone (MCH), present in the hypothalamus of all vertebrates, was first discovered in the pituitary of chum salmon, Oncorhynchus keta, because of its role in the regulation of skin pallor (Kawauchi et al. 1983). In in vitro bioassays, MCH directly affects the concentration of melanin granules in the rainbow trout, Oncorhynchus mykiss, carp, Cyprinus carpio, black rockfish, Sebastes schlegeri, and fat greenling, Hexagrammos otakii. MCH-immunoreactive (ir) fibers ap-

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peared to be absent from the posterior neurohypophysis of the polypteriformes, but were evident in this region in the chondrosteans, the holosteans, and all teleosts, suggesting its use as a neurohypophysial hormone (Baker and Bird 2002). In teleost fish, MCH has been suggested to also play a role as a central neurotransmitter or neuromodulator in addition to its role as a neurohypophysial hormone, since MCH-ir fibers have been reported in extrahypothalamic areas, such as the olfactory bulb, pretectal thalamus, optic tectum and midbrain (Naito et al. 1985; Batten and Baker 1988; Batten et al. 1990). Since very little MCH peptide can be found in the mammalian pituitary or circulation (Bittencourt et al. 1992; Takahashi et al. 1995), it has been suggested that MCH peptide has been conserved principally as a central neuromodulator or neurotransmitter. MCH is considered to regulate food intake (Qu et al. 1996; Presse et al. 1996; Schwartz et al. 2000). In addition, MCH has been suggested to be involved in many physiological controls, including lactation (Knollema et al. 1992), water intake (Presse and Nahon 1993), osmoregulation (Parkes and Vale 1992; Hervieu et al. 1996; Parkes 1996), stress (Baker 1994; Nahon 1994) and various forms of behavior (Miller et al. 1993; Gonzalez et al. 1996; Sanchez et al. 1997). The localization of MCH-ir neuronal somata and fibers has been examined by immunocytochemistry in several species of teleosts: chum salmon, rainbow trout (Naito et al. 1985), molly, Poecilia latipinna (Batten and Baker 1988), chinook salmon, Oncorhynchus tshawytscha (Minth et al. 1989), carp (Bird et al. 1989), gilthead seabream, Sparus auratus (Mancera and FernándezLlebrez 1995; Baker and Bird 2002), golden-eyes, Hiodon alosoids, butterfly fish, Pantodon berkholzei, European eel, Anguilla anguilla, grass carp, Ctenopharyngodon idellus, and European flounder, Pleuronectes flesus (Baker and Bird 2002). It was originally supposed that MCH-ir neuronal somata were located only in the nucleus tuberis lateralis (NLT). NLT-MCH neuronal somata project to the pituitary and their synthetic activity alters in response to the color of the environment (Bird and Baker 1989; Groneveld et al. 1995a; Suzuki et al. 1995). However, a second group of parvocellular MCH neuronal somata has recently been identified in the dorsal hypothalamus, located in the area above the lateral ventricular recess (LVR) of the third ventricle in the rainbow trout, Table 1 Sampling date, days after hatching (days), mean total length (TL), mean body weight (BW), and stage of development of barfin flounder (NT not tested)

by in situ hybridization and immunocytochemistry. LVRMCH neuronal somata do not project to the pituitary but project centrally (Baker et al. 1995). Existence of LVRMCH neurons has also been reported in golden-eyes, butterfly fish, European eel, grass carp, European flounder and gilthead seabream (Baker and Bird 2002). The ontogenic development of the MCH system has been examined only in chum salmon (Naito et al. 1993) and gilthead seabream (Mancera and Fernández-Llebrez 1995). According to these reports, the MCH system is fully developed by the time of hatching in chum salmon, whereas MCH-ir fibers do not appear in the neurohypophysis until 2 days posthatching in gilthead seabream. Thus, it is suggested that MCH plays some role in the early development of teleost fish. Studies are needed to examine the ontogenic development of MCH neurons in teleost fish for a better understanding of its function. Barfin flounder, Verasper moseri, is a large pleuronectiform fish inhabiting cold sea basins around eastern Hokkaido, Japan. This species is promising for aquaculture and resource enhancement in northern Japan due to its high commercial value. The existence of MCH in the brain of barfin flounder has been confirmed by molecular cloning of MCH; the deduced amino acid sequence was the same as that of chum salmon except for one amino acid (Takahashi et al., in preparation). Indeed, injection of salmonid MCH into barfin flounder induced concentration of melanin granules and whitened the body color (unpublished data). It has recently become a problem that the side with no eyes blackens under rearing conditions in the barfin flounder, because this blacking diminishes the commercial value of the fish. MCH may be involved in this. In this study, as a first step, we examined the distribution of MCH-ir neuronal somata and fibers in adult barfin flounder by immunocytochemistry. Then, we examined the ontogenic development of the MCH system in the barfin flounder.

Materials and methods Fish One-year-old barfin flounder (body weight 50–120 g) were obtained from the Iwate Fisheries Technology Center, Iwate Prefecture, Japan. The fish were reared under a natural photoperiod in seawater of natural temperature.

Sampling date

Days

TL

BW

Stage of development

7 April 2000 14 April 2000 21 April 2000 28 April 2000 5 May 2000 12 May 2000 19 May 2000 26 May 2000 2 June 2000 14 June 2000

0 7 14 21 28 35 42 49 56 68

0.34 0.51 0.54 0.59 0.99 1.24 1.85 2.53 2.99 3.58

NT NT NT NT NT 0.03 0.10 0.22 0.31 0.49

Newly hatched yolk-sac larva Yolk-sac larva Preflexion larva Flexion larva Postflexion larva, onset of metamorphosis Postflexion larva, early metamorphosis Postflexion larva, late metamorphosis Juvenile Juvenile Juvenile

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Fig. 1 Schematic illustration of the distribution of MCH-ir neuronal somata in the NLT (large dots), MCH-ir neuronal somata in the area above the LVR (small dots) and MCH-ir fibers in a parasagittal section of adult fish (C cerebellum, LVR lateral ventricular recess, M medulla oblongata, OB olfactory bulb, ON optic nerve, OT optic tectum, PIT pituitary, T telencephalon). Bar 1 mm For the ontogenic study, barfin flounder that hatched on April 7, 2000, were used. As shown in Table 1, fish were sampled on April 7 (day 0), 14 (day 7), 21 (day 14), and 28 (day 21), May 5 (day 28), 12 (day 35), 19 (day 42), and 26 (day 49), and June 2 (day 56) and 14 (day 68). For the developmental stage of larva, we referred to Aritaki et al. (2000). Metamorphosis, characterized by the migration of the eye from the left to right side of the body, began on day 28, and was complete by day 49. Immunocytochemistry Fish were anesthetized by immersing them in 0.05% 2-phenoxyethanol. Total length and body weight were measured. Brains or head regions were fixed with Bouin's fluid for 24 h at 4°C and subsequently rinsed in cold 70% ethanol, dehydrated through a graded series of ethanol concentrations and embedded in Paraplast (Monoject, Sherwood Medical, St. Louis, MO). Sagittal or frontal sections were cut at 8 µm, and mounted on gelatinized slides. Antibody to salmon MCH was used. The antibody was diluted 6,500fold with 0.1 M phosphate buffer (pH 7.4) containing 0.75% NaCl and 0.3% Triton X-100. For immunocytochemical reactions, a Histofine immunostaining kit (Nichirei, Tokyo, Japan) was used. To test the specificity of immunoreactions, the control sections were incubated in antisera that were preabsorbed overnight at 4°C with an excess amount of synthetic MCH (10 µg MCH in 1 ml of antiserum). The subsequent procedure was the same as in the experimental sections. For the histological identification of the nuclear boundaries, adjacent sections were stained with cresyl violet. We followed the terminology of Rodríguez-Gómez et al. (1999).

Results Distribution of MCH-ir neuronal somata and fibers in adult fish The distribution of MCH-ir neuronal somata and fibers in the adult fish is summarized in Fig. 1. MCH-ir neuronal somata were located in the NLT in the basal hypothalamus (Figs. 2A, B, 3A, 4A). Projections of MCH-ir fibers from the neuronal somata of the NLT (NLT-MCH neurons) to the pituitary were observed (Fig. 2B). MCHir neuronal somata were also present in dorsally projecting parvocellular neurons, located more posteriorly in the area above the LVR (Figs. 2A, 3B, 4B). LVR-MCH neurons did not seem to project to the pituitary.

Fig. 2 A Sagittal section through the NLT and LVR. Large arrowheads indicate MCH-ir neuronal somata in the NLT and small arrowheads indicate MCH-ir neuronal somata in the area above the LVR. B Sagittal section through the NLT and the pituitary. Large arrowheads indicate MCH-ir neuronal somata in the NLT. MCH-ir fibers (small arrowheads) were observed in the neurohypophysial tissues within the pars intermedia and, to a lesser extent, in the pars distalis. Bars 100 µm

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Fig. 3 A Schematic illustration of the distribution of MCH-ir neuronal somata in a frontal section through the NLT. B Schematic illustration of the distribution of MCH-ir neuronal somata in a frontal section through the LVR. Bar 1 mm

Fig. 4 A Frontal section through the NLT. Large arrowheads indicate MCH-ir neuronal somata in the NLT. B Frontal section through the LVR. Small arrowhead indicates MCH-ir neuronal soma in the area above the LVR. Bars 50 µm

MCH-ir neuronal somata in the NLT (about 23 µm in diameter) were larger than those in the area above the LVR (about 16 µm in diameter). In the pituitary, MCH-ir fibers were mainly distributed in the neurohypophysial tissues within the pars intermedia and, to a lesser extent, into the pars distalis (Fig. 2B). In the brain, MCH-ir fibers were detected not only in the hypothalamus but also in areas such as the optic tectum and thalamus (Fig. 1). No immunoreactive neuronal somata and fibers were observed when the antiserum was preabsorbed overnight at 4°C with an excess amount of synthetic salmonid MCH (data not shown).

(Fig. 5B). The distribution of MCH-ir neuronal somata and fibers showed a pattern similar to that in the adult fish 35–42 days after hatching (Fig. 5C–G).

Ontogenic development of the MCH system MCH-ir neuronal somata and fibers were not detected on the day of hatching (data not shown). MCH-ir neuronal somata and fibers were first detected in the hypothalamus and the pituitary, respectively, 7 days after hatching (Fig. 5A). Subsequently, they were seen in the NLT and in the area above the LVR 14 days after hatching

Discussion MCH-ir neuronal somata were detected in two nuclei of the hypothalamus, in the NLT and in the area above the LVR, in the adult barfin flounder. These results are consistent with those obtained in the rainbow trout (Baker et al. 1995), golden-eyes, butterfly fish, European eel, grass carp, European flounder and gilthead seabream (Baker and Bird 2002). Moreover, projections of MCH neurons in the NLT (NLT-MCH neurons) to the pituitary were observed, indicating that NLT-MCH neurons function as a neurohypophysial hormone in barfin flounder. This has been confirmed in all the teleost fish species examined to date (Naito et al. 1985; Batten and Baker 1988; Minth et al. 1989; Bird et al. 1989; Mancera and FernándezLlebrez 1995). MCH has been named after its function as a skin color-regulating hormone, causing pallor by a

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Fig. 5 A Sagittal section through the hypothalamus and the pituitary (day 7). MCH-ir neuronal somata were detected in the hypothalamus (large arrowhead) and MCH-ir fibers (small arrowhead) were observed in the pituitary. B Sagittal section through the hypothalamus (day 14). MCH-ir neuronal somata were detected in the NLT (large arrowhead) and in the area above the LVR (small arrowhead). C Sagittal section through the hypothalamus (day 21). MCH-ir neuronal somata were detected in the NLT (large arrowheads). D Sagittal section through the hypothalamus (day 21). MCH-ir neuronal somata were detected in the area above the LVR (small arrowheads). E Sagittal section through the hypothalamus (day 28). MCH-ir neuronal somata were detected in the NLT (large arrowheads) and in the area above the LVR (small arrowheads). F Sagittal section through the hypothalamus (day 35). MCH-ir neuronal somata were detected in the NLT (large arrowheads) and in the area above the LVR (small arrowheads). G Sagittal section through the pituitary (day 35). MCH-ir fibers were observed in the neurohypophysial tissues within the pars intermedia (large arrowhead). Bars 50 µm

direct hormonal action on the chromatophores as well as by depressing the release of α-melanophore-stimulating hormone (α-MSH) from the pars intermedia in teleost fish. In the brain of barfin flounder, MCH-ir fibers were detected not only in the hypothalamus but also in areas such as the optic tectum and thalamus. Since LVR-MCH neurons did not seem to project to the pituitary in barfin flounder or in rainbow trout (Suzuki et al. 1995), LVRMCH neurons have been suggested to send fibers into the brain and function as a neurotransmitter or neuromodulator. Whether all the NLT-MCH neurons project to the pituitary or not is not clear, and the possibility cannot

be ruled out that NLT-MCH neurons also function as a neurotransmitter or neuromodulator in the brain. Do NLT-MCH and LVR-MCH neurons have different functions in the barfin flounder? It has been shown that the functions of NLT-MCH neurons and LVR-MCH neurons differ in tilapia, Oreochromis mossambicus; changes in background color and treatment with low pH induced pronounced differences in MCH mRNA expression in the NLT, but not in the nucleus recessus lateralis (NRL), and a specific increase in MCH mRNA levels following repeated disturbance stress was observed only in the NRL (Groneveld et al. 1995b). In contrast, this is not the case in the rainbow trout. In the rainbow trout (18 months old), MCH mRNA levels were fourfold higher in white-reared fish than in black-reared fish in both the NLT- and the LVR-MCH neurons (Suzuki et al. 1995). Recently, MCH in the brain of barfin flounder has been identified by isolating MCH cDNA (Takahashi et al., in preparation), which enabled the examination of MCH mRNA levels by in situ hybridization. Thus, it will also be necessary to examine the function of NLT- and LVR-MCH neurons for barfin flounder, by comparing the changes in MCH mRNA levels in response to background color and stress. The ontogenic development of the MCH system has been examined in only three species: chum salmon (Naito et al. 1993), gilthead seabream (Mancera and Fernández-Llebrez 1995) and barfin flounder (this study). In barfin flounder, MCH-ir neuronal somata and fibers were first detected in the hypothalamus and the pituitary, respectively, 7 days after hatching. Subsequently,

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MCH-ir neuronal somata were observed in the NLT and in the area above the LVR 14 days after hatching. The distribution of MCH-ir neuronal somata and fibers showed a pattern similar to that in the adult fish 35–42 days after hatching. The timing of the appearance of MCH in the early developmental stage of barfin flounder differs from that of chum salmon and gilthead seabream. In chum salmon, the MCH system is fully developed by the time of hatching (Naito et al. 1993). The early projection of MCH-ir fibers may also indicate active secretion of MCH into the circulation to control the skin melanophores against background adaptation, because a remarkable increase in melanophores occurs in salmon alevins immediately after hatching (see Naito et al. 1993). In gilthead seabream, MCH-ir fibers first appeared in the neurohypophysis 2 days posthatching (Mancera and Fernández-Llebrez 1995). Since newly hatched gilthead seabream larvae remain floating upward at the surface of the water, darkening of the skin would be disadvantageous because of predation, as Mancera and Fernández-Llebrez (1995) have pointed out. In barfin flounder, melanophores already exist just after hatching (Aritaki et al. 2000), suggesting that MCH regulates skin pallor at this stage. It would be interesting to examine when the first regulation of skin pallor occurs in the barfin flounder. MCH has been shown to function not only as a neurohypophysial hormone but also as a neuromodulator in the brain, as mentioned above. Taking into consideration that the MCH system is developed 35–42 days after hatching, it is suggested that MCH may play some other physiological role in the early development of the barfin flounder. A knowledge of the ontogenic development of the MCH system in various fish species is considered to be helpful in understanding the functional role of MCH in the early developmental stages. In conclusion, we showed that MCH neurons were located in the NLT and in the area above the LVR and that NLT-MCH neurons project to the pituitary in barfin flounder. We also showed that MCH neurons were first detected 7 days after hatching, suggesting that MCH plays some physiological role in the early development of barfin flounder. Acknowledgements We thank Ms. Sakura Tokumo and Ms. Kiyoe Fujishiro, School of Fisheries Sciences, Kitasato University, for their help in this study. This study was supported in part by a Kitasato University Research Grant for Young Researchers.

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