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FMRFamide-like immunoreactivity in the brain and pituitary of the goldfish, Carassius auratus Ken Fujii and Hideshi Kobayashi Research Laboratory, Zenyaku Kogyo Co. Ltd., 2-33-7 Ohizumi-machi, Nerima-ku, Tokyo 178, Japan

Summary.

The distribution of FMRFamide-like immunoreactivity was determined in the brain and the pituitary of the goldfish, Carassius auratus. Immunoreactive perikarya were observed in the olfactory nerve, nucleus entopeduncularis, nucleus anterioris periventricularis, nucleus posterioris periventricularis, lateral part of the nucleus lateralis tuberis pars posterioris, midbrain tegmentum, and medulla oblongata. Immunoreactive fibers were widely distributed in the brain, in particular in the ventral telencephalon and the hypothalamus. A few immunoreactive nerve fibers were observed in the pituitary. The findings are discussed in relation to male sexual behavior and the involvement of FMRFamide-like peptide in pituitary functions in the goldfish.

Key words: FMRFamide -

Immunohistochemistry -

Goldfish brain

Introduction The presence of FMRFamide (Phe-Met-Arg-Phe-NH 2 , a molluscan cardioexcitatory tetrapeptide) has been demonstrated immunohistochemically in the nervous system, gastrointestinal tract, and pancreas in vertebrate species (Bore et al. 1980; Dockray et al. 1981), in particular in serveral mammalian species (see review by Raffa 1988). The biological actions of FMRFamide and related peptides in mammals have also been discussed in some detail (Raffa 1988). In teleosts, perikarya with FMRFamide-like immunoreactivity have been found in the telencephalon and the hypothalamus, and immunoreactive fibers have been observed in the brain of the black molly (Bore et al. 1980). The immunoreactivity has also been observed in the terminal nerve (Stell et al. 1984) and the retina (Stell et al. 1984; Muske et al. 1987) of the goldfish, and in the retina and the pineal organ of the stickleback and the salmon (Ekstrom et al. 1988). Further-

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more, colocalization of FMRFamide- and gonadotropinreleasing hormone-like immunoreactivities was observed in the terminal nerve, and the efficacy of these peptides with respect to the retinal ganglion cells has been demonstrated in the goldfish (Stell et al. 1984). Thus, most such investigations have been concerned with the retinal pathway. The present paper describes the distribution of FMRFamide-like immunoreactivity in the goldfish brain, with emphasis on the possible involvement of FMRFamide-like peptide in pituitary functions. During the preparation of this report, a report by others describing FMRFamide immunoreactivity in the brain and pituitary of the goldfish was published (Bonn and Konig 1989). Our results confirm the recently published data while supplementing them with additional information.

Ann. Anat. (1992) 174: 217-222 Gustav Fischer Verlag Jena

Materials and methods Goldfish, Carassius auratus, (both sexes, ranging from 5 to 11 cm in body length) were purchased from a commercial source. Each fish was decapitated after anesthesia with ethyl 3-arninobenzoate, methanesulfonate (Aldrich Chemical Company, Inc., Milwaukee, Wisconsin). The whole head was immediately immersed in 4 % paraformaldehyde in phosphate buffer (0.1 M, pH 7.4) for about 1 hr at room temperature. Subsequently, the brain with pituitary was dissected out and kept in the same fixative for about 3 hrs at room temperature. After rinsing in 0.01 M phosphate-buffered saline (PBS; pH 7.2) which contained 20 % sucrose for 1 to 4 days at 4 DC, the brain was cut with a cryostat at 15 flm and the sections were transferred onto gelatin-coated slides. The sections were immunostained by the peroxidase-antiperoxidase (PAP) method of Sternberger et al. (1970). Rehydrated sections were soaked in 0.3 % H20 2 in PBS for 30 min and rinsed for 15 min in PBS that contained 0.3 % Triton X-I 00. They were preincubated in 1 % normal goat serum (Polysciences Inc., Warrington, Pennsylvania) prepared in PBS that contained 0.3% Triton X-IOO. The sections were then incubated with FMRFarnide-specific antiserum raised in rabbit (1: 4000; Immuno Nuclear Corp., Stillwater, Minnesota) overnight

at 4°C. Then the sections were washed in PBS that contained 0.3 % Triton X-lOO for 15 min and incubated with antiserum raised in goat against rabbit IgG (1 : 200; Polysciences) for 60 min. After the sections were washed in PBS that contained Triton X-1OO for 15 min, they were incubated with PAP complex (1 : 100; Dako Corp., Copenhagen, Denmark). Each incubation was performed in a moist chamber at room temperature. Finally, the sections were stained with 0.02 % 3,3-diaminobenzidine tetrahydroch1oride (DAB; Wako Pure Chemicals, Osaka, Japan) in 0.05 M Tris-HCI buffer (pH 7.6) that contained 0.005 % HzOz for about 5 to 10 min, and then slides were washed in PBS and distilled water for 5 min each. The sections were dehydrated through an ethanol series, cleared in xylene, and mounted with Eukitt (0. Kinder, Germany). The specificity of the immunoreaction was checked by a preabsorption test in which the antiserum was incubated for 24 hr with synthetic FMRFamide (5 [.lg/ml: Peptide Institution, Osaka, Japan). To identify the nucleus in the goldfish brain, we referred to the anatomical atlas of Peter and Gill (1975).

Results All of the positive immunoreactions mentioned below were completely eliminated after preabsorption of the FMRFamide-specific antiserum with the synthetic FMRFamide. Therefore, the immunoreactions were specific within the parameters used.

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Perikarya showing FMRFamide-like immunoreactivity were found in the olfactory nerve, telencephalon, diencephalon, mesencephalon and medulla oblongata. Immunoreactive fibers were widely distributed in the brain and a few were seen in the pituitary of the goldfish. The distribution of immunoreactive perikarya and their fibers is summarized schematically in Figures 1 and 2. FMRFamide-like immunoreactivity in the goldfish brain showed almost the same pattern of distribution regardless of the size of the fish. In the olfactory nerve (OLN) , the immunoreactive perikarya projected fibers caudally through the olfactory bulb and the olfactory tract (OLT) to the telencephalon (Figs. 1, 3). A few immunoreactive fibers were also observed to run rostrally, but their terminals were not detected. Thick immunoreactive fiber bundles originating in the OLN branched off into roughly two directions in the middle region of the ventral telencephalon and proceeded dorsally and ventrally (Figs. 1, 4). In the telencephalon, immunoreactive perikarya were rarely found in the caudomedial part of the OLT (COLT; Figs. 1, 2a, 5) and they sent fibers posteriorly (Fig. 5). Weakly immunostained perikarya were found in the nucleus entopeduncularis (NE; Figs. 1, 2c, 6). Many immunoreactive fibers were distributed in the ventromedial telencephalon (VT) (Figs. 1, 2b, 4). Most of these fibers seemed to

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Fig. 1. Schematic representation of perikarya (filled circles) and fibers (dotted lines) showing FMRFamide-like immunoreactivity projected on the median sagittal plane of the goldfish brain and pituitary. Arrows (a - j) at the bottom of the Figure indicate the transverse planes of the sections depicted in Fig. 2a-2j. Scale bar, 1 mm. Abbreviations: AC, anterior commissure; C, cerebellum; COLT, caudomedial part of the olfactory tract; DT, dorsal telencephalon; HOC, horizontal commissure; LR, lateral recess; MO, medulla oblongata; MT, midbrain tegmentum; NAPv, nucleus anterioris periventricularis; NDLI, nucleus diffusus lobi inferioris; NE, nucleus entopeduncularis; NH, nucleus habenularis; NI, neurointermediate lobe; NLTp, nucleus lateralis tuberis pars posterioris; NPPv, nucleus posterioris periventricularis; OB, olfactory bulb; OLN, olfactory nerve; OLT, olfactory tract; OPN, optic nerve; OTec, optic tectum; POA, preoptic area; PPD, proximal pars distalis; RPD, rostral pars distalis; PI-PIll, fiber pathways; VT, ventral telencephalon.

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CD Fig. 2. Schematic representation of the distribution of immunoreactive perikarya and fibers in the transverse sections cut at the levels indicated by a-j in Fig. 1. The left side of each sections shows immunorective perikarya (filled circles) and fibers (dots) and the right side shows areas of the brain. Scale bar, 1 mm. Abbreviations : NAT, nucleus anterior tuberis; NDM, nucleus dorsomedialis thalami; NDTL, nucleus diffusus tori lateralis; NLTa, nucleus lateral is tuberis pars anterioris ; NPO, nucleus preopticus ; NPP, nucleus preopticus periventricularis ; NPT , nucleus posterior tuberis; NRL, nucleus recessus lateralis; NVM, nucleus ventromedialis thalami; OT, optic tract ; P, pituitary; PC, posterior commissure; SV , saccus vasculosus . Other abbreviations are explained in the legend to Fig. 1.

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originate in the OLN. Few immunoreactive fibers were found in the dorsal telencephalon (DT ; Figs. 1, 2 d). In the diencephalon, immunoreactive perikarya were localized in the nucleus anterioris periventricularis (NAPv; Figs. 1, 2d, 2e, 7), nucleus posterioris periventricularis (NPPv ; Figs . 1, 2f, 7, 8a, 8b, 8c) and lateral part of nucleus lateralis tuberis pars posterioris (NLTp ; Figs. 1, 2f, 2g, 9). Immunoreaction was strong in the perikarya of the NPPv , but weak in those of the NAPv and of the lateral part of the NLTp. There were three major pathways of immunoreactive fibers . One pathway, including mainly fibers that originated in the OLN, ran from the ventral telencephalon to the NLTp through the lateral preoptic area, as described by Koyama et al. (1984) , and antero-Iateral hypothalamus (PI in Figs. 1, 2e) . Another pathway, followed by a dense array of immunoreactive fibers, proceeded from the ventral telencephalon to the NLTp through the lateral preoptic area and postero-lateral hypothalamus . These fibers included the fibers that originated in the NAPv and NPPv (PH in Figs. 1, 2f, 2g, 2h) . This pathway branched off at the lateral preoptic area and continued caudally to the medulla oblongata (MO; PIll in Fig. 1, 2i, 2j). In addition to these three pathways, immunoreactive fibers proceeded from the NPPv to the NLTp through the periventricular region of the hypothalamus (Fig. 2f, 8a) . Some perikarya of the NPPv sent their processes towards the ventricle (Fig. 8c). Thus, the NLTp area contained dense arrays of imunoreactive fibers. Eight to ten large perikarya were localized in a group in the dorsal part of both sides of the midbrain tegmentum (MT; Figs. 1, 2i, 10). A few , small immunoreactive

perikarya were scattered in the dorsal part of the midbrain, just behind the large perikarya (Figs. 1, 2j). Immunoreactive perikarya were also found bilaterally in the MO (Fig. 1). Immunoreactive fibers were also observed in the optic nerve (Fig . 1), optic tectum (Figs. 1, 2 e - 2 j), nucleus habenula (Figs . 1, 2e), saccus vasculosus (Figs. 2h, 11) and spinal cord (Fig. 1). The origins of these immunoreactive fibers were unclear. A few immunoreactive fibers were observed to enter the pituitary. Most of these fibers were observed together with aldehyde fuchsin-positive neurosecretory fibers that proceeded to the neurohypophysis (Figs. 1, 2f, 12). They were widely distributed in the rostral pars distalis, proximal pars distalis and neurointermediate lobe, but the nature of the target cells of these immunoreactive fibers could not be determined.

Discussion FMRFamide-like immunoreactivity in the brain and pituitary of the goldfish was reported by Bonn and Konig (1989) during the preparation of our present paper. They described the presence of immunoreactive perikarya in the ON, NE, NPO, NVM , NPPv , NRL, NRP and rhombencephalon, and reported that immunoreactive fibers were widely distributed throughout the brain. We detected immunoreactive perikarya in the ON, NE, NAPv , NPPv , NLTp, MT, but not in the NPO, NVM and NRL in this study. The discrepancy is probably due to differences in the antisera used and physiological conditions of the fish .

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Fig. 3. Sagittal section through the olfactory nerve (OLN) and the olfactory bulb (OB). Immunoreactive perikarya are visible in the OLN. Arrows, immunoreactive fibers. Scale bar, lOOflm. Fig. 4. Sagittal section through the ventral telencephalon (VT). Thick immunoreactive fibers that originate in the olfactory nerve proceed through the olfactory tract (OLT) and branch off into approximately dorsal (arrow) and ventral (arrowheads) directions in the VT. Scale bar, 200flm. Fig. 5. Sagittal section through the ventral telencephalon. A few immunoreactive perikarya are visible in the caudomedial part of the olfactory tract (COLT). Their fibers proceed posteriorly. Scale bar, 100 flill. Fig. 6. (a) Transverse section through the telencephalon at the level of the anterior commissure (AC). Numerous immunoreactive fibers can be observed in the lateral preoptic area (LPA). The rectangular area includes the nucleus entopeduncularis (NE). (b) Immunoreactive perikarya (arrowheads) in the NE. Scale bar, (a) 500 flm, (b) 50 flm. Fig. 7. Sagittal section of the hypothalamus through the optic nerve and near the third ventricle. Immunoreactive perikarya showing FMRFamide-like immunoreactivity in the nucleus anterioris periventricularis (NAPv) and the nucleus posterioris periventricularis (NPPv) are shown. LR, lateral recess; OPN, optic nerve. Scale bar, 200 flm.

It is known that antiserum raised against FMRFamide shares characteristics with antiserum raised against neuropeptide Y (NPY) (Shen and Cheng 1989). Therefore, we compared the distribution of FMRFamide with that of NPY, which was reported by Kah et al. (1989), in the goldfish brain. According to Kah et al. (1989), perikarya with NPY-like immunoreactivity are localized in the NDM, NVM and OTec and immunoreactive fibers are distributed in the lateral part of the telencephalon. However, FMRFamidelike immunoreactivities were not found in these regions in our study. In addition, there is a difference in the overall pattern of distribution of immunoreactive fibers between those that contain FMRFamide- and those that contain NPYimmunoreactive material. However, since perikarya showing FMRFamide-like and NPY-like immunoreactivity were localized in the NE and NPPv, antiserum against FMRFamide might have cross-reacted with NPY in certain regions of the brain. To resolve this problem, it will be

necessary to examine whether or not the same cells react with antisera raised against FMRFamide and against NPY. Futhermore, highly specific antisera will be required for such experiments. The terminal nerve (TN) has perikarya in the olfactory nerve and bulb, and these perikarya send fibers to the telencephalon through the olfactory tract and to the optic nerve. The TN is the pathway for pheromonal triggering of sexual arousal and the TN fibers also play a role in the chemosensory modulation of the release of sperm in the goldfish (Demski and Northcutt 1983). The TN appears to be involved in the sexual behavior of the male, since such behavior is disrupted by sectioning of the olfactory tract (Partidge et al. 1976; Stacey and Kyle 1983). However, Koyama et al. (1984) reported that bilateral ablation of the olfactory bulb, destroying the ganglion cells of the TN, had no effect on male sexual behavior. It is still too early to conclude, therefore, that the TN is involved in male sexual

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Fig. 8. Transverse section of the hypothalamus at the level of the nucleus posterioris periventricularis (NPPv), nucleus anterior tuberis (NAT), and nucleus lateralis tuberis pars posterioris (NLTp). (a) Immunoreactive perikarya in the NPPv project immunoreactive fibers to the NLTp through the postero-Iateral hypothalamus (single arrows) and periventricular region of the hypothalamus (double arrows), (b) Immunoreactive perikarya in the NPPv. (c) Immunoreactive perikaryon in the NPPv sending a process (arrow) toward the ventricle (V). Scale bar, (a) 500 J.lm, (b) 50 J.lm, (c) 50 J.lm. Fig. 9. Transverse section of the hypothalamus at the level of the nucleus lateralis tuberis pars posterioris (NLTp). Perikarya showing weak FMRFamide-like immunoreactivity (arrowheads) can be found among the dense arrays of immunoreactive fibers. Scale bar, 100 J.lm.

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Fig. 10. Transverse section through the dorsal part of the midbrain tegmentum (MT) showing immunoreactive perikarya (arrowheads). Arrows, immunoreactive fibers. Scale bar, 100 J.lm. Fig. 11. Transverse section through the saccus vasculosus (SV). Arrows, immunoreactive material, NRP, nucleus recessus posterioris. Scale, 100 J.lm. Fig. 12. Sagittal section through the pituitary showing immunoreactive fibers (arrows) entering in the pars distalis and pars neurointermedia (NI). PPD, proximal pars distalis; RPD, rostral pars distalis. Scale bar, 200 J.lm.

behavior. Although we detected FMRFamide-like peptide in the TN of the golfish, as reported by Stellet al. (1984), elucidation of the physiological role of the peptide in the TN awaits further studies. We observed dense arrays of fibers that contained FMRFamide-like immunoreactivity in the ventral telencephalon and the lateral preoptic area (LPA) of the goldfish. Koyama et al. (1984) have shown by an electrolytic technique that the area ventralis telencephali pars supracommissuralis and/or the posterior area ventralis telencephali pars

ventralis (Vs-pVv) and NPP play important roles in the sexual behavior of male goldfish, while the olfactory bulb and anterior part of the telencephalic hemispheres and the LPA have no role in sexual behavior. Therefore, it seems that FMRFamide-like peptide in the fibers detected in the region ofthe ventral telencephalon except areas of the Vs-pVv and the LPA may not be involved in male sexual behavior. Further studies are needed to clarify the relationship between sexual behavior and FMRFamide-like peptide. It has been demonstrated in some species of fishes that

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several hypothalamic hormones and many neuropeptides are carried down by fibers to the pituitary through the neurohypophysis (see Moons et al. 1989). We found FMRFamidelike peptide in the hypothalamo-pituitary system of the goldfish. In the hypothalmus, perikarya showing FMRFarnidelike immunoreactivity were localized characteristically in the NAP, NPPv and the lateral part of the NLTp, and moderate numbers of immunoreactive fibers, which may originate from these immunoreactive nuclei, were found to invade the pituitary. Immunoreactive fibers from the telencephalon and from cells of the NAPv and NPPv were found to run to the NLTp. In addition, dense arrays of fibers, which may innervate the cells of the NLTp, were observed in the NLTp. Since it has been suggested that the NLTp is involved in control of pituitary functions in teleosts (Ball 1981), fibers with FMRFamide-like immunoreactivity in the NLTp and in the pituitary may take part in the control of pituitary functions. As mentioned above, the functions of FMRFamide-like peptide in fishes have not been well established. Further physiological and ultrastructural studies on fish with brain lesions will be necessary to elucidate the physiological actions of FMRFamide-like peptide in fishes. Acknowledgements. We wish to thank Mr. K. Hashimoto, Administrative Director of the Department of Research and Development of Zenyaku Kogyo, Co, Ltd. and Dr. M. Takase, Vice-Director of the Research Laboratory of the Company for giving permission for this work to be performed.

References Ball IN (1981) Hypothalamic control of the pars distalis in fishes, amphibians, and reptiles. Gen Comp Endocrinol 44: 135-170 Boer HH, Schot LPC, Veenstra JA, Reichelt D (1980) Immunocytochemical identification of neural elements in the central nervous systems of a snail, some insects, a fish, and a mammal with an antiserum to the molluscan cardio-excitatory tetrapeptide FMRFamide. Cell Tissue Res 213: 21-27 Bonn U, Konig B (1989) FMRFarnide immunoreactivity in the brain and pituitary of Carassius auratus (Cyprinidae, teleostei). J Himforsch 30: 361 - 370 Demski LS, Northcutt RG (1983) The terminal nerve: a new chemosensory system in vertebrates? Science 220: 435 -437

Dockray GJ, Vaillant V, Williams RG (1981) New vertebrate brain-gut peptide related to a molluscan neuropeptide and an opioid peptide. Nature 293: 656-657 Ekstrom, P, Honkanen T, Ebbesson SOE (1988) FMRFarnide-like immunoreactive neurons of the nervus terminalis of teleosts innervate both retina and pineal organ. Brain Res 460: 68-75 Kah 0, Pontet A, Danger JM, Dobourg P, Pelletier G, Vaudry H, Calas A (1989) Characterization, cerebral distribution and gonadotropin release activity of neuropeptide Y (NPY) in the goldfish. Fish Physiology and Biochemistry. Amsterdam, Vol. 7 nos 1-4, pp 69-76 Koyama Y, Satou M, Oka Y, Ueda K (1984) Involvement of the telencephalic hemispheres and the preoptic area in sexual behaviour of the male goldfish, Carassius auratus: a brainlesion study. Behav Neural Bioi 40: 70-86 Moons L, Cambre M, Ollevier F, Vandesande F (1989) Immunocytochemical demonstration of close relationships between neuropeptidergic nerve fibres and hormone-producing cell types in the adenohypophysis of the sea bass (Dicentrarchus labrax). Gen Comp Endocrinol 73: 270-283 Muske LE, Dockray GJ, Chohan KS, Stell WK (1987) Segregation of FMRFarnide-immunoreactive efferent fibers from NPYimmunoreactive amacrine cells in goldfish retina. Cell Tissue Res 247: 299-307 Partridge BL, Liley NR, Stacey NE (1976) The role of pheromones in the sexual behaviour of the goldfish. Anim Behav 24: 291-299 Peter RE, Gill VE (1975) A stereotaxic atlas and technique for forebrain nuclei of the goldfish, Carassius auratus. J Comp Neuro1159: 69-102 Raffa RB (1989) The action of FMRFamide (Phe-Met-Arg-PheNH2) and related peptides on mammals. Peptides 9: 915-922 Shen CL, Cheng JT (1989) Cross-reaction of FMRFamide with a neuropeptide Y(NYP) antiserum: histological and radioimmunochemical evidence. Biomed Res 10: 517-520 Stacey NE, Kyle AL (1983) Effects of olfactory tract lesions on sexual and feeding behavior in the goldfish.Physiol Behav 30: 621-628 Stell WK, Walker SE, Chohan KS, Ball AK (1984) The goldfish nervus terminalis: a luteinizing hormone-releasing hormone and molluscan cardioexcitatory peptide immunoreactive olfactoretinal pathway. Proc Natl Acad Sci USA 81: 940-944 Sternberger LA, Hardy PH jr., Cuculis n, Meyer HG (1970) The unlabeled antibody enzyme method for immunohistochemistry: preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-antihorseradish peroxidase) and its use in identification of spirochetes. J Histochem Cytochem 18: 315-333. Accepted June 28, 1990

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FMRFamide-like immunoreactivity in the brain and pituitary of the goldfish, Carassius auratus.

The distribution of FMRFamide-like immunoreactivity was determined in the brain and the pituitary of the goldfish, Carassius auratus. Immunoreactive p...
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