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Localization of atrial natriuretic factor (ANF)-related peptides in the central nervous system of the elasmobranch fish Scyliorhinus canicula
Peptides, Vol. 11, pp. 1175-1181. ©Pergamon Press plc, 1990. Printed in the U.S.A.
0196-9781/90 $3.00 + .00
Localization of Atrial Natriuretic Factor (ANF)-Related Peptides in the Central Nervous System of the Elasmobranch Fish Scyliorhinus canicula M . V A L L A R I N O , * M . F E U I L L O L E Y , t J. G U T K O W S K A , : I : M CANTIN:~ A N D H. V A U D R Y t
*Institute of Comparative Anatomy, University of Genova, Viale Benedetto XV, 5, 16132 Genova, Italy ?Groupe de Recherche en Endocrinologie Mol#culaire, CNRS URA 650, Unit# Affili#e ~t I'INSERM Facult# des Sciences, Universit# de Rouen, 76134 Mont-Saint-Aignan, France 4:Clinical Research Institute of Montreal, Montr#al, Qu#bec, Canada H2W1R7 R e c e i v e d 6 M a r c h 1990
VALLARINO, M., M. FEUILLOLEY, J. GUTKOWSKA, M. CANTIN AND H. VAUDRY. Localization of atrial natriuretic factor (ANF)-relatedpeptides in the central nervous system of the elasmobranchfish Scyliorhinus canicula. PEPTIDES 11(6) 11751181, 1990.--We have investigated the localization of atrial natriuretic factor (ANF)-like immunoreactivity in the central nervous system of the cartilaginous fish, Scyliorhinus canicula, using the indirect immunofluorescence technique. Immunoreactive perikarya and fibers were observed in two regions of the telencephalon, the area superficialis basalis and the area periventricularis ventrolateralis. In the diencephalon, the hypothalamus exhibited a moderate number of ANF-containing neurons and fibers located in the preoptic and periventricular nuclei and in the nucleus lateralis tuberis. The most important group of ANF-immunoreactive cells was observed in the nucleus tuberculi posterioris of the diencephalon. In contrast, the mesencephalon showed only a few ANF-positive nerve processes located in the tegmentum mesencephali. Numerous fine fibers and nerve terminals were found in the dorsal area of the neurointermediate lobe of the pituitary. These results provide the first evidence for the presence of ANF-related peptides in the brain of a cartilaginous fish. The widespread distribution of ANF-positive cells and fibers in the brain and pituitary suggests that this peptide may act both as a neurotransmitter and (or) a neurohormone in fish. Atrial natriuretic factor
Brain
Pituitary
Immunocytochemistry
Neuropeptides
Dogfish
Scyliorhinus canicula
THE atrial natriuretic factor (ANF) family consists of a series of regulatory peptides, exhibiting strong natriuretic and diuretic activity, which were initially characterized in atrial extracts (19). In the rat, the major circulating form of ANF is a 28 amino acid peptide corresponding to the sequence Ser99-Tyr126 of the ANF prohormone (25). The occurrence of ANF-related peptides has been demonstrated in various organs including the urogenital tract (16) and the salivary gland (1). Neuropeptides of the ANF family are also synthesized in the central nervous system (9,20) and related structures, including the eye (22), the autonomic ganglia (2) and the pituitary (5,8). In nonmarnmalian vertebrates immunohistochemical and biochemical data indicate that both atrium and ventricle cardiocytes contain ANF-like peptides (14). The sequence of ANF has recently been determined in the frog (11) and in the eel (24). These studies show that the molecules expressed in amphibian and fish heart exhibit a high degree of homology with mammalian ANF. Antibodies against rat ANF have been successfully employed to
localize ANF-containing neurons and fibers in the brain and pituitary of amphibians (12,13). The distribution of ANF-like peptides has also been investigated in the central nervous system of the hagfish Myxine glutinosa (18). Although the existence of ANF-related peptides has been reported in the heart of both teleost and elasmobranch fish (3,7), the occurrence of ANF-containing neurons has not been investigated in the brain of cartilaginous fish. In the present study, we have studied the distribution of ANF-like peptides in the brain of the dogfish Scyliorhinus canicula, using the indirect immunofluorescence technique. METHOD
Animals Twelve adult small spotted dogfish Scyliorhinus canicula (1.4 to 2.4 kg body weight) were captured in June by travel net in the Ligurian sea (Western Mediterranean sea). The animals were kept
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for one week before use in an aquarium, at constant temperature (13 ±0.5°C) and under natural illumination.
Immunofluorescence Procedure The fish were anesthetized by immersion in tricaine methanesulfonate 100 mg/1 (MS 222, Sandoz) and perfused via the ventral aorta with 0.05 M phosphate buffer saline (PBS, pH 7.4), followed immediately by paraformaldehyde solution (4% in PBS) or Bouin's fixative. The brains, with the attached pituitary, were quickly dissected and postfixed overnight in the same fixative solution. The tissues treated by the Bouin's fixative were dehydrated by immersion in increasing concentrations of ethanol. The samples were embedded in paraffin and cut in 5-p,m thick sections. The brains fixed with paraformaldehyde were rinsed for 24 hr in PBS containing 40% sucrose and frozen on dry ice. Frontal or parasagittal sections (20 Ixm) were cut in a cryostat. The slices were processed according to the indirect immunofluorescence technique, using a rabbit antiserum raised against the synthetic ANF fragment (Arglm-Tyr 126) (5). Sections were rinsed in 0.05 M PBS and preincubated for 20 min in normal goat serum (Dakopatts, DK) at a dilution 1:50. The incubation buffer consisted of 0.05 M PBS containing 0.3% Triton X-100 and 1% human serum albumin. Thereafter, incubation was performed at 4°C for 18 hr with the ANF antiserum diluted 1:400 in the incubation buffer. The sections were rinsed in PBS and incubated for 1 hr at room temperature in fluorescein isothiocyanateconjugated swine anti-rabbit ~/-globulins (Dakopatts, DK) diluted 1:100 in PBS. Finally, the sections were rinsed three times in PBS and mounted in a glycerol-PBS (1:1) solution. All preparations were examined on a Zeiss epifluorescence microscope. Some sections were counterstained with cresyl violet to localize the immunoreactive cells. Sections used as controls were incubated either with nonimmunized rabbit serum or with antiserum preadsorbed with synthetic (Argm~-Tyr 126) ANF (10 6 M). Nomenclature of brain areas was based on the work of Smeets et al. (21). RESULTS
The regional distribution of ANF-like immunoreactive cells and fibers in the brain and pituitary of Scyliorhinus canicula is presented in Fig. 1. ANF-containing neurons were mainly located in the telencephalon and diencephalon. Immunoreactive fibers and nerve terminals were also observed in the mesencephalon and the neurointermediate lobe of the pituitary. In the telencephalon, moderate to high number of ANFcontaining cell bodies and fibers were noted in the ventral and caudal regions of the subpallium. The anterior part of the area superficialis basalis contained numerous large and oval immunoreactive perikarya; more caudally, the ANF-containing perikarya
G
H
ABeD EF
G H
FIG. 1. Schematic coronal sections through the brain of Scyliorhinus canicula depicting the distribution of ANF-like immunoreactive perikarya (A) and fibers (-). The position of the sections (noted from A to H) is indicated on the sagittal scheme of the brain on the bottom of the figure. A: nucleus A; Ap: area periventricularis ventrolateralis; Asb: area superficialis basalis; En: nucleus entopeduncularis; Et: eminenthia thalami; Fb: fasciculus basalis telencephali; Hab: ganglion habenulae; Hy: hypophysis; Ic: nucleus interstitialis commissurae anterioris; LI: nucleus lobi lateralis; Lt: nucleus lateralis tuberis; M: nucleus M; Mh: nucleus medius hypothalami; P: nucleus periventricularis hypothalami; Pd: pallium dorsale; Pdcl: pallium dorsale, pars centralis lateralis; Pds: pallium dorsale, pars superficialis: PI: pallium laterale; Pm: pallium mediale; Po: nucleus preopticus; Sc: nucleus septi caudoventralis; Sin: nucleus septi medialis, pars posterioris; Td: thalamus dorsalis; Tect: tectum mesencephali; Tv: thalamus ventralis; Tp: nucleus tuberculi posterioris.
FACING PAGE FIG. 2. Immunofluorescence photomicrograph showing ANF-like positive perikarya and fibers in the area superficialis basalis of the telencephalon. Sagittal section ( x 360). FIG. 3. Coronal section of the caudo-lateral part of the area superficialis basalis demonstrating the presence of ANF-positive cells and beaded fibers ( x 360). FIG. 4. Typical large ANF-containing neuron of the rostral area superficialis basalis. Coronal section ( x 320). FIG. 5. Micrograph showing ANF-like immunoreactive cells and fibers in the area pefiventficularis ventrolateralis of the subpallium. Sagittal section ( × 320). FIG. 6. Coronal section through the antero-lateral part of the nucleus preopticus illustrating a group of positive perikarya (V: telencephalic ventricle) ( X 320). FIG. 7. View of ANF-positive neurons in the nucleus pefiventficulafis hypothalami (I: infundibulum). Coronal section ( x 180).
A N F IN THE BRAIN OF THE DOGFISH
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FIGS. 2-7
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I,
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FIGS. 8-13
appeared somewhat smaller (Figs. 2, 3, 4). The area periventricularis ventrolateralis of the subpallium exhibited scattered ANFpositive cell bodies, mainly located in its lateral part (Fig. 5). Dense bundles of nerve fibers coursing through both regions and
extending more caudally in the basal forebrain bundle were also observed. Frequently, fine ANF-containing beaded fibers were found surrounding nonimmunoreactive cell bodies (Figs. 3, 5). In the medial subpallium ANF-positive nerve processes were noted in
ANF IN THE BRAIN OF THE DOGFISH
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FACING PAGE FIG. 8. Sagittal section showing ANF-containing neurons and fibers in the nucleus lateralis tuberis (I: infundibulum) ( x 380). FIG. 9. Parasagittal section through the nucleus tuberculi posterioris showing a group of ANF-like immunoreactive cells ( x 420). FIG. 10. Typical ANF-positive cell observed in the caudal part of the posterior recess. Coronal section ( x 380). FIG. 11. Long beaded positive fibers crossing the anterior hypothalamus (I: infundibulum). Sagittal section (x 185). FIG. 12. Coronal section demonstrating the accumulation of fine beaded fibers in the caudal region of the hypothalamus ( x 380). FIG. 13. Close-up view of ANF-like immunoreactive fibers in the dorsal side of the neurointermediate lobe of the pituitary. Sagittal section (x 380).
the pars posterioris of the nucleus septi medialis. More caudally, scattered fibers were located in the nucleus interstitialis commissurae anterioris. The different areas of the pallium appeared to be totally devoid of ANF-like immunoreactive cells or fibers. In the diencephalon, the rostral hypothalamus exhibited a moderate number of positive perikarya restricted to the dorso-lateral part of the anterior preoptic nucleus. Most of these neurons were round or oval (Fig. 6). Occasionally, cerebrospinal fluidcontacting cells exhibiting ANF-like immunoreactivity were noted in the subependymal layers of the preoptic recess. In the postchiasmatic region of the hypothalamus, two groups of ANF-like immunoreactive cells were identified in the nucleus periventricularis hypothalami and in the nucleus lateralis tuberis. These cells, which surrounded the infundibular recess, were respectively found in the dorso-lateral periventricular area and in the ventrocaudal region of the hypothalamus (Figs. 7, 8). The caudal diencephalon contained a group of immunoreactive cells in the dorso-caudal infundibular region within the posterior tuberculum. These cells, located in the nucleus tuberculi posterioris, represent the most prominent group of stained neurons found in the brain of S. canicula. They were intensely fluorescent, round or oval in shape (Fig. 9). A few stained perikarya were also visualized more caudally within the posterior recess (Fig. 10). The preoptic area, the retrochiasmatic region and the caudal hypothalamus were densely loaded with ANF-positive beaded fibers (Figs. 11, 12). Nerve processes were also noted in the ventral thalamus and in the posterior tuberculum. The mesencephalon did not contain any ANF-immunoreactive perikarya. A moderate number of fibers was visualized in the tegmentum mesencephali. All other structures of the brain were totally devoid of ANF-like immunoreactive cells or fibers, In the pituitary., numerous ANF-positive fibers were observed in the neurointermediate lobe. These nerve processes were mainly distributed in the dorsal and caudal parts of the intermediate lobe. Many fibers and nerve terminals were observed in close vicinity of pituitary cells (Fig. 13). Preadsorption of the antiserum with synthetic ANF (10 - 6 M) resulted in complete loss of the immunoreaction. Similarly, no staining was observed when the ANF antiserum was replaced by normal rabbit serum or PBS. No significant differences in the distribution and density of ANF-containing neurons and fibres were noted between the tissues treated with paraformaldehyde or Bouin's fixative. DISCUSSION
The present results provide evidence for the occurrence of ANF-like immunoreactive peptides in the central nervous system of the cartilaginous fish Seyliorhinus canicula. The antiserum used in this study has previously been employed to localize ANF in mammalian cardiocytes (1) and in the heart and brain of the frog (12-14). The presence of ANF-like immunoreactive cells and
fibers in the central nervous system of S. canicula is consistent with data previously obtained in the rat (5, 8, 9, 20), frog (12,13) and the hagfish Myxine glutinosa (Cyclostomata) (18). In the rat brain, the highest concentration of ANF-containing elements is located in the hypothalamus, at the level of the periventricular preoptic nucleus, paraventricular nucleus and arcuate nucleus (4, 9, 20). Similarly, in amphibians, ANF-like immunoreactive cells are found in the preoptic and infundibular nuclei (13). In mammals, the neurons located in the anterior hypothalamus are involved in cardiovascular regulation (6) and in the control of electrolyte and water homeostasis (4). The occurrence of ANF-positive cells in the anterior hypothalamus of S. canicula suggests that, in cartilaginous fish, the rostral hypothalamic area may participate in the regulation of vascular functions and saline balance. The presence of dense populations of ANF-containing neurons in the preoptic and periventricular areas of the hypothalamus is of particular interest. In the preoptic complex, immunocytochemical techniques have shown the occurrence of a variety of regulatory neuropeptides including neuropeptide Y (NPY) (28), bombesin (29), corticotropin-releasing hormone (CRF) (30), 13-endorphin (33) and vasotocin (32). Neurons located in this area send a dense plexus of neurosecretory processes through the basal hypothalamus towards the hypophysial system (34), suggesting that ANF, like several other regulatory peptides originating from the preoptic nucleus, may be involved in the control of pituitary functions. The hypothalamus of S. canicula contains a rich network of ANF-immunoreactive fibers mainly concentrated in the nucleus lateralis tuberis. We have previously reported that this nucleus also contains other regulatory peptides such as adrenocorticotropin (ACTH) (33), melanin-concentrating hormone (MCH) (27) and NPY (28). ANF-positive fibers, emerging from the nucleus lateralis tuberis, were found coursing along the hypothalamus and invaded the neurointermediate lobe of the pituitary. In both the nucleus lateralis tuberis and pituitary, ANF-immunoreactive fibers made intimate contacts with cell bodies. In agreement with these findings, ANF has been detected in the neurointermediate lobe of frog pituitary (12), while in the rat, ANF-immunoreactive material is found in both anterior (8) and posterior pituitary (5). The distribution of ANF-containing fibers in the neurointermediate lobe of the dogfish suggests that the peptide may control the secretion of pituitary hormones. In support of this hypothesis, a high density of ANF binding sites has been observed in the pituitary of amphibians (26) and mammals (17). In addition, in the frog, ANF has been shown to stimulate o~-melanotropin release from neurointermediate lobes in vitro (10). Thus our data support the concept that, in fish, as in other vertebrates, ANF may act as a hypophysiotropic neurohormone. In the caudal diencephalon, we found an important group of ANF-positive cells located in the nucleus tuberculi posterioris. This nucleus is interconnected with other diencephalic structures
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(21) and projects towards the periventricularis ventrolateralis nucleus of the telencephalon (15). ANF is also widely distributed in the telencephalon and the mesencephalon, as previously described in the rat (20) and the frog (13). Thus the presence of ANF in such regions, which are not involved in cardiovascular regulation, suggests that the neuropeptide ANF may also exert neuromodulator or neurotransmitter functions. This hypothesis is supported by recent observations showing a high density of ANF binding sites in the central nervous system of mammals (17) and amphibians (26). Studies performed in the spiny dogfish Squalus acanthias have shown that the area superficialis basilis, the nucleus periventricularis ventrolateralis and the nucleus tuberculi posterioris contain a large number of tyrosine hydroxylase (TH) immunoreactive cells and fibers (15). In S. canicula, these three regions are also densely loaded with ANF-like immunoreactive structures. Moreover, we have previously reported the presence of MCH (27), ~x-melanocyte-stimulating-hormone (o~-MSH) (33) and sauvagine/urotensin I (31) in the neurons of the nucleus tuberculi posterioris. Therefore, possible colocalization of ANF-like material with other neuropeptides or classical neurotransmitters deserves further investigations. ANF immunoreactivity in the mesencephalon of S. canicula was restricted to sparse fibers located in the tegmentum. In contrast, rat (20) and frog (13) mesencephalon contain a high
number of ANF-positive cells and fibers. The scarcity of ANFcontaining structures in the mesencephalon of S. canicula reveals the existence of species differences in the regional distribution of ANF in the brain of vertebrates. In contrast, in all groups studied, other areas such as the hypothalamus are always densely loaded with ANF-related peptides (9, 15, 20). Recently, a new peptide, termed brain natriuretic peptide (BNP), has been isolated from porcine brain (23). This neuropeptide exhibits similar pharmacological properties as ANF (23). Since BNP possesses a high degree of sequence homology with ANF, the antiserum used in the present study might have revealed a BNP-related peptide rather than authentic ANF. The function of ANF-immunoreactive peptides in the brain remains unknown. The distribution of ANF-like neurons and fibers reported herein suggests that ANF-like substances may act as neuroregulators in a variety of functions. In particular, the presence of immunoreactivity in the hypothalamus and within the neurointermediate lobe of the hypophysis supports the view that in fish, as in other vertebrates (10), ANF-like peptides may play a role in the control of pituitary hormone secretion. ACKNOWLEDGEMENTS This research was supported by grants from Italian M.P.I. (40%,60%), CNRS (URA 650), INSERM-CNR exchange program and by the Conseil R6gional de Haute-Normandie.
REFERENCES I. Cantin, M.; Gutkowska, J.; Thibault, G.; Milne, R. W.; Ledoux, S.; Min Li, S.; Chapeau, C.; Garcia, R.; Hamet, P.: Genest, J. lmmunocytochemical localization of atrial natriuretic factor in the heart and salivary glands. Histochemistry 80:113-127; 1984. 2. Debinski, W.; Gutkowska, J.; Kuchel, O.; Racz, K.; Buu, N. T.; Cantin, M.; Genest, J. ANF-like peptide(s) in the peripheral autonomic nervous system. Biochem. Biophys. Res. Commum 134: 279-284; 1986. 3. Duff, D. W.; Olson, K. R. Trout vascular and renal responses to atrial natriuretic factor and heart extracts. Am. J. Physiol. 251:R639-R642; 1986. 4. Geiger, H.; Bahner, U.; Palkovits, M.; Hugo, C.; Niklas, E.; Heidland, A. Atrial natriuretic peptide in brain preoptic areas: Implication for fluid and salt homeostasis. J. Cardiovasc. Pharmacol. 13:$20-$23; 1989. 5. Gutkowska, J.; Racz, K.; Debinski, W.; Thibault, G.; Garcia, R.: Kuchel, O.; Cantim M.; Genest, J. An atrial natriuretic factor-like activity in rat posterior hypophysis. Peptides 8:461-465; 1987. 6. Hartle, D. K.; Brody, M. J. The angiotensin II pressor system of the rat forebrain Circ. Res. 54:355-366; 1984. 7. Hirohama, T.; Uemura, H.; Nakamura, S.; Aoto, T. Atrial natriuretic peptide (ANF)-immunoreactivity of cardiocytes in fish. Zool Sci. 5:833-845; 1988. 8. Inagaki, S.; Kubota, Y.; Kito, S.; Kangawa, K.; Matsuo, H. Atrial natriuretic polypeptide-like immunoreactivity in the rat pituitary: Light and electron microscopic studies. Regul. Pept. 14:101-111; 1986. 9. Kawata, M.; Nakao, K.; Morii, N.; Kiso, Y.; Yamashita, H.; Imura, H.; Sano, Y. Atrial natriuretic polypeptide: Topographical distribution in the rat brain by radioimmunoassay and immunohistochemistry. Neuroscience 16:521-546; 1985. 10. Lamacz, M.; Netchit~/flo, P.; Tonon, M. C.; Feuilloley, M.; Ling, N.; Pelletier, G.; Vaudry, H. Atrial natriuretic factor (ANF) stimulates the release of ~-MSH from frog neurointermediate lobes in vitro. Interaction with dopamine, GABA, and neuropeptide Y. Life Sci. 40: 1853-1857; 1987. 11. Lazure, C.; Ong, H.; Mac Nicoll, N.; Netchitailo, P.; Chr~tien, M.; De I_dan, A.; Vaudry, H. The aminoacid sequences of frog heart atrial natriuretic-like peptide and mammalian ANF are closely related. FEBS Lett. 238:300-306; 1988. 12. Netchita'flo, P.; Feuilloley, M.; Pelletier, G.; Cantin, M.; Leboulenger, F.; Andersen, A.; Vaudry, H. Localization of atrial natriuretic
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factor (ANF)-immunoreactive material in the hypothalamo-pituitary complex of the frog. Neurosci. Lett. 72:141-146; 1986. Netchit~/flo, P.; Feuilloley, M.; Pelletier, G.; Leboulenger, F.; Cantin, M.; Gutkowska, J,; Vaudry, H. Atrial natriuretic factor-like immunoreactivity in the central nervous system of the frog. Neuroscience 22:341-359; 1987. Netchita'flo, P.; Feuilloley, M.; Pelletier, G.; De Ldan, A.; Ong, H.; Cantin, M.; Gutkowska, J. ; Leboulenger, F. ; Vaudry, H. Localization and identification of immunoreactive atrial natriuretic factor (ANF) in the frog ventricle. Peptides 9:1-6; 1988. Northcutt, R. G.; Reiner, A.; Karter, H. J. Immunocytochemical study of the telencephalon of the spiny dogfish Squalus acanthias. J. Comp. Neurol. 277:250-267; 1988. Papka, R. E.; Traurig, H. H.; Wekstein, M. Localization of peptides in nerve terminals in the paracervical ganglion of the rat by light and electron microscopic immunocytochemistry: Enkephalin and atrial natriuretic factor. Neurosci. Lett. 61:285-290; 1985. Quirion, R. Receptor sites for atrial natriuretic factors in brain and associated structures: An overview. Cell. Mol. Neurobiol. 9:45-55; 1989. Reinecke, M.; Betzler, D.; Forssmann, W. G. Immunocytochemistry of cardiac polypeptide hormones (cardiodilatin/atrial natriuretic polypeptide) in brain and heart of Mvxine glutinosa (Cyclostomata). Histochemistry 86:233-239; 1987. Seidah, N. G.; Lazure, C.; Chr6tien, M.; Thibault, G.; Garcia, R.; Cantin, M.; Genest, J.; Nutt, R. F.; Brady, S. F.; Lyle, T. A.: Paleveda, W. J.; Colton, C. D.; Ciccarone, T. M.; Veber, D. F. Amino-acid sequence of homologous rat atrial peptides: Natriuretic activity of native and synthetic forms. Proc. Natl. Acad. Sci. USA 81:2640-2644; 1984. Skofitsch, G.; Jacobowitz, D. M. Atrial natriuretic peptide in the central nervous system of the rat. Cell. Mol. Neurobiol. 8:339-391; 1988. Smeets, W. J. A. J.; Nieuwenhuys, R.; Roberts, B. L. The central nervous system of cartilaginous fish. Berlin: Springer-Verlag; 1983. Stone, R. A.; Glembotsky, C. C. Immunoreactive atrial natriuretic peptide in the eye: Molecular forms in anterior uvea and retina. Biochem. Biophys. Res. Commun. 134:1022-1028; 1986. Sudoh, T.; Kangawa. K.; Minamino, N.; Matsuo, H. A new natriuretic peptide in porcine brain. Nature 332:78-81; 1988. Takei, Y.; Takahashi, A.; Watanabe, T. X.; Nakajima, K.; Sakakibara, S. Amino acid sequence and relative biological activity of eel
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atrial natriuretic peptide. Biochem. Biophys. Res. Commun. 164: 537-543; 1989. Thibault, G.; Lazure, C.; Schiffrin, E. L.; Gutkowska, J.; Chartier, L.; Garcia, R.; Seidah, N. G.; Chr6tien, M.; Genest, J.; Cantin, M. Identification of a biologically active circulating form of rat natriuretic factor. Biochem. Biophys. Res. Commun. 130:981-986; 1985. Tong, Y.; Netchitai'lo, P.; Leboulenger, F.; Vaudry, H.; Pelletier, G. Localization of atrial natriuretic factor (ANF) binding sites in the central nervous system of the frog. J. Comp. Neurol. 281:384-396; 1989. Vallarino, M.; Andersen, A. C.; Delbende, C.; Ottonello, I.; Eberle, A. N.; Vaudry, H. Melanin-concentrating-hormone (MCH) immunoreactivity in the brain and pituitary of the dogfish Scyliorhinus canicula. Colocalization with alpha-melanocyte-stimulating-hormone (a-MSH) in hypothalamic neurons. Peptides 10:375-382; 1989. Vallarino, M.; Danger, J. M.; Fasolo, A.; Pelletier, G.; Saint-Pierre, S.; Vaudry, H. Distribution and characterization of neuropeptide Y in the brain of an elasmobranch fish. Brain Res. 448:67-76; 1988. Vallarino, M.; D'Este, L.; Negri, L.; Ottonello, I.; Renda, T. Occurrence of bombesin-like immunoreactivity in the brain of the cartilaginous fish, Scyliorhinus canicula. Cell Tissue Res. 259:177181; 1990.
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30. Vallarino, M.; Fasolo, A.; Ottonello, I.; Perroteau, I.; Tonon, M. C.; Vandesande, F.; Vaudry, H. Localization of corticotropinreleasing hormone (CRF)-like immunoreactivity in the central nervous system of the elasmobranch fish, Scyliorhinus canicula. Cell Tissue Res. 258:541-546; 1989. 31. Vallarino, M.; Ottonello, I.; D'Este, L.; Renda, T. Sauvagine/urotensin I-like immunoreactivity in the brain of the dogfish, Scyliorhinus canicula. Neurosci. Lett. 95:119-124; 1988. 32. Vallarino, M.; Ottonello, I.; Panzica, G. C.; Viglietti-Panzica, C. Immunocytochernical localization of vasotocin in the hypothalamus of a cartilaginous fish. Proc. Xlth Int. Symp. Comp. Endocrinol., in press; 1990. 33. Vallarino, M.; Tranchand-Bunel, D.; Delbende, C.; Ottonello, I.; Vaudry, H. Distribution of the pro-opiomelanocortin-derived peptides, alpha-melanocyte-stimulating hormone (et-MSH), adrenocorticotropic hormone (ACTH), and beta-endorphin in the brain of the dogfish Scyliorhinus canicula: An immunocytochemical study. J. Exp. Zool. Suppl. 2:112-121; 1989. 34. Van de Kamer, J. C.; Zandberger, M. A. The hypothalamichypophyseal system and its evolutionary aspects in Scyliorhinus canicula. Cell Tissue Res. 214:575-582; 1981.
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Localization of Atrial Natriuretic Factor (ANF)-Related Peptides in the Central Nervous System of the Elasmobranch Fish Scyliorhinus canicula M . V A L L A R I N O , * M . F E U I L L O L E Y , t J. G U T K O W S K A , : I : M CANTIN:~ A N D H. V A U D R Y t
*Institute of Comparative Anatomy, University of Genova, Viale Benedetto XV, 5, 16132 Genova, Italy ?Groupe de Recherche en Endocrinologie Mol#culaire, CNRS URA 650, Unit# Affili#e ~t I'INSERM Facult# des Sciences, Universit# de Rouen, 76134 Mont-Saint-Aignan, France 4:Clinical Research Institute of Montreal, Montr#al, Qu#bec, Canada H2W1R7 R e c e i v e d 6 M a r c h 1990
VALLARINO, M., M. FEUILLOLEY, J. GUTKOWSKA, M. CANTIN AND H. VAUDRY. Localization of atrial natriuretic factor (ANF)-relatedpeptides in the central nervous system of the elasmobranchfish Scyliorhinus canicula. PEPTIDES 11(6) 11751181, 1990.--We have investigated the localization of atrial natriuretic factor (ANF)-like immunoreactivity in the central nervous system of the cartilaginous fish, Scyliorhinus canicula, using the indirect immunofluorescence technique. Immunoreactive perikarya and fibers were observed in two regions of the telencephalon, the area superficialis basalis and the area periventricularis ventrolateralis. In the diencephalon, the hypothalamus exhibited a moderate number of ANF-containing neurons and fibers located in the preoptic and periventricular nuclei and in the nucleus lateralis tuberis. The most important group of ANF-immunoreactive cells was observed in the nucleus tuberculi posterioris of the diencephalon. In contrast, the mesencephalon showed only a few ANF-positive nerve processes located in the tegmentum mesencephali. Numerous fine fibers and nerve terminals were found in the dorsal area of the neurointermediate lobe of the pituitary. These results provide the first evidence for the presence of ANF-related peptides in the brain of a cartilaginous fish. The widespread distribution of ANF-positive cells and fibers in the brain and pituitary suggests that this peptide may act both as a neurotransmitter and (or) a neurohormone in fish. Atrial natriuretic factor
Brain
Pituitary
Immunocytochemistry
Neuropeptides
Dogfish
Scyliorhinus canicula
THE atrial natriuretic factor (ANF) family consists of a series of regulatory peptides, exhibiting strong natriuretic and diuretic activity, which were initially characterized in atrial extracts (19). In the rat, the major circulating form of ANF is a 28 amino acid peptide corresponding to the sequence Ser99-Tyr126 of the ANF prohormone (25). The occurrence of ANF-related peptides has been demonstrated in various organs including the urogenital tract (16) and the salivary gland (1). Neuropeptides of the ANF family are also synthesized in the central nervous system (9,20) and related structures, including the eye (22), the autonomic ganglia (2) and the pituitary (5,8). In nonmarnmalian vertebrates immunohistochemical and biochemical data indicate that both atrium and ventricle cardiocytes contain ANF-like peptides (14). The sequence of ANF has recently been determined in the frog (11) and in the eel (24). These studies show that the molecules expressed in amphibian and fish heart exhibit a high degree of homology with mammalian ANF. Antibodies against rat ANF have been successfully employed to
localize ANF-containing neurons and fibers in the brain and pituitary of amphibians (12,13). The distribution of ANF-like peptides has also been investigated in the central nervous system of the hagfish Myxine glutinosa (18). Although the existence of ANF-related peptides has been reported in the heart of both teleost and elasmobranch fish (3,7), the occurrence of ANF-containing neurons has not been investigated in the brain of cartilaginous fish. In the present study, we have studied the distribution of ANF-like peptides in the brain of the dogfish Scyliorhinus canicula, using the indirect immunofluorescence technique. METHOD
Animals Twelve adult small spotted dogfish Scyliorhinus canicula (1.4 to 2.4 kg body weight) were captured in June by travel net in the Ligurian sea (Western Mediterranean sea). The animals were kept
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for one week before use in an aquarium, at constant temperature (13 ±0.5°C) and under natural illumination.
Immunofluorescence Procedure The fish were anesthetized by immersion in tricaine methanesulfonate 100 mg/1 (MS 222, Sandoz) and perfused via the ventral aorta with 0.05 M phosphate buffer saline (PBS, pH 7.4), followed immediately by paraformaldehyde solution (4% in PBS) or Bouin's fixative. The brains, with the attached pituitary, were quickly dissected and postfixed overnight in the same fixative solution. The tissues treated by the Bouin's fixative were dehydrated by immersion in increasing concentrations of ethanol. The samples were embedded in paraffin and cut in 5-p,m thick sections. The brains fixed with paraformaldehyde were rinsed for 24 hr in PBS containing 40% sucrose and frozen on dry ice. Frontal or parasagittal sections (20 Ixm) were cut in a cryostat. The slices were processed according to the indirect immunofluorescence technique, using a rabbit antiserum raised against the synthetic ANF fragment (Arglm-Tyr 126) (5). Sections were rinsed in 0.05 M PBS and preincubated for 20 min in normal goat serum (Dakopatts, DK) at a dilution 1:50. The incubation buffer consisted of 0.05 M PBS containing 0.3% Triton X-100 and 1% human serum albumin. Thereafter, incubation was performed at 4°C for 18 hr with the ANF antiserum diluted 1:400 in the incubation buffer. The sections were rinsed in PBS and incubated for 1 hr at room temperature in fluorescein isothiocyanateconjugated swine anti-rabbit ~/-globulins (Dakopatts, DK) diluted 1:100 in PBS. Finally, the sections were rinsed three times in PBS and mounted in a glycerol-PBS (1:1) solution. All preparations were examined on a Zeiss epifluorescence microscope. Some sections were counterstained with cresyl violet to localize the immunoreactive cells. Sections used as controls were incubated either with nonimmunized rabbit serum or with antiserum preadsorbed with synthetic (Argm~-Tyr 126) ANF (10 6 M). Nomenclature of brain areas was based on the work of Smeets et al. (21). RESULTS
The regional distribution of ANF-like immunoreactive cells and fibers in the brain and pituitary of Scyliorhinus canicula is presented in Fig. 1. ANF-containing neurons were mainly located in the telencephalon and diencephalon. Immunoreactive fibers and nerve terminals were also observed in the mesencephalon and the neurointermediate lobe of the pituitary. In the telencephalon, moderate to high number of ANFcontaining cell bodies and fibers were noted in the ventral and caudal regions of the subpallium. The anterior part of the area superficialis basalis contained numerous large and oval immunoreactive perikarya; more caudally, the ANF-containing perikarya
G
H
ABeD EF
G H
FIG. 1. Schematic coronal sections through the brain of Scyliorhinus canicula depicting the distribution of ANF-like immunoreactive perikarya (A) and fibers (-). The position of the sections (noted from A to H) is indicated on the sagittal scheme of the brain on the bottom of the figure. A: nucleus A; Ap: area periventricularis ventrolateralis; Asb: area superficialis basalis; En: nucleus entopeduncularis; Et: eminenthia thalami; Fb: fasciculus basalis telencephali; Hab: ganglion habenulae; Hy: hypophysis; Ic: nucleus interstitialis commissurae anterioris; LI: nucleus lobi lateralis; Lt: nucleus lateralis tuberis; M: nucleus M; Mh: nucleus medius hypothalami; P: nucleus periventricularis hypothalami; Pd: pallium dorsale; Pdcl: pallium dorsale, pars centralis lateralis; Pds: pallium dorsale, pars superficialis: PI: pallium laterale; Pm: pallium mediale; Po: nucleus preopticus; Sc: nucleus septi caudoventralis; Sin: nucleus septi medialis, pars posterioris; Td: thalamus dorsalis; Tect: tectum mesencephali; Tv: thalamus ventralis; Tp: nucleus tuberculi posterioris.
FACING PAGE FIG. 2. Immunofluorescence photomicrograph showing ANF-like positive perikarya and fibers in the area superficialis basalis of the telencephalon. Sagittal section ( x 360). FIG. 3. Coronal section of the caudo-lateral part of the area superficialis basalis demonstrating the presence of ANF-positive cells and beaded fibers ( x 360). FIG. 4. Typical large ANF-containing neuron of the rostral area superficialis basalis. Coronal section ( x 320). FIG. 5. Micrograph showing ANF-like immunoreactive cells and fibers in the area pefiventficularis ventrolateralis of the subpallium. Sagittal section ( × 320). FIG. 6. Coronal section through the antero-lateral part of the nucleus preopticus illustrating a group of positive perikarya (V: telencephalic ventricle) ( X 320). FIG. 7. View of ANF-positive neurons in the nucleus pefiventficulafis hypothalami (I: infundibulum). Coronal section ( x 180).
A N F IN THE BRAIN OF THE DOGFISH
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FIGS. 2-7
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appeared somewhat smaller (Figs. 2, 3, 4). The area periventricularis ventrolateralis of the subpallium exhibited scattered ANFpositive cell bodies, mainly located in its lateral part (Fig. 5). Dense bundles of nerve fibers coursing through both regions and
extending more caudally in the basal forebrain bundle were also observed. Frequently, fine ANF-containing beaded fibers were found surrounding nonimmunoreactive cell bodies (Figs. 3, 5). In the medial subpallium ANF-positive nerve processes were noted in
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FACING PAGE FIG. 8. Sagittal section showing ANF-containing neurons and fibers in the nucleus lateralis tuberis (I: infundibulum) ( x 380). FIG. 9. Parasagittal section through the nucleus tuberculi posterioris showing a group of ANF-like immunoreactive cells ( x 420). FIG. 10. Typical ANF-positive cell observed in the caudal part of the posterior recess. Coronal section ( x 380). FIG. 11. Long beaded positive fibers crossing the anterior hypothalamus (I: infundibulum). Sagittal section (x 185). FIG. 12. Coronal section demonstrating the accumulation of fine beaded fibers in the caudal region of the hypothalamus ( x 380). FIG. 13. Close-up view of ANF-like immunoreactive fibers in the dorsal side of the neurointermediate lobe of the pituitary. Sagittal section (x 380).
the pars posterioris of the nucleus septi medialis. More caudally, scattered fibers were located in the nucleus interstitialis commissurae anterioris. The different areas of the pallium appeared to be totally devoid of ANF-like immunoreactive cells or fibers. In the diencephalon, the rostral hypothalamus exhibited a moderate number of positive perikarya restricted to the dorso-lateral part of the anterior preoptic nucleus. Most of these neurons were round or oval (Fig. 6). Occasionally, cerebrospinal fluidcontacting cells exhibiting ANF-like immunoreactivity were noted in the subependymal layers of the preoptic recess. In the postchiasmatic region of the hypothalamus, two groups of ANF-like immunoreactive cells were identified in the nucleus periventricularis hypothalami and in the nucleus lateralis tuberis. These cells, which surrounded the infundibular recess, were respectively found in the dorso-lateral periventricular area and in the ventrocaudal region of the hypothalamus (Figs. 7, 8). The caudal diencephalon contained a group of immunoreactive cells in the dorso-caudal infundibular region within the posterior tuberculum. These cells, located in the nucleus tuberculi posterioris, represent the most prominent group of stained neurons found in the brain of S. canicula. They were intensely fluorescent, round or oval in shape (Fig. 9). A few stained perikarya were also visualized more caudally within the posterior recess (Fig. 10). The preoptic area, the retrochiasmatic region and the caudal hypothalamus were densely loaded with ANF-positive beaded fibers (Figs. 11, 12). Nerve processes were also noted in the ventral thalamus and in the posterior tuberculum. The mesencephalon did not contain any ANF-immunoreactive perikarya. A moderate number of fibers was visualized in the tegmentum mesencephali. All other structures of the brain were totally devoid of ANF-like immunoreactive cells or fibers, In the pituitary., numerous ANF-positive fibers were observed in the neurointermediate lobe. These nerve processes were mainly distributed in the dorsal and caudal parts of the intermediate lobe. Many fibers and nerve terminals were observed in close vicinity of pituitary cells (Fig. 13). Preadsorption of the antiserum with synthetic ANF (10 - 6 M) resulted in complete loss of the immunoreaction. Similarly, no staining was observed when the ANF antiserum was replaced by normal rabbit serum or PBS. No significant differences in the distribution and density of ANF-containing neurons and fibres were noted between the tissues treated with paraformaldehyde or Bouin's fixative. DISCUSSION
The present results provide evidence for the occurrence of ANF-like immunoreactive peptides in the central nervous system of the cartilaginous fish Seyliorhinus canicula. The antiserum used in this study has previously been employed to localize ANF in mammalian cardiocytes (1) and in the heart and brain of the frog (12-14). The presence of ANF-like immunoreactive cells and
fibers in the central nervous system of S. canicula is consistent with data previously obtained in the rat (5, 8, 9, 20), frog (12,13) and the hagfish Myxine glutinosa (Cyclostomata) (18). In the rat brain, the highest concentration of ANF-containing elements is located in the hypothalamus, at the level of the periventricular preoptic nucleus, paraventricular nucleus and arcuate nucleus (4, 9, 20). Similarly, in amphibians, ANF-like immunoreactive cells are found in the preoptic and infundibular nuclei (13). In mammals, the neurons located in the anterior hypothalamus are involved in cardiovascular regulation (6) and in the control of electrolyte and water homeostasis (4). The occurrence of ANF-positive cells in the anterior hypothalamus of S. canicula suggests that, in cartilaginous fish, the rostral hypothalamic area may participate in the regulation of vascular functions and saline balance. The presence of dense populations of ANF-containing neurons in the preoptic and periventricular areas of the hypothalamus is of particular interest. In the preoptic complex, immunocytochemical techniques have shown the occurrence of a variety of regulatory neuropeptides including neuropeptide Y (NPY) (28), bombesin (29), corticotropin-releasing hormone (CRF) (30), 13-endorphin (33) and vasotocin (32). Neurons located in this area send a dense plexus of neurosecretory processes through the basal hypothalamus towards the hypophysial system (34), suggesting that ANF, like several other regulatory peptides originating from the preoptic nucleus, may be involved in the control of pituitary functions. The hypothalamus of S. canicula contains a rich network of ANF-immunoreactive fibers mainly concentrated in the nucleus lateralis tuberis. We have previously reported that this nucleus also contains other regulatory peptides such as adrenocorticotropin (ACTH) (33), melanin-concentrating hormone (MCH) (27) and NPY (28). ANF-positive fibers, emerging from the nucleus lateralis tuberis, were found coursing along the hypothalamus and invaded the neurointermediate lobe of the pituitary. In both the nucleus lateralis tuberis and pituitary, ANF-immunoreactive fibers made intimate contacts with cell bodies. In agreement with these findings, ANF has been detected in the neurointermediate lobe of frog pituitary (12), while in the rat, ANF-immunoreactive material is found in both anterior (8) and posterior pituitary (5). The distribution of ANF-containing fibers in the neurointermediate lobe of the dogfish suggests that the peptide may control the secretion of pituitary hormones. In support of this hypothesis, a high density of ANF binding sites has been observed in the pituitary of amphibians (26) and mammals (17). In addition, in the frog, ANF has been shown to stimulate o~-melanotropin release from neurointermediate lobes in vitro (10). Thus our data support the concept that, in fish, as in other vertebrates, ANF may act as a hypophysiotropic neurohormone. In the caudal diencephalon, we found an important group of ANF-positive cells located in the nucleus tuberculi posterioris. This nucleus is interconnected with other diencephalic structures
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(21) and projects towards the periventricularis ventrolateralis nucleus of the telencephalon (15). ANF is also widely distributed in the telencephalon and the mesencephalon, as previously described in the rat (20) and the frog (13). Thus the presence of ANF in such regions, which are not involved in cardiovascular regulation, suggests that the neuropeptide ANF may also exert neuromodulator or neurotransmitter functions. This hypothesis is supported by recent observations showing a high density of ANF binding sites in the central nervous system of mammals (17) and amphibians (26). Studies performed in the spiny dogfish Squalus acanthias have shown that the area superficialis basilis, the nucleus periventricularis ventrolateralis and the nucleus tuberculi posterioris contain a large number of tyrosine hydroxylase (TH) immunoreactive cells and fibers (15). In S. canicula, these three regions are also densely loaded with ANF-like immunoreactive structures. Moreover, we have previously reported the presence of MCH (27), ~x-melanocyte-stimulating-hormone (o~-MSH) (33) and sauvagine/urotensin I (31) in the neurons of the nucleus tuberculi posterioris. Therefore, possible colocalization of ANF-like material with other neuropeptides or classical neurotransmitters deserves further investigations. ANF immunoreactivity in the mesencephalon of S. canicula was restricted to sparse fibers located in the tegmentum. In contrast, rat (20) and frog (13) mesencephalon contain a high
number of ANF-positive cells and fibers. The scarcity of ANFcontaining structures in the mesencephalon of S. canicula reveals the existence of species differences in the regional distribution of ANF in the brain of vertebrates. In contrast, in all groups studied, other areas such as the hypothalamus are always densely loaded with ANF-related peptides (9, 15, 20). Recently, a new peptide, termed brain natriuretic peptide (BNP), has been isolated from porcine brain (23). This neuropeptide exhibits similar pharmacological properties as ANF (23). Since BNP possesses a high degree of sequence homology with ANF, the antiserum used in the present study might have revealed a BNP-related peptide rather than authentic ANF. The function of ANF-immunoreactive peptides in the brain remains unknown. The distribution of ANF-like neurons and fibers reported herein suggests that ANF-like substances may act as neuroregulators in a variety of functions. In particular, the presence of immunoreactivity in the hypothalamus and within the neurointermediate lobe of the hypophysis supports the view that in fish, as in other vertebrates (10), ANF-like peptides may play a role in the control of pituitary hormone secretion. ACKNOWLEDGEMENTS This research was supported by grants from Italian M.P.I. (40%,60%), CNRS (URA 650), INSERM-CNR exchange program and by the Conseil R6gional de Haute-Normandie.
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30. Vallarino, M.; Fasolo, A.; Ottonello, I.; Perroteau, I.; Tonon, M. C.; Vandesande, F.; Vaudry, H. Localization of corticotropinreleasing hormone (CRF)-like immunoreactivity in the central nervous system of the elasmobranch fish, Scyliorhinus canicula. Cell Tissue Res. 258:541-546; 1989. 31. Vallarino, M.; Ottonello, I.; D'Este, L.; Renda, T. Sauvagine/urotensin I-like immunoreactivity in the brain of the dogfish, Scyliorhinus canicula. Neurosci. Lett. 95:119-124; 1988. 32. Vallarino, M.; Ottonello, I.; Panzica, G. C.; Viglietti-Panzica, C. Immunocytochernical localization of vasotocin in the hypothalamus of a cartilaginous fish. Proc. Xlth Int. Symp. Comp. Endocrinol., in press; 1990. 33. Vallarino, M.; Tranchand-Bunel, D.; Delbende, C.; Ottonello, I.; Vaudry, H. Distribution of the pro-opiomelanocortin-derived peptides, alpha-melanocyte-stimulating hormone (et-MSH), adrenocorticotropic hormone (ACTH), and beta-endorphin in the brain of the dogfish Scyliorhinus canicula: An immunocytochemical study. J. Exp. Zool. Suppl. 2:112-121; 1989. 34. Van de Kamer, J. C.; Zandberger, M. A. The hypothalamichypophyseal system and its evolutionary aspects in Scyliorhinus canicula. Cell Tissue Res. 214:575-582; 1981.