Localization of CRF-like immunoreactivity in the brain and pituitary of teleost fish

Peptides, Vol. 9, pp. 13--21.©PergamonJournals Ltd., 1988. Printedin the U.S.A.

0196-9781/88$3.00 + .00

Localization of CRF-Like Immunoreactivity in the Brain and Pituitary of Teleost Fish MADELEINE

OLIVEREAU AND JACQUELINE

OLIVEREAU

Laboratoire de Physiologie, Institut Oc~anographique 195, rue Saint-Jacques, F-75005 Paris, France R e c e i v e d 6 A u g u s t 1987 OLIVEREAU, M. AND J. OLIVEREAU. Localization of CRF-like immunoreactivity in the brain and pituitary of teleost fish. PEPTIDES 9(1) 13-21, 1988.--Immunocytochemical techniques were applied to brain and pituitary sections of eleven teleost species. A corticotropin-releasing factor (CRF)-antiserum allowed the identification of a CRF-like system in these species. Perikarya were labeled in the preoptic nucleus. Labeled fibers were traced laterally, then ventrally close to the optic chiasma, forming two symmetrical tracts running through the basal hypothalamus. These ended in the rostral neurohypophysis (NH) close to ACTH cells as shown by double immunostaining. Other fibers, often more variquous, ended in the caudal NH close to melanocorticotropic cells. In Salmo fario, small perikarya also stained in the nucleus lateralis tuberis. The CRF-like system appears distinct from that of somatostatin. In Anguilla, adjacent sections stained with CRF- and vasotocin (AVT)-antisera respectively showed that these two peptides coexist in some perikarya. As few fibers containing only AVT end in the rostral NH, they probably do not control ACTH cells directly. AVT fibers terminate mostly in the caudal NH close to melanocorticotropic cells. Some extra-hypothalamic fibers suggest that CRF may also act as a neurotransmitter. The plurality of hormones showing a CRF-like activity in teleosts is considered. Corticotropin-releasing factor Teleosts

Vasotocin

Somatostatin

Immunocytochemistry

Brain

Pituitary

THE presence of a corticotropin-releasing factor (CRF)-like peptide was demonstrated by immunohistochemistry in the brain of a few teleost species [4-6, 8, 9, 34-36, 4%51]. Immunoreactive perikarya located in the nucleus preopticus (NPO) send axons which reach the pituitary stalk and end in two different areas of the neurohypophysis (NH), the rostral portion facing the corticotropic cells, and the caudal portion along the melanocorticotropic cells (MSH) of the intermediate lobe (IL). The topography of the CRF system of teleosts shows several similarities with the preopticohypophysial neurosecretory system described in the eel [3, 28, 42] after staining with the aldehyde-fuchsin or the in situ technique of Braak. This neurosecretory pathway contains arginine vasotocin (AVT) according to biochemical, ultrastructural and immunocytochemical criteria [23-25]. In the present paper, the distribution of CRF-like immunoreactivity will be described in eleven teleost species, mainly in the eel and in the elver, its small size allowing the observation of complete sets of brain serial sections. The CRF-like system will be compared with those demonstrated with anti-AVT and anti-somatostatin (SRIF) sera in Anguillidae.

the pituitary was studied), 5 Sahno irideus (1 kg BW), 10 Salmo salar collected in the Atlantic Ocean near Greenland, 30ncorhynchus tshawytscha and 5 0 . keta collected in November during spawning. Freshwater eels (20 Anguilla anguilla L., 60-110 g BW, 4 A. rostrata, 25-50 g BW, 6 A. japonica 180-200 g BW) and 7 elvers ofA. anguilla were also

METHOD

Immunocytochemical techniques described previously [35] were performed on 4- or 5/zm-thick sections. The primary antiserum against synthetic CRF 1-41 was raised in rabbits and used at a dilution of 1/1000 or 1/2000 for 18 hours at room temperature. Labeling was demonstrated with the peroxidase-antiperoxidase complex (PAP) and 3,3'-

studied; among them, three eels received an injection of 100 /zg of colchicine 36 hr before killing by rapid decapitation. In some eels, the brain was removed after anesthesia in MS 222 and rapid bleeding. Four Myoxocephalus octodecimspinosus (200--700 g BW collected in Maine through the courtesy of the Mount Desert Island Biological Laboratory and Dr. G. V. Callard) and 3 Mugil ramada collected in brackish water were also investigated. The brain with the pituitary was fixed in sublimated Bouin-Hollande solution and embedded in paraffin. Sagittal, frontal or horizontal sections were serially cut in the eel. The different pituitary cell types were identified on a few slides by staining with Herlant's tetrachrome or immunocytochemistry.

lmmunocytochemistry

Animals The following species were studied: 6 Salmo gairdneri (50-100 g body weight), 30 Salmo fario (250-700 g BW) at different stages of the sexual cycle (in half the animals, only

13

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Bulbus olfactorius Commissura anterioris Cerebellum Commissura horizontalis area dorsalis telencephali pars centralis area dorsalis telencephali pars dorsalis area dorsalis telencephali pars lateralis dorsalis area dorsalis telencephali pars lateralis ventralis area dorsalis telencephali pars medialis Habenula Mesencephalon Medulla oblongata nucleus anterior paraventricularis nucleus dorsomedialis thalami nucleus lateralis tuberis nucleus lateralis tuberis pars rostralis nervus opticus

diaminobenzidine (DAB) technique. Double immunostainings were also p e r f o r m e d [44]. The C R F - a n t i s e r u m was followed by an A C T H 1-24-antiserum or by a vasotocin- or somatostatin (SRIF)-antiserum, the second labeling being revealed with the 4-chloro-1-naphthol. In the eel, sets of two adjacent sections w e r e stained with C R F - and A V T - a n t i s e r a respectively. S o m e sections were stained with isotocin (IT) antiserum. C R F - a n t i s e r u m was twice e x h a u s t e d by solid phase i m m u n o a d s o r p t i o n on a S e p h a r o s e - 4 B - A V T c o m p l e x and then on a Sepharose-4B-IT. A V T - a n t i s e r u m was exhausted by S e p h a r o s e - 4 B - C R F c o m p l e x . Tests for specificity of the m e t h o d s and antisera were p e r f o r m e d [35,44] by omitting the primary antiserum or using an antiserum exhausted by its antigen by solid phase i m m u n o a d s o r p t i o n . In all cases, reactions w e r e negative. RESULTS

Sahnonids As the C R F - l i k e system appeared similar in trout, Atlantic and Pacific salmon, its m o r p h o l o g y will be described simultaneously in the different salmonid species investigated. Large perikarya were labeled in the preoptic area. T h e y w e r e often elongated in the magnocellular portion of the N P O with a maximal length of 40-60 p~m and a width of 2 5 - 2 8 / z m in S a h n o f a r i o (Fig. 1). In the parvocellular portion, perikarya

NPO NPOppc NPOpmc NRL NRP NVM OC P RH RI RL RO RP T TL TO SV V

nucleus praeopticus nucleus praeopticus pars parvocellularis nucleus praeopticus pars magnocellularis nucleus recessus lateralis nucleus recessus posterioris nucleus ventromedialis thalami Optic chiasma pituitary Rhombencephalon recessus infundibularis recessus lateralis recessus opticus recessus posterioris Telencephalon torus Iongitudinalis tectum opticum saccus vasculosus third ventricle

were more rounded (Fig. 2) (maximal size 20-25 × 25-30 /zm), the intensity of labeling being quite variable. In the periventricular preoptic nucleus (NPP) perikarya were few in number, smaller and elongated. Fibers arising from the parvocellular N P O extended laterally first, then ventrally towards the optic chiasma where they formed two rather large tracts of fine fibers with s o m e varicosities (Fig. 3). The two symmetrical tracts b e c a m e d e n s e r and e x t e n d e d caudally along the basal hypothalamus. Fibers issued from the N P P formed a thinner tract running ventrally close to the NPO-pituitary tract. These fibers reached the pituitary stalk from a rostro-caudal direction. Fibers issued from the magnocellular N P O did not form a thick bundle and ran more dorsally. T h e s e were most probably turning around the nucleus o f the recessus lateralis ( N R L ) and posterioris (NRP) where cross sections of fibers w e r e often o b s e r v e d in sagittal sections, mainly in the ventral area. T h e s e fibers reached the pituitary stalk from a caudo-rostral direction. Double immunostaining with C R F - and S R I F - a n t i s e r a showed that CRF-like perikarya were most often larger than those containing S R I F . Indices of a c o e x i s t e n c e o f these two peptides were not o b s e r v e d . The fine varicose CRF-like fibers reached the tuberal region and ran a m o n g the large perikarya of the nucleus lateralis tuberis ( N L T ) pars rostralis and lateralis. T h e y possibly made contacts with these large cells which were

FACING PAGE PLATE I. CRF in salmonids: Salmo filrio (Figs. 1 to 7) and Salmo salar (Figs. 8 and 9). FIG, 1. Perikarya and their axons (arrows) are labeled with CRF anti-serum (AS) in the magnocellular portion of the nucleus preopticus (NPO). ×375. FIG. 2. Parvocellular portion of the NPO. Some perikarya are stained with CRF-AS, others remain unlabeled. ×590. FIG. 3. Tract of fine CRF-immunoreactive fibers (arrows) close to the optic chiasma, x 150. FIG. 4. a and b. Small perikarya stained with CRF-AS are located in the nucleus lateralis pars ventrolateralis. Some perikarya show endings (arrows) contacting the lumen of the third ventricle (V). ×940. FIG. 5. Fine CRF-like fibers (arrows) end close to the rostral pars distalis (RPD). Other fibers enter the neurohypophysis (NH) along the recessus infundibutaris (RI) and terminate in the caudal NH among the digitations of the intermediate lobe (IL). ×60. FIG. 6. Double immunostaining. Fine CRF-like fibers end in the RPD (small arrows). Large and more strongly labeled somatostatin (SRIF) fibers (arrow heads) end in the neural digitations penetrating the proximal pars distalis (PPD). × 150. FIG. 7. Double immunostaining. ACTH cells (c) are stained with ACTH 1-24-AS. CRF-like fibers (small arrows) end close to the corticotropic layer. Some elongated cells or the material present in the follicular lumen are stained with CRF-AS (arrow heads). Follicles (F) of prolactin cells, x 150. FIG. 8. Double immunostaining. CRF-like fibers (arrows) are located in the NH close to the corticotropic cells (c) labeled with ACTH-1-24-AS. Follicles (F) of prolactin cells. × 150. FIG. 9. Double immunostaining. CRF-like fibers (arrows) at the periphery of the NH are close to the melanocorticotropic cells faintly labeled with ACTH-1-24-AS in the IL. × 150.

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FIG. 10. Line drawing of a paramedian sagittal section of the brain of an elver (Anguilla anguilla L.). Dots: CRF-like perikarya. Dashed lines and points: CRF-like fibers. Arrow: rostral direction. always unstained. In the ventrolateral portion of the N L T c o m p o s e d o f small cell bodies, a few perikarya (7-8 × 10 /zm) were stained (Figs. 4a, b). Those located close to the third ventricle sent short processes ending on the e p e n d y m a l surface. T h e s e images suggested that a CRF-like material could be released into the cerebrospinal fluid (CSF). A few small cells (4×5 tzm) were also stained in the ventral part of the N R L in the same brain. These neurons were o b s e r v e d in Sahno fario irrespective of the sex of the fish and the month it was killed. In the N L T ofSahno salar, Oncorhynchus and Anguilla, labeled perikarya were not detected. The two symmetrical tracts of fibers joined at the pituitary stalk and after entering the gland separated into smaller tracts of fine fibers. The fibers s h o w e d rather thick varicosities close to the recessus infundibularis. Fine fibers penetrated into the rostral pars distalis (RPD) and ended in close contact with the basal lamina adjacent to the corticotropic cell layer along the digitations of the rostral N H (Fig. 5). This localization is similar in the different salmonid species. N o clear changes in the a m o u n t of C R F fibers were discernible in trout killed at various stages during the annual sexual cycle. Double immunostaining showed that CRF-like fibers ran always more closely to the A C T H cells and appeared finer than SRIF-like fibers which spread mainly in the proximal pars distalis (PPD) (Fig. 6) and exceptionally in the neurointermediate lobe. Double immunostaining allowed the visualization of the proximity of CRF-like fibers and A C T H cells, for e x a m p l e in Salmo Mdeus (Fig. 7) and Salmo salar (Fig. 8). H o w e v e r , in S. irMeus killed after several w e e k s of starvation, an unusual finding was o b s e r v e d . A CRF-iike material was s o m e t i m e s present b e t w e e n two prolactin cells or in small cells at the periphery of a prolactin cell follicle or staining the c o n t e n t o f the lumen. The labeled structures s e e m e d similar to the agranular or stellate cells occurring

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FIG. I 1. Line drawings of frontal sections at different levels in the telencephalon and mesencephalon of the brain in Anguilla an~,,uilla. Dots: CRF-like perikarya. Dotted lines: CRF-like fibers. among prolactin cells and other cell types in the pituitary of teleosts. The caudal N H was highly ramified in salmonids. It contained a rather dense network of CRF-like fibers, often as fine as those occurring in the rostral N H (Figs. 5, 9). These fibers ended close to the M S H cells or around the blood vessels o f N H . In some areas, varicose fibers entered the IL and isolated fibers could be seen surrounding M S H cells.

Anguillidae The distribution of C R F was similar in E u r o p e a n , J a p a n e s e and A m e r i c a n eels. This study was facilitated by

FACING PAGE PLATE 2. CRF in Anguilla anguilla or A. rostrata or A. japonica. FIG. 12. Frontal section of the NPO containing CRF-like perikarya and fibers (arrow). Third ventricle (V). × 150. FIG. 13. Double immunostaining. Some perikarya (arrows) close to the optic recessus (long arrow) are labeled with SRIF-AS (S). Other perikarya often larger are labeled with CRF-AS (arrow heads), x 150. FIG. 14. Some CRF-like perikarya (arrows) contact the lumen of the third ventricle (V). x590. FIGS. 15 and 16. Adjacent sections stained with CRF- and AVT-AS respectively. Several perikarya contain both CRF and AVT. Two small perikarya contain AVT only (arrows). Blood vessel (bv). × 390. FIG. 17. Endings of CRF-like fibers in the rostral NH facing the RPD mainly composed of follicles (F) of prolactin cells and ACTH cells (c). CRF-like fibers surround blood vessels (+). ×375. FIG. 18. Double immunostaining of a frontal section of the RPD. CRF-like fibers occur in the rostral NH close to the ACTH cells (arrows) stained with ACTH 1-24-AS. CRF fibers do not contact prolactin cells (F). ×375. FIGS. 19 and 20. Sections of the neurointermediate lobe stained with CRF- and AVT-AS respectively. Labeled fibers end on the basal lamina of the IL. Some AVT fibers show large varicosities. × 150.

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18 the small size of elvers available forAnguilla anguilla. Small labeled perikarya were observed in the preoptic area of the elver. They were rounded or elongated and sometimes bipolar. Some fibers ran rostrally into the basal part of the telencephalon, but their endings could not be identified. Other fibers passed along the optic chiasma and through the basal hypothalamus reached the pituitary. Other fibers coming from some larger perikarya ran posteriorly under the nucleus periventricularis anterior, close to the horizontal commissure and reached the pituitary more caudally. From the pituitary stalk, fine fibers entered the RPD facing the corticotropic layer. In the caudal NH, CRF-like fibers were thicker with some varicosities. In some samples, a few labeled perikarya occurred in the dorsal part of the mesencephalic area, under the cerebellum and sent axons anteriorly into the thalamus. Their endings could not be identified. Fine fibers ran also posteriorly into the mesencephalic area and the spinal cord, mainly in the ventral area (Fig. 10). Male silver and female yellow eels showed a similar distribution of CRF. Figure 11 summarized the location of CRF-like perikarya and fibers in frontal sections of the brain of a silver eel. Perikarya were observed in the NPO (Figs. 12, 13) and in a small number in the NPP. The tracts issued from these cell bodies have a route similar to that of elvers and ended in the pituitary. A small network of fibers occurred in the anterodorsal mesencephalic area in some fish, but perikarya were not detected. In the N L T , nuclei were not labeled, but fines fibers were occasionally seen among cell bodies, mainly in the ventrolateral part of the NLT. Cells bordering the recessus infundibularis and those of the N R L and NRP were also unlabeled. In frontal sections, sections of scattered fibers were seen in the ventral telencephalic area. This tract was denser close to the anterior part of the third ventricle. Along the ventricle, numerous unipolar perikarya were labeled. Some bipolar perikarya made contact with the CSF (Fig. 14) and showed processes up to 40-50/zm length. Fibers ran perpendicularly to the ventricular wall, then lateroventrally towards the optic chiasma and continued ventrally in the basal hypothalamus. After the horizontal commissure, these two symmetric tracts became median and joined to enter the pituitary stalk. AVT perikarya were more numerous in the magnocellular than in the parvocellular portion of the NPO. They were occasionally contacting CSF. They gave rise to two tracts of fibers often intermingled with CRF fibers in the ventral hypothalamus. However AVT axons extended more dorsally than CRF fibers at the level of the optic chiasma. The comparison of Figs. 14 and 15 clearly showed that several perikarya contained both CRF and AVT. Some perikarya containing A V T only were smaller than those containing AVT and CRF (Fig. 16) or CRF only. Perikarya containing a single peptide had a more intense immunostaining than those labeled with the two antisera. Some AVT fibers possessed thick varicosities. In the rostral NH, fine C R F fibers extended laterally contacting the basal lamina along the corticotropic layer (Figs. 17, 18). Fibers did not enter the very few neural digitations of the PPD, but formed thick bundles in the caudal N H (Fig. 19). They showed large varicosities and were sometimes in close contact with the basal lamina of the IL. Occasional large Herring bodies close to the recessus infundibularis and in the caudal N H were labeled with CRF- and AVT-antisera. A small number of AVT fibers entered the rostral NH, but did not appear to contact the corticotropic layer. They showed no relation with prolactin and thyrotropic cells

OLIVEREAU AND OLIVEREAU which did not receive a direct aminergic or peptidergic innervation in the eel. The main trunk penetrated into the caudal NH. AVT fibers often showed larger varicosities mainly at the origin of the caudal N H close to the pituitary stalk (Fig. 20). AVT fibers ended on the basal lamina of the IL as MSH cells did not receive a direct innervation in the eel. Labeling with AVT was more intense at the periphery of some neural digitations than in the center in agreement with data obtained with a neurophysin-antiserum (unpublished data). IT perikarya were identified in the NPO. IT fibers were thicker than CRF fibers in the preoptic area and N H and more widely spread than CRF and AVT fibers in the brain. A coexistence of CRF and IT remained uncertain. Double immunostaining with CRF- and SRIF-antisera showed that SRIF-like perikarya were smaller, less abundant and often more spherical than CRF-like perikarya in the NPO (Fig. 13). Indication of a colocalization of S R I F and CRF was not observed. In the pituitary, SRIF fibers ended in the NH digitations along the PPD. A few scattered fibers occurred in the caudal NH. These various immunostainings were not clearly affected in eels receiving a cholchicine injection before killing.

Myoxocephalus Octodecimspinosus Few immunoreactive cell bodies were observed in the NPP; they were more numerous and larger in the NPO. The fiber tracts along the optic chiasma came down perpendicularly to the ventral border of the hypothalamus and entered the pituitary stalk located at a short distance from the optic chiasma. Rather thick immunoreactive fibers entered the NH which showed large digitations surrounding islets of ACTH cells. Fibers extended into the NH close to the IL located rostrally in this species.

Magil Ramada Immunoreactive perikarya were detected in the NPO. They gave rise to two tracts running obliquely along the optic chiasma and then in the ventral hypothalamus. CRF-like fibers occurred close to the ACTH cells mixed with some prolactin cells. In the caudal NH, CRF-like fibers showed much larger varicosities. DISCUSSION A CRF-like system extending from the preoptic area to the NH has been identified in eleven teleost species, in agreement with that previously described in few other species (see introduction). However, minor specific differences are apparent. The presence of two symmetrical tracts close to the fourth ventricle and of endings on large rhombencephalic perikarya described in the mackerel [8] has not been observed in the present study. CRF-like perikarya are restricted to the preoptic area in most species studied. However, in Acipenser ruthenus, a CRF-like immunoreactivity predominates in tuberal nuclei, being less important in the NPO [6]. Some parvocellular neurons in the caudo-ventral tuberal region and ventral telencephalon also contain CRF in Catostomus commersoni [50,51]. In Salmo fario CRF occurs in some small cells of the NLT, but it never predominates versus CRF in NPO. Some CRF-like perikarya (eel, trout) are in a subependymal location. Their occasional contacts with the ventricular lumen suggest that CRF may be released into the CSF.

CRF IN T E L E O S T B R A I N A N D P I T U I T A R Y The thickness of the main tract of CRF fibers is variable. It is easy to identify in the basal hypothalamus on sagittal sections and distinct from the S R I F tract which runs somewhat more centrally in the hypothalamus. In the pituitary of some species [35], CRF-like fibers of the rostral N H are much thinner than those of the caudal N H which possess numerous varicosities. Ultrastructural studies of the pituitary of Poecilia latipinna confirm that CRF fibers of the distal NH are larger than those of the rostral N H [4]. This statement is not evident in salmonids in which CRF-like fibers seem to be fine with small varicosities in both parts of NH. Similarly, the aldehyde-fuchsin positive material appears finely granulated in salmonids whereas it makes larger masses in other species. The hypothalamo-neurohypophysial peptidergic system has been described in the eel (see introduction) and the presence of AVT demonstrated by immunocytochemistry [25,33] as well as in Salmo gairdneri [43] and Carassius auratus [11]. The release of AVT into the CSF occurs in different teleost species ([21,43], present study) and may also involve the participation of CSF-contacting neurons [45]. AVT is commonly described as being restricted to the caudal N H [20,21]. In the eels and trouts (present study), the goldfish [8] and Poecilia latipinna [4] occasional AVT fibers are observed in the rostral NH. In the caudal N H fibers are identified as AVT only according to their varicosities [8]. However the criterion of size seems insufficient to differentiate AVT fibers from CRF-like fibers. Differences in granule size are also modest: the average diameter of C R F granules (116 nm) is slightly lower than that of AVT granules (135 nm in Poecilia [4] and 150 nm in Carassius [11]). Only small poorly defined CRF-like perikarya are observed in the parvocellular NPO ofPoecilia. AVT perikarya are more numerous and also present in the magnocellular portion [5]. CRF and AVT perikarya also occur in the NPO of Dicentrarchus labrax [30] and Salmo gairdneri [36] and are distinct. In the eel, however, staining of alternate consecutive sections of the NPO with CRF- and AVT-antisera suggests that the two peptides coexist in some perikarya [34]. Double immunostaining and scanning microdensitometric studies also demonstrate this coexistence [33]. The percentage of perikarya showing this colocalization is variable among different species of Anguilla and probably influenced by the physiological condition of the fish. Such a coexistence was reported in Catostomus: all CRF perikarya of the NPO contain AVT simultaneously whereas CRF perikarya of the N L T do not contain AVT [51]. In this respect, Catostomus seems to differ from the eel. A similar coexistence is demonstrated in intact or adrenalectomized mammals as discussed elsewhere [33]. Effects of ovine CRF on the release of ACTH by the pituitary or of corticosterone in vivo [13,38] and in vitro [2, 7, 27, 31] in the adult rat and fetus [12] are potentiated by VP. In vitro CRF stimulates the release of VP by the neurointermediate lobe [1]. In the duck, AVT potentiates the effect of CRF [10]. In the goldfish, AVT, IT and CRF stimulate ACTH release by dispersed anterior pituitary cells. CRF and AVT show additive effects but no potentiation [19]. VP, AVT and IT increase cortisol plasma levels [18]. Urotensin I (U I), a hormone isolated from the caudal neurosecretory system of teleosts [29] and localized by immunocytochemistry in most neurons of the urophysis [48] promotes A C T H release in vivo and in vitro in the rat [39] and in vitro in the goldfish [19]. CRF and U I seem equipotent, an activity also shared by sauvagine found in

19 frog skin. In addition urophysis shows a CRF-like immunoreactivity which coexists in some neurons with U I in Cyprinus carpio [47], Porichthys notatus [37] and Catostomus [51]. Anatomical data suggest that U I as well as CRF secreted in the most caudal part of the body are unable to stimulate quickly the release of pituitary ACTH. In Catostomus U I is also present in dorsal telencephalon, area pretectalis and two unidentified brain stem nuclei. In the NLT, the number of small neurons containing U I shows a 4-fold increase after urophysectomy [49]. Hypothalamic U I content increases in urophysectomized goldfish and may be involved in the simultaneous increase of plasma cortisol and pituitary ACTH content [46]. In the adenohypophysis, however, U I fibers are restricted to the PPD [51] which does not contain ACTH. This localization of U I in the PPD suggests that our CRF antiserum does not cross react with U I and that U I does not coexist with CRF in the pituitary whereas it does in the urophysis. The control of ACTH release appears more complex in fishes than in mammals as fish brain synthesizes AVT and IT showing an intrinsic ACTH-releasing activity, a CRF-like peptide and U I having a CRF-like activity. In all species of the present study, rostral CRF fibers, which do not contain AVT, arise from the NPO. This hypothalamic CRF system may control directly ACTH synthesis and release from the RPD. These data agree with the stimulation of NPO in eels [32] and goldfish [17] treated with metopyrone which blocks the l l/3-hydroxylation and induces a strong stimulation of ACTH cells. Similarly, lesions of the NPO reduce the cortisol increase in stressed goldfish although its basal secretion remains unaffected [16]. In Catostomus, the NPO does not seem to control ACTH release from the RPD, as fibers containing CRF only originate exclusively from the N L T [50]. Moreover, aH-GABA is incorporated into the N H mainly along the ACTH cells of Gasterosteus [14]. In the goldfish, whose pituitary possesses a GABAergic innervation [26], G A B A reduces ACTH release [15]. Perikarya containing both CRF and AVT are located in the NPO in Anguilla as in Catostomus. IT perikarya also occurs in the NPO of the eel. They give rise to fibers terminating in the caudal NH. CRF and AVT may then participate in the regulation of MSH cells in addition to dopamine and serotonine. Despite the lack of potentiation of CRF by AVT [19], their additive effect may increase the release of intermediate ACTH if they are released simultaneously by the same nerve endings. The coexistence of two neuropeptides is most probably not restricted to the case of CRF and AVT in teleosts. In the rat, an intragranular colocalization of CRF and Metenkephalin-8 is observed in nerve terminals of the median eminence [22]. A subpopulation of CRF perikarya contains dynorphin 1-8 [40] and CRF seems to coexist with enkephalin, PHI and neurotensin in subsets of parvocellular neurons [41]. Various neuropeptides, neurotransmitters and enzymes involved in amine biosynthesis are detected in the NPO and/or the pituitary ofteleosts. They may also interfere with the complex regulation of ACTH by CRF, AVT, IT, U I and G A B A in teleost fishes. ACKNOWLEDGEMENTS We gratefully acknowledge Pr. F. Vandesande from the Catholic University of Leuven, Belgium, who kindly donated most antisera used in this work, and Mrs. Lieve Moons (Leuven) for the adsorption of various antisera. This research was supported in part by research grants from CNRS and INSERM (U 223).

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OLIVEREAU

AND OLIVEREAU

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