Distribution of FMRFamide-like immunoreactivity in the forebrain of the catfish, Clarias batrachus (Linn.)

Peptides,Vol. 13, pp. 183-191, 1992

0196-9781/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd.

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Distribution of FMRFamide-Like Immunoreactivity in the Forebrain of the Catfish, Clarias batrachus (Linn.) N. S. R A M A K R I S H N A A N D N I S H I K A N T K. S U B H E D A R 1 Department o f Pharmaceutical Sciences, Nagpur University, Nagpur-440 010, India R e c e i v e d 5 A p r i l 1991 RAMA KRISHNA, N. S. AND N. K. SUBHEDAR. Distribution of FMRFamide-like immunoreactivity in the forebrain of the catfish, Clarias batrachus (Linn.). PEPTIDES 13(1) 183-191, 1992.--The anatomical distribution of FMRFamide-like immunoreactivity in the forebrain and pituitary of the catfish, Clarias batrachus, was investigated. Immunoreactive cells were observed in the ganglion cells of the nervus terminalis (NT) and in the medial olfactory tracts. In the preoptic area, FMRFamide-containing perikarya were restricted to the lateral preoptic area, paraventricular subdivision of the nucleus preopticus, nucleus suprachiasmaticus and nucleus preopticus periventricularis posterior. In the postoptic area, some cells of the nucleus postopticus lateralis and nucleus of the horizontal commissure showed moderate immunoreactivity. In the tuberal area, immunoreactivity was observed in few cells of the nucleus hypothalamicus ventralis and nucleus arcuatus hypothalamicus (NAH). Nucleus ventromedialis thalami was the only thalamic nucleus with FMRFamide immunoreactivity. Immunoreactive processes were traceable from the NT through the medial as well as lateral olfactory tracts into the telencephalon and the area ventralis telencephali pars supracommissuralis (Vs). Further caudally, the immunoreactive fibers could be traced into discrete areas, including habenular and posterior commissures, neurohypophysis and pituitary; isolated fibers were also observed in the pineal stalk. A loose network of immunoreactive processes was observed in the olfactory bulbs and the entire telencephalon, with higher densities in some areas, including Vs. A dense plexus of immunoreactive fibers was seen in the pre- and postoptic areas and around the paraventricular organ, while relatively few were observed in the thalamus. A high concentration of fiber terminals was found in the caudal tuberal area. FMRFamide

Forebrain

Immunohistochemistry

Neuropeptides

Catfish, Clarias batrachus

and substance P [see (34,35)], and on the other, displays connectivities to the olfactory epithelium, retina and also possibly to the pineal and pituitary (13, 15, 29, 30, 34, 35). Although much remains to be learned about the functional significance of the NT, it is suspected to modulate the sensory signals (35) with reference to pheromone reception and to influence the reproduction of fish (10,12). In view of the increasing interest generated by the peptide, and the limited information available as to its distribution in the brain of teleosts, it is hoped that the present work on the distribution of the neuropeptide, in the forebrain of the catfish, Clarias batrachus, might be of value for the comparative analysis of the vertebrate nervous system.

FMRFamide (Phe-Met-Arg-Phe-NH2), a molluscan cardioexcitatory tetrapeptide, was first isolated, purified and sequenced from the central ganglia of the Venus clam, Macrocallista nimbosa (26). In recent years, FMRFamide or its related peptides have been demonstrated in the central nervous system of animals from different taxa (3,14) and were shown to be extensively distributed in the brains of mammals [rat (9, 16, 42, 43); guinea pig (39); monkey (7)], birds (11,44), amphibians (21, 41, 45), teleosts (4, 5, 24) and lamprey (23). Although the physiological significance of the presence of this peptide in various parts of the central nervous system and peripheral tissues is poorly understood, its wide distribution within the brain suggests that it may play a role as a neurotransmitter (9, 22, 43) and also possibly as a neurohormone (23). Recent studies have also suggested that this peptide may have a role in neuroendocrine functions (8), vasopressor responses (1), and also possibly in modulation of pain (17). The study of FMRFamide draws its special significance in its occurrence in the ganglion cells of nervus terminalis (NT) or its homologue nucleus olfactoretinalis (NOR) of teleosts (13, 24, 34, 35, 40), amphibians (21, 41, 45) and birds (44). This is an exclusive system, that, on one hand, shows the coexistence of FMRFamide, luteinizing hormone-releasing hormone (LH-RH)

METHOD Adult catfish, Clarias batrachus (Linn.), of both sexes, weighing between 80 and 100 g, were used in the present study. Each fish was anesthetized with an overdose of tricaine methane sulfonate (MS 222; Sandoz), perfused transcardially with 50 ml of cold phosphate buffer (pH 7.4) followed by 50 ml of 4% paraformaldehyde fixative in phosphate buffer. The brains and olfactory bulbs, along with the organs, were dissected out and

~Requests for reprints should be addressed to Dr. N. K. Subhedar.

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postfixed overnight in fresh fixative at 4°C. The tissues were cryoprotected with 30% sucrose in phosphate buffer at 4°C. The brains were rapidly frozen with expanding CO2, cut with a cryostat at 15-20 Ixm thickness in transverse (6 brains), sagittal (6 brains) and horizontal (4 brains) planes. The sections were mounted on poly-L-lysine-coated slides, air dried, and processed for immunocytochemistry. The olfactory bulbs, along with olfactory organs and the retinae, were also processed. The sections were rehydrated in Tris buffer (pH 7.4) and processed for unlabeled antibody peroxidase-antiperoxidase (PAP) method of Sternberger et al. (36) as described earlier (37). The sections were preincubated with 1% normal goat serum for 30-60 min to avoid the background staining. Primary antiserum against FMRFamide (INCSTAR Corp. Inc., USA) at the dilution of 1:1000 was applied to each section and the slides were incubated in a closed moist chamber for 24-36 h at 4°C. All the dilutions and rinses were carried out in 0.1 M Tris buffer-HCl and Tris-HC1 containing Triton X-100 (pH 7.4). After rinsing, the sections were incubated in goat anti-rabbit IgG (National Institute of Immunology, New Delhi, India) at 1:100 dilution for 2 h at room temperature. The sections were washed for 10 min in two changes and incubated in PAP complex (Sigma, USA) at 1:100 dilution for 2 h followed by 10-min wash in Tris buffer (pH 7.6). Sections were then treated with 0.05% 3,3'-diaminobenzidine tetrahydrochloride (DAB; Sigma) with 0.03% H202 in 0.05 M Tris buffer (pH 7.6) for 5-10 min, rinsed in distilled water, dehydrated through graded series of alcohols, cleared in xylene and coverslipped with DPX. To verify the specificity of the antiserum used in the studies, several control procedures were performed. They included omission of one step of reaction (secondary antibody/PAP), replacing the anti-FMRFamide with normal goat serum and/or absorbing 1 ml of the dilution of the antiserum with 40 I~M FMRFamide (Sigma) for 24 h at 4°C prior to the incubation. To identify the brain areas and nuclei, additional brains were processed for routine histology and stained with the Kltiver and Barrera (18) method. The nomenclature used in the present study was from the earlier reports (2, 6, 25, 28, 38). RESULTS In the forebrain of C. batrachus, the FMRFamide-like immunoreactive (FMRFamide-ir) perikarya are confined to certain well-defined loci in the olfactory system, preoptic area, postoptic area, hypothalamus and thalamic regions. The fibers are distributed throughout the forebrain, with higher densities in certain circumscribable areas. The distribution of cell bodies and fibers is diagrammatically represented in Figs. 1 and 2.

FMRFamide-Like Immunoreactive Perikarya Olfactory system. The olfactory bulbs of C. batrachus are of

185

the pedunculated type; each bulb is an oval body communicating rostrally with the olfactory epithelium via a short olfactory nerve and caudally with the telencephalon by the extended peduncles consisting of medial (MOT) and lateral (LOT) olfactory tracts. The FMRFamide-ir perikarya of nervus terminalis (NT) are located rostrally at the junction of olfactory bulb (OB) and olfactory nerve (Fig. 3), along the midventral margin of the OB (Figs. 3, 4) and caudally at the junction between the OB and olfactory tracts (Fig. 5). The large and moderately sized cells stain intensely with anti-FMRFamide, while the small cells exhibit moderate immunoreactivity. In addition, few immunoreactive cell bodies are also observed within the MOT (Fig. 6). Telencephalon. With the exception of 2-3 FMRFamide-ir cells located within the MOT at the rostral pole of the telencephalon (Fig. 8), no immunoreactive perikarya are observed in this region of the forebrain. Diencephalon. Although the immunoreactive perikarya and processes are widely distributed in the diencephalon, a high degree of regional preference is observed; the preoptic area shows the highest concentration of FMRFamide-ir structures. In the lateral preoptic area, slightly caudal to the level of anterior commissure and below the lateral forebraln bundles (LFB), a subset of 8-12 moderately stained FMRFamide-ir perikarya is observed (Fig. 1). The cells are bipolar or multipolar and seem to be closely associated with the optic chiasma; the processes are directed either dorsolaterally through the narrow margin between the LFB and the optic tracts or extend medially towards the preoptic recess. No FMRFamide-ir perikarya are observed around the rostral preoptic recess. At midchiasmal level, on either side of the preoptic recess, 10-15 moderately stained FMRFamide-ir cells are clearly visible in the nucleus suprachiasmaticus (NSC) (Figs. 1, 10); their processes extend laterally. The nucleus preopticus (NPO) of C. batrachus consists of two welldefined cytoarchitectonic subdivisions, viz., the paraventricular group (PV) located adjacent to the preoptic recess and the supraoptic group (SO) situated above the optic chiasma (38). Whereas the SO shows no immunoreactive perikarya, few isolated large FMRFamide-ir cells are present in the PV (Figs. 1, 11). The unstained cells of both the subdivisions are profusely traversed by the immunoreactive processes. In the caudal preoptic area, few cells are also observed in the nucleus preopticus periventricularis posterior (NPPp) (Figs. 1, 2). The postoptic area of C. batrachus is a short zone of transition between the caudal limit of the optic chiasma and horizontal commissure (28). Laterally in this region, the nucleus postopticus lateralis (NPlp) shows the presence of some spindleshaped immunoreactive cells equipped with processes extending caudally into the tuberal area (Figs. 1, 2, 12). In the vicinity of the horizontal commissure, some deeply immunostained neurons partly encircle the horizontal commissure; these are identified as belonging to the nucleus of the horizontal commissure (NHC) (Figs. 1, 2, 13). Some immunoreactive processes from these

FIGS. 1 AND 2. Chartings of transverse sections through the forebrain showing the FMRFamide-like immunoreactive cells (black circles) and processes. The inset figure of the sagittal view of the brain at the upper right in Fig. 1 shows the level of the corresponding transverse sections. Abbreviations: AC: anterior commissure; C: cerebellum; DC: central zone of the area dorsalis telencephali (D); Dd: dorsal zone of the D; DLd: dorsal part of the lateral zone of the D; DLv: ventral part of the lateral zone of the D; DM: medial zone of the D; HbC: habenular commissure; HbG: habenular ganglion; HC: horizontal commissure; IL: inferior lobe; LFB: lateral forebrain bundles; LOT: lateral olfactory tract; LPA: lateral preoptic area; LR: lateral recess; MOT: medial olfactory tract; NAIl: nucleus arcuatus hypothalamicus; NHC: nucleus of the horizontal commissure; NHv: nucleus hypothalamicus ventralis; NPlp: nucleus postopticus lateralis; NPPp: nucleus preopticus periventricularis posterior; NSC: nucleus suprachiasmaticus; NVT: nucleus ventromedialis thalami; OC: optic chiasma; ON: optic nerve; OT: olfactory tract; P: pituitary gland; PC: posterior commissure; POA: preoptic area; POR: preoptic recess; PS: pineal stalk; PV: paraventricular subdivision of the nucleus preopticus; PVO: paraventricular organ; RI: recess of the inferior lobe; RP: recessus posterioris; SE: sulcus extemus; T: telencephalon; TO: optic tectum; V: ventricle; Vs: area ventralis telencephall pars supracommissuralis; Vv: ventral nucleus of area ventralis telencephali.

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cells intersect the horizontal commissure and descend into the rostral tuberal area; few fibers decussate in the commissure (Figs. 1, 2). In the rostral tuberal area, slightly away from the third ventricle, isolated FMRFamide-ir cells are encountered in the nucleus hypothalamicus ventralis (NHv). The cells are usually fusiform and bipolar, orientated in dorsoventral directions (Figs. 2, 14), with the processes extending dorsally into the nucleus anterior tuberis and ventrally towards the hypothalamo-hypophyseal tract. In the caudal tuberal area, at the level of the rostral margin of the pituitary gland, some small and bipolar FMRFamide-ir perikarya (Figs. 2, 15) are observed in the nucleus arcuatus hypothalamicus (NAH). In the thalamus, the nucleus ventromedialis thalami (NVT) is the only nucleus with FMRFamide immunoreactivity (Fig. 2). It shows several small and multipolar FMRFamide-ir perikarya equipped with immunoreactive processes extending dorsally or descending into the tuberal area. FMRFamide-Like Immunoreactive Fibers and Terminals Most of the immunoreactive fibers are thin and beaded and appear to be widely distributed throughout the forebrain of C. batrachus. In the olfactory system, thin FMRFamide-ir fibers arising from the NT are seen to course rostrally along the olfactory nerves and terminate among the olfactory lamellae. Several thick immunoreactive fibers are concentrated along the ventral margin of the bulbs; some are also seen along the dorsal surface. The substance of the bulb shows a network of thin as well as beaded processes (Figs. 3, 5). From the OB, thick, thin and beaded immunoreactive processes travel caudally through the MOT (Fig. 7); however, the LOT shows the presence of thick processes exclusively. The immunoreactive processes of the MOT penetrate the telencephalon at the rostromedial aspect (Fig. 8), and the LOT enters laterally at the level of sulcus externus (Fig. 1). The FMRFamide-ir processes in the LOT can be traced via the region above the sulcus externus caudally up to the level marked by the commissure of Goldstein; thereafter, these fibers radiate into the dorsolateral telencephalon. The immunoreactive processes of the MOT ascend caudally (Figs. 1, 9), and split into smaller fascicles, just prior to the level of the commissure of Goldstein. The majority of these fibers travel through the ventral (Vv) and central (Vc) nuclei of the area ventralis telencephali and can be traced caudally up to the level of the anterior commissure and the area ventralis telencephali pars supracommissuralis (Vs). Some of the fibers are seen to decussate via the commissure of Goldstein and anterior commissure (Fig. 1).

187

From the region of anterior commissure and Vs, the FMRFamide-ir fibers of the olfactory system diverge and seem to follow one of the three courses. Some immunoreactive processes run dorsocaudally, above the horizontal commissure to the habenular region (Figs. 1, 2, 17) and decussate in the habenular and posterior commissures (Figs. 2, 18). Although we cannot be certain, some of these fibers might extend into the pineal stalk. The processes are invariably thin and beaded (Fig. 18). The second path consists of thick immunoreactive processes which descend into the preoptic area, swing caudally and run in close association with the forebrain bundles and pass into the tuberal region. Some fibers could be traced medially into the hypothalamo-hypophyseal tract and the neurohypophysis (Figs. 1, 16). The third pathway extends from the area Vs, straight into the tegmental areas via the diencephalon. In addition to the above, a loose network of thin and beaded FMRFamide-ir fibers is widely distributed throughout the forebrain, particularly in the telencephalon. The area dorsalis telencephali (D) shows a high concentration of fibers in the dorsal (DLd) and ventral (DLv) parts of the lateral zone (Fig. 1), and in the caudal part of the posterior zone; the medial zone (DM) and area Vs showed an even higher density of fibers. The central (DC) zone of the D, on the contrary, shows a restricted number of fibers (Figs. 1, 2). Several beaded FMRFamide-ir fibers are directed towards the pial surface or run parallel to it. Sagittal sections reveal several thin and beaded immunoreactive processes ascending through the LFB and medial forebrain bundle (MFB) and radiate into the DM, DLd, DLv and DC. The preoptic area shows the highest concentration of FMRFamide-ir processes; fibers are particularly abundant at the chiasmal level and also around the LFB and MFB (Fig. 1). The neuropil around the PV and SO divisions of the NPO, NSC and lateral preoptic area displays high concentrations of immunoreactive processes. Some FMRFamide-ir fibers from the lateral preoptic area ascend into the telencephalon; few are also seen to enter the optic tracts. The retina, however, does not reveal any immunoreactivity. The postoptic area shows a fairly dense network of FMRFamide-ir processes (Figs. 1, 2, 12). While the rostral tuberal area contains relatively fewer immunoreactive processes, the FMRFamide-ir fibers and terminals are abundant, particularly around the NAH in the caudal region. In the immediate vicinity of the third ventricle, a large number of long beaded immunoreactive fibers is observed, particularly around the paraventricular organ, running parallel to the ventricular surface (Fig. 2). Further caudally, several FMRFamide-ir processes are seen near the mamillary nuclei and recessus posterioris. Occasionally, isolated and beaded immunoreactive fibers are observed in the inferior lobes.

FIG. 3. Sagittal section through the olfactory bulb (OB) showing the unipolar (short arrow) and bipolar (long arrow) FMRFamide-ir cells of the nervus terminalis and processes. Note that the periphery of the OB contains thick fibers and the substance shows thin and beaded processes exclusively. Bar = 300 p~m. FIG. 4. Sagittal section through the olfactory bulb showing a large multipolar FMRFamide-ir cell of the nervus terminalis located at the ventral margin. Bar = 50 I~m. FIG. 5. Sagittal section through the olfactory bulb (OB) and medial olfactory tract (MOT) showing the conspicuously stained large and multipolar FMRFamide-ir cells of the nervus terminalis (NT) located at the junction of the OB and MOT. Bar = 200 tzm. FIG. 6. Sagittal section through the medial olfactory tract (MOT) showing a FMRFamide-ir cell located within the MOT. Bar = 100 ~m. FIG. 7. Horizontal section through the medial olfactory tract (MOT) showing thick (large arrows) and thin (small arrows) FMRFamide-ir processes. Bar = 150 p,m. FIG. 8. Sagittal section through the junction of the medial olfactory tract (MOT) and the telencephalon (T). Arrow indicates a bipolar FMRFamide-ir cell located at the junction between MOT and T. Bar = 150 p,m. FIG. 9. Sagittal section through the telencephalon (T) showing the thick FMRFamide-ir fibers of the medial olfactory tract (MOT). Bar = 150 tzm.

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In the pituitary, thick and thin as well as beaded FMRFamide-ir processes are richly distributed throughout the neurohypophysis (Figs. 2, 12) and its arborizations. No immunoreactive processes are found in the rostral pars distalis of the pituitary. In the thalamus, a limited number of FMRFamide-ir processes is encountered in the periventricular region (Fig. 2) and around the nucleus ventromedialis thalami. The number of FMRFamide-ir processes is relatively very low in this region of the forebrain. Control reaction with 1) omission of secondary antibody or PAP, 2) nonimmune serum instead of anti-FMRFamide serum,

RAMA KRISHNA AND SUBHEDAR

or 3) immune serum preabsorbed with FMRFamide resulted in loss of immunoreactivity.

DISCUSSION

The present study describes the distribution of FMRFamidecontaining neuronal perikarya and fibers in the forebrain of a teleost, C. batrachus, as revealed by the antisera against FMRFamide. It should, however, be mentioned that FMRFamide antisera may also recognize other peptides like pancreatic

FMRFamide IN THE FOREBRAIN OF CATFISH

polypeptide-like peptides, neuropeptide Y, as well as the peptides with an amidated C-terminal sequence Arg-Phe-NH 2 [see (19, 20, 39)]. A detailed biochemical analysis and a variety of preabsorption control studies would be required to ascertain the precise nature of the antigen peptide in question. Our results demonstrate the presence of several FMRFamidelike immunoreactive perikarya in the NT and in the diencephalon. While the immunoreactive fibers are abundantly present in the olfactory tracts, they ate seen throughout the telencephalon with higher densities in certain areas. In the diencephalic, preoptic and postoptic areas, a well-organized pattern of fibers extending towards the optic tracts, paraventricular organ and the pituitary was observed. More dorsally, discrete fibers are seen to extend to the habenular and posterior commissures and towards the pineal stalk. Most of the available literature on FMRFamide in the teleosts dwells on the occurrence of the peptide in the cells of NT (5, 34, 35) or its homologue, the NOR (4, 13, 40). While the presence of FMRFamide has been reported in the NT of amphibians (21,45) and birds (44), it was absent in the NT of mammals (33). The NT of C. batrachus showed the presence of several FMRFamide-ir perikarya, located at the rostral and caudal poles and near the ventral surface of the olfactory bulbs; isolated immunoreactive cells are also seen along the MOT and at the junction between the MOT and the anterior pole of the telencephalon. The immunoreactive fibers of the NT can be traced rostrally as far as the olfactory lamellae, as also reported in goldfish (34,35) and salmon (24). Caudally from the olfactory bulbs, the FMRFamide-ir fibers of the NT travel via both the LOT and MOT as far as the Vs. The overall distribution of FMRFamide in the olfactory system is similar to that of LH-RH in the same fish (37), thus suggesting the possibility of coexistence of the two peptides. In support of this possibility, in both goldfish (34,35) and whitespotted greenling (40), FMRFamide was reported to colocalize with LH-RH within the NT/NOR. The immunoreactive processes of the NT origin, at the level of the Vs, seem to diverge along three different directions. The first pathway extends dorsocaudally to the habenular region; some fascicles decussate in habenular and posterior commissures, whereas isolated FMRFamide-ir fibers are observed in the pineal stalk. The possibility of FMRFamide-containing processes of the NOR projecting to the pineal gland has recently been suggested (13). The second pathway shows fascicles of immunoreactive fibers extending via the preoptic area caudally to the pituitary gland; these processes are conspicuously thick and easily traceable. The pathway could be compared with the LH-RH

189

containing thick processes of the NOR, which were shown to extend to the nucleus preopticus periventricularis, nucleus lateralis tuberis (NLT) and the pituitary in the platyfish, Xiphophorus maculatus (30-32). Bilateral lesioning of the olfactory tracts in C. batrachus resulted in abolition of the immunoreactivity in the pituitary (29) and also in the above-mentioned pathways (our unpublished data). The results support the presence of FMRFamide-containing NT innervation to the preoptic area, tuberal hypothalamus, pituitary and the habenular region. Besides, the third pathway extending caudally from the Vs might be similar to the NT innervation of the tegmental areas in salmon (24). It therefore seems likely that the NT might be influencing a wide range of brain loci employing FMRFamide and LH-RH as the regulatory peptides. Although the telencephalon of C. batrachus, with the exception of few cells of the NT, did not show the presence of any FMRFamide-ir perikarya, the immunoreactive fiber population is quite rich in the telencephalon. The processes are particularly concentrated in the DM and Vs. In the telencephalon of other teleosts (4,24), no cells but several fibers have been observed. While the amphibian telencephalon revealed a similar pattern, FMRFamide immunoreactivity was also reported in the medial telencephalic and septal regions and in the anterior commissure of amphibians (21, 41, 45). The telencephalon of mammals showed a wide distribution of FMRFamide-ir cells and fibers (7, 9, 43). In the preoptic area of C. batrachus, immunoreactive cells are observed in the PV group of the NPO, lateral preoptic area, NSC and NPPp. FMRFamide-ir perikarya in similar locations were also reported in other teleosts (4,5). In the preoptic area of lamprey, Lampetra japonica, the periventricular region showed the presence of FMRFamide-ir cells; however, these were shown to be different from magnocellular (aldehyde fuchsin-positive) neurons (23). In amphibians, the preoptic area showed immunoreactive cells in the bed nucleus of the anterior commissure, anterior preoptic area and caudal portion of the periventricular preoptic nucleus, which were considered as laminae terminalis or the junctional septo-preoptic area (21, 41, 45). Few FMRFamide-ir neurons were also observed on the dorsal edge of the optic chiasma in Rana pipiens (45). The cells might represent the nucleus suprachiasmaticus and may be comparable with the NSC in C. batrachus. In mammals, also, the preoptic area showed wide distribution of FMRFamide-ir cells in the nucleus paraventricularis and nucleus ventromedialis (7, 9, 43). The preoptic area of C. batrachus showed a high density of immunoreactive processes. High concentration of immunoreactivity was

FIG. 10. Transverse section through the preoptic area showing two FMRFamide-ir cells of the nucleus suprachiasmaticus. Bar = 50 ~m. FIG. 11. Transverse section through the preoptic area showing a large FMRFamide-ir cell in the paraventricular subdivision of the nucleus preopticus. Bar = 25 p.m. FIG. 12. Sagittal section through the postoptic area showing numerous thin and beaded FMRFamide-ir processes and the cells of the nucleus postopticus lateralis (NPlp). OC: optic chiasma. Bar = 150 Ixm. FIG. 13. Sagittal section through the postoptic area showing the FMRFamide-ir cells of the nucleus of the horizontal commissure (NHC) in the vicinity of the horizontal commissure (HC). Bar = 50 ~m. FIG. 14. Sagittal section through the tuberal area showing bipolar FMRFamide-ir cell of the nucleus hypothalamicus ventralis. Bar = 50 Ixm. FIG. 15. Transverse sections through the tuberal area showing bipolar FMRFamide-ir cell of the nucleus arcuatus hypothalamicus. Bar = 25 p.m. FIG. 16. Sagittal section through the tuberal area (TA) and the pituitary gland showing thick (large arrow) and beaded (small arrows) FMRFamide-ir processes within the hypothalamo-hypophysial tract (HHT) and neurohypophysis (NH). RPD: rostral pars distalis. Bar = 50 ~m. FIG. 17. Transverse section through the habenular region showing the long FMRFamide-ir processes ascending over the habenular ganglion (HbG). V: ventricle. Bar = 50 i~m. FIG. 18. Transverse section through the habenular commissure (HbC) showing long FMRF-ir processes (small arrows) traversing through the HbC. Thick arrow points to a small beaded immunoreactive process in the pineal stalk (PS). Bar = 150 Ixm.

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also observed in the hypothalamus of amphibians (41,45), birds (11) and mammals (7, 9, 43). The postoptic area of C. batrachus consists of two nuclei, viz., NPlp and NHC (28). Both the groups revealed isolated FMRFamide-ir cell bodies surrounded by a dense network of immunoreactive processes. However, no comparable immunoreactive cells were reported in earlier studies (4, 5, 24). While the hypothalamus of salmon showed many cerebrospinal fluid (CSF)contacting cells in the periventricular region (24), these were not observed in C. batrachus. Previous reports mentioned the presence of FMRFamide-ir perikarya in the NLT, CSF-contacting cells of nucleus recessus lateralis and in the nucleus posterior tuberis (4,5). The NLT of C. batrachus consists of very large perikarya which, however, did not show any immunoreactivity; neither was any immunolabeling seen in the nucleus recessus lateralis. The immunoreactivity is observed in the nucleus hypothalamicus ventralis in the rostral tuberal area; these cells are small and could be easily distinguished from the NLT (28). The nucleus arcuatus hypothalamicus (NAH) of C. batrachus consists of a compact group of cells situated just rostral to the pituitary gland (28); while the nucleus showed isolated FMRFamide-ir perikarya, it has been shown to send processes to the pituitary (27). Besides, the neuropil in the NAH and paraventricular organ also showed high densities of immunoreactive fibers and terminals. In mammals, also, the cells of the nucleus arcuatus were shown to contain abundant FMRFamide immunoreactivity (7-9, 43). Based on these findings, the possible involvement of FMRFamide in neuroendocrine regulation has been suggested (8). The suggestion is

further supported by the fact that FMRFamide-ir fibers are reported in the pituitary of lamprey (23) and teleosts [(4, 5, 24) and the present study] and in the median eminence of mammals (7, 9, 43). While the retina of most of the teleosts studied so far revealed the presence of FMRFamide-ir processes (24,35) and cells (presumably amacrine cells) (24), the retina of the catfish, C. batrachus, showed no immunoreactivity. While the reason for this discrepancy may be attributed to the species-specific differences, it may be recalled that in the retina of channel catfish also no FMRFamide immunoreactivity was observed [see (35)]. In summary, the overall organization of FMRFamide in C. batrachus has much in common with that in other vertebrates, in spite of some conspicuous species-specific differences. The NT seems to be the major immunoreactive center with wide neuronal connectivities. While the extensive distribution of FMRFamide in the forebrain suggests a neurotransmitter role for the peptide, its presence in the hypothalamus and the pituitary underscores a neuroendocrine implication. ACKNOWLEDGEMENTS We are grateful to Professor G. Bagavant for laboratory facilities and National Institute of Immunology, New Delhi, India for providing the anti-IgG. One of the authors (N.S.R.K.) is grateful to Council of Scientific and Industrial Research, New Delhi for the award of Research Associateship. This study was partially supported by the grants from Indian National Science Academy and Indian Council of Agricultural Research [No. 4(18)/87-ASR(I)].

REFERENCES 1. Bamard, C. S.; Dockray, G. J. Increases in arterial blood pressure in the rat in response to a new vertebrate neuropeptide, LPLRFamide, and a related molluscan peptide, FMRFamide. Regul. Pept. 8:209-215; 1984. 2. Bass, A. H. Organization of the telencephalon in the channel catfish, lctalurus punctatus. J. Morphol. 169:71-90; 1981. 3. Boer, H. H.; Schot, L. P. C.; Veenstra, J. A.; Reichelt, D. Immunocytochemical identification of neuronal elements in the central nervous system of a snail, some insects, a fish, and a mammal with an antiserum to the molluscan cardioexcitatory tetrapeptide FMRFamide. Cell Tissue Res. 213:21-27; 1980. 4. Bonn, U.; KiSnig, B. FMRFamide-like immunoreactivity in the brain and pituitary of the teleost, Eigenmannia lineata (Gymnotiformes). Z. Mikrosk. Anat. Forsch. 103:221-236; 1989. 5. Bonn, U.; K6nig, B. FMRFamide-like immunoreactivity in the brain and pituitary of Carassius auratus (Cyprinidae, Teleostei). J. Himforsch. 30:361-370; 1989. 6. Braford, M. R., Jr.; Northcutt, R. G. Organization of the diencephalon and pretectum of the ray-finned fishes. In: Davis, R. E.; Northcutt, R. G., eds. Fish neurobiology, vol. 2, higher brain areas and functions. Ann Arbor: The University of Michigan Press; 1983: 117-163. 7. Chen, S. T.; Tsai, M. S.; Shen, C. L. Distribution of FMRFamidelike immunoreactivity in the central nervous system of the Formosan monkey (Macaca cyclopsis). Peptides 10:825-834; 1989. 8. Chronwall, B. M. Anatomy and physiology of the neuroendocrine arcuate nucleus. Peptides 6:1-1 l; 1985. 9. Chronwall, B. M.; Olschowka, J. A.; O'Donohue, T. L. Histochemical localization of FMRFamide-like immunoreactivity in the rat brain. Peptides 5:569-584; 1984. 10. Demski, L. S.; Northcutt, R. G. The terminal nerve: A new chemosensory system in vertebrates? Science 220:435-437; 1983. 11. Dockray, G. J.; Dimaline, R. FMRFamide- and gastrirdCCK-like peptides in birds. Peptides 6:333-337; 1985. 12. Dulka, J. G.; Stacey, N. E.; Sorensen, P. W.; Van Der Kraak, G. J.; Marchant, T. A. A sex pheromone system in goldfish: Is the

13. 14.

15.

16.

17.

18. 19.

20.

21.

nervus terminalis involved? Ann. NY Acad. Sci. 519:411-420; 1987. Ekstr6m, P.; Honkanen, T.; Ebbesson, S. O. E. FMRFamide-like immunoreactive neurons of the nervus terminalis of teleosts innervate both retina and pineal organ. Brain Res. 460:68-75; 1988. Greenberg, M. J.; Price, D. A.; Lehman, H. K. FMRFamide-like peptides of molluscs and vertebrates: Distribution and evidence of function. In: Kobayashi, H.; Bern, H.; Urano, A., eds. Neurosecretion and the biology of neuropeptides. Tokyo: Japan Sci. Soc. Press; 1985:370-376. Grober, M. S.; Bass, A. H.; Burd, G.; Marchaterre, M. A.; Segil, N.; Scholz, K.; Hodgson, T. The nervus terminalis ganglion in Anguilla rostrata: An immunocytochemical and HRP histochemical analysis. Brain Res. 436:148-152; 1987. H6ckfelt, T.; Lundberg, J. M.; Tatemoto, K.; Mutt, V.; Terenius, L.; Polak, J.; Bloom, S.; Sasek, C.; Elde, R.; Goldstein, M. Neuropeptide Y (NPY)- and FMRFamide neuropeptide-like immunoreactivities in catecholamine neurons of the rat medulla oblongata. Acta Physiol. Scand. 117:315-318; 1983. Kavaliers, M. Inhibitory influences of mammalian FMRFamide (PheMet-Arg-Phe-amide)-related peptides on nociception and morphineand stress-induced analgesia in mice. Neurosci. Lett. 115:307-312; 1990. Kltiver, H.; Barrera, E. A method for the combined staining of cells and fibers in the nervous system. J. Neuropathol. Exp. Neurol. 12: 400-403; 1953. Moore, R. Y.; Gustafson, E. L.; Card, J. P. Identical immunoreactivity of afferents to the rat suprachiasmatic nucleus with antisera against avian pancreatic polypeptide, molluscan cardioexcitatory peptide and neuropeptide Y. Cell Tissue Res. 236:41-46; 1984. Muske, L. E.; Dockray, G. J.; Chohan, K. S.; Stell, W. K. Segregation of FMRFamide-immunoreactive efferent fibers from NPYimmunoreactive amacrine cells in goldfish retina. Cell Tissue Res. 247:299-307; 1987. Muske, L. E.; Moore, F. L. The nervus terminalis in amphibians: Anatomy, chemistry and relationship with the hypothalamic gona-

F M R F a m i d e IN THE F O R E B R A I N O F C A T F I S H

22.

23. 24.

25.

26. 27.

28.

29.

30.

31.

32.

33.

dotropin-releasing hormone system. Brain Behav. Evol. 32:141150; 1988. Newton, B. W.; Maley, B.; Sasek, C.; Traurig, H. Distribution of FMRF-NH2-1ike immunoreactivity in rat and cat area postrema. Brain Res. Bull. 13:391-399; 1984. Ohtomi, M.; Fujii, K.; Kobayashi, H. Distribution of FMRFamidelike immunoreactivity in the brain and neurohypophysis of the lamprey, Lampetra japonica. Cell Tissue Res. 256:581-584; 1989. Ostholm, T.; Ekstr6m, P.; Ebbesson, S. O. E. Distribution of FMRFamide-like immunoreactivity in the brain, retina and nervus terminalis of the sockeye salmon parr, Oncorhynchus nerka. Cell Tissue Res. 261:403-418; 1990. Peter, R. E.; Gill, V. E. A stereotaxic atlas and technique for forebrain nuclei of the goldfish, Carassius auratus. J. Comp. Neurol. 159:69-102; 1975. Price, D. A.; Greenberg, M. J. Structure of molluscan cardioexcitatory neuropeptide. Science 197:670-671; 1977. Rama Krishna, N. S.; Subhedar, N. Hypothalamic innervation of the pituitary in the catfish, Clarias batrachus (L.): A retrograde horseradish peroxidase study. Neurosci. Lett. 107:39--44; 1989. Rama Krishna, N. S.; Subhedar, N. Cytoarchitectonic pattern of the hypothalamus in the catfish, Clarias batrachus (Linn.). J. Hiruforsch., in press; 1991. Rama Krishna, N. S.; Subhedar, N.; Schreibman, M. P. FMRFamide-like immunoreactive nervus terminalis innervation to the pituitary in the catfish, Clarias batrachus (Linn.): Demonstration by lesion and immunocytochemical techniques. Gen. Comp. Endocrinol., in press; 1991. Schreibman, M. P.; Margolis-Nunno, H. Reproductive biology of the terminal nerve (nucleus olfactoretinalis) and other LHRH pathways in teleost fishes. Ann. NY Acad. Sci. 519:60--68; 1987. Schreibman, M. P.; Margolis-Kazan, H.; Halpern-Sebold, L.; O'Neill, P. A.; Caracheo, F. An LHRH-containing center in the brain, connecting olfactory and reproductive systems. Am. Zool. 22:856; 1982. Schreibman, M. P.; Margolis-Kazan, H.; Halpern-Sebold, L.; O'Neill, P. A.; Silverman, R. C. Structural and functional links between olfactory and reproductive systems: Puberty-related changes in olfactory epithelium. Brain Res. 302:180-183; 1984. SchwanzeI-Fukuda, M.; Morrell, J. I.; Pfaff, D. W. Localization of choline acetyltransferase and vasoactive intestinal polypeptide-like immunoreactivity in the nervus terminalis of the fetal and neonatal rat. Peptides 7:899-906; 1986.

191

34. Stell, W. K.; Walker, S. E.; Chohan, K. S.; Ball, A. K. The goldfish nervus terminalis: A luteinizing hormone-releasing hormone and molluscan cardioexcitatory peptide immunoreactive olfactoretinal pathway. Proc. Natl. Acad. Sci. USA 81:940-944; 1984. 35. Stell, W. K.; Walker, S. E.; Ball, A. K. Functional-anatomical studies on the terminal nerve projection to the retina of bony fishes. Ann. NY Acad. Sci. 519:80-96; 1987. 36. Sternberger, L. A.; Hardy, P. H., Jr.; Cuculis, J. J.; Meyer, H. G. The unlabeled antibody enzyme method for immunocytochemistry: 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; 1970. 37. Subhedar, N.; Rama Krishna, N. S. Immunocytochemical localization of LH-RH in the brain and pituitary of the catfish, Clarias batrachus (Linn.). Gen. Comp. Endocrinol. 72:431-442; 1988. 38. Subhedar, N.; Rama Krishna, N. S.; Prasada Rao, P. D. The intrinsic organization of the nucleus preopticus in the catfish, Clarias batrachus (Linn.). J. Hirnforsch. 31:25-40; 1990. 39. Triepel, J.; Grimmelikhuijzen, C. J. P. Mapping of neurons in the central nervous system of the guinea pig by use of antisera specific to the molluscan neuropeptide FMRFamide. Cell Tissue Res. 237: 575-586; 1984. 40. Uchiyama, H. Immunohistochemical subpopulations of retinopetal neurons in the nucleus olfactoretinalis in a teleost, the whitespotted greenling (Hexagrammos stelleri). J. Comp. Neurol. 293:54-62; 1990. 41. Uchiyama, H.; Reh, T. A.; Stell, W. K. Immunocytochemical and morphological evidence for a retinopetal projection in anuran amphibians. J. Comp. Neurol. 274:48-59; 1988. 42. Weber, E.; Evans, C. J.; Samuelsson, S. J.; Barchas, J. D. Novel peptide neuronal system in the rat brain and pituitary. Science 214: 1248-1251; 1981. 43. Williams, R. G.; Dockray, G. J. Immunohistochemical studies of FMRFamide-like immunoreactivity in rat brain. Brain Res. 276: 213-229; 1983. 44. Wirsig-Wiechmann, C. R. The nervus terminalis in the chick: A FMRFamide-immunoreactive and AChE-positive nerve. Brain Res. 523:175-179; 1990 45. Wirsig-Wiechmann, C. R.; Basinger, S. F. FMRFamide-immunoreactive retinopetal fibers in the frog, Rana pipiens: Demonstration by lesion and immunocytochemical techniques. Brain Res. 449:116134; 1988.