Localization of atrial natriuretic factor (ANF)-related peptides in the central nervous system of the elasmobranch fish Scyliorhinus canicula

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.     

Distribution of FMRFamide-like immunoreactivity in the brain of the elasmobranch fish Scyliorhinus canicula.

The distribution of FMRFamide-like-immunoreactive peptides was investigated in the brain and pituitary of the elasmobranch fish Scyliorhinus canicula using the indirect immunofluorescence technique. FMRFamide-immunoreactive cells and fibers were mainly observed in the telencephalon and the diencephalon, while other brain structures were almost unstained. In the telencephalon, FMRFamide-like-containing neurons were seen in the caudal part of the area periventricularis pallialis, in the posterior area of the nucleus septi medialis and in the nucleus septi caudoventralis. In the diencephalon, numerous FMRFamide-positive cell bodies were observed in the hypothalamus, ventral thalamus and posterior tuberculum. The highest density of immunofluorescent perikarya was found in the nucleus lobi lateralis hypothalami and in the nucleus periventricularis hypothalami. More caudally, the mesencephalon and the caudal brainstem only contained scattered varicose FMRFamide-immunoreactive fibers. Stained fibers were also identified in the median eminence and several FMRFamide-like-positive cells were detected in the dorsal and rostral parts of the neurointermediate lobe of the pituitary. These data indicate that substances related to the molluscan cardioexcitatory peptide FMRFamide are widely distributed in the brain of S. canicula, suggesting their implication in neuroendocrine and/or neuromodulatory functions.     

Localization of corticotropin-releasing hormone (CRF)-like immunoreactivity in the central nervous system of the elasmobranch fish,Scyliorhinus canicula

Summary The occurrence and localization of immunoreactive corticotropin-releasing factor (CRF) in the brain and pituitary of the elasmobranch fish Scyliorhinus canicula, were studied by means of specific radioimmunoassay and immunohistochemistry using the indirect immunofluorescence method. Brain and pituitary extracts showed a good cross-reactivity with the ovine CRF antiserum, but serial dilutions of tissue samples did not completely parallel the standard curve. Relatively high concentrations of CRF-like material were found within the pituitary, diencephalon, and telencephalon. CRF-like immunoreactive perikarya were observed in the preoptic nucleus and in the nucleus lateralis tuberis. Numerous immunoreactive cells appeared to be of the CSF-contacting type. CRF-like immunopositive fibers were seen to run through the hypothalamus within the ventro-medial floor of the infundibular region. A dense plexus of immunoreactive nerve endings terminated in the median eminence and the neurointermediate lobe of the pituitary. These results indicate that a neurosecretory system containing CRF-like immunoreactivity exists in the brain of elasmobranchs, a group of vertebrates which has diverged early from the evolutionary line leading to mammals. In addition, our data support the notion that a CRF-like molecule is involved in the regulation of corticotropic and melanotropic cell activity in this primitive species of fish.     

The structure of the gills of the elasmobranch,Scyliorhinus canicula (L.)

Summary The anatomy of the blood supply to the gills of the dogfish, Scyliorhinus canicula, is described. The anatomical basis for a counter-current exchange system at the respiratory surfaces is reported. Within the interbranchial septum there is a capillary network joining all the afferent branchial arterioles of the gill. The structure of the walls of the corpus cavernosum is found to be of smooth muscle cells supported by a basal lamina and connective tissue and lined by endothelial cells containing phagocytic vesicles. Both the capillary network and corpus cavernosum are suggested to function in smoothing the pressure pulses of the blood flow. Pre- and post-lamellar vessels and pre- and post-lamellar sphincters are described. The sphincters are thought to control the number of secondary lamellae physiologically in the respiratory circuit, and by retaining blood within nonperfused lamellae to act in conjunction with pillar cells (contracting in antagonism to the hydrostatic skeleton of the blood) to maintain the rigidity of secondary lamellae in the water current.Whorls of cells of unknown function are found within the interbranchial septum. In the epithelium lining the water channel large cells having a complexly branching plasma membrane and a very large central vacuole occurs. The cytoplasm lining the lumen contains numerous vacuoles each surrounded by a double membrane.This work formed part of a thesis submitted for the degree of Master of Science at the University of Bristol. I should like to thank Professor G.M. Hughes for the use of facilities in the Department of Zoology, University of Bristol.     

GABAergic system of the pineal organ of an elasmobranch (Scyliorhinus canicula): a developmental immunocytochemical study

The present immunocytochemical study provides evidence of a previously unrecognized, rich, γ-aminobutyric acid (GABA)-ergic innervation of the pineal organ in the dogfish (Scyliorhinus canicula). In this elasmobranch, the pineal primordium is initially detected at embryonic stage 24 and grows to form a long pineal tube by stage 28. Glutamic acid decarboxylase (GAD)-immunoreactive (-ir) fibers were first observed at stage 26, and by stage 28, thin GAD-ir fibers were detectable at the base of the pineal neuroepithelium. In pre-hatchling embryos, most fibers gave rise to GAD-ir boutons that were localized in the basal region of the neuroepithelium, although a smaller number of labeled terminals ascended to the pineal lumen. A few pale GAD-ir perikarya were observed within the pineal organ of stage 29 embryos, but GAD-ir perikarya were not observed at other developing stages or in adults. In contrast, GABA immunocytochemistry revealed the presence of GABAergic perikarya and fibers in the pineal organ of late stage embryos and adults. Although high densities of GABAergic cells were observed in the paracommissural pretectum, posterior tubercle, and tegmentum of dogfish embryos (regions previously demonstrated to contain pinealopetal cells), the presence of GABA-ir perikarya in the pineal organ strongly suggests that the rich GABAergic innervation of the elasmobranch pineal organ is intrinsic. This contrasts with the central origin of GABAergic fibers in the pineal gland of some mammals. This work was supported by the Spanish Education and Science Ministry and FEDER (BXX2000-0453-C02 and BFU2004-03313/BF1), the Xunta de Galicia (PGIDT99BIO20002), and NIH/NIDCD awards R01 DC01705 and P01 DC01837 (to G.R.H.).     

Myelinated synapse-bearing cell bodies in the central nervous system of Scyliorhinus canicula (L.)

Summary Some neurons of the anterior lateral-line lobe of dogfish (Scyliorhinus) have synapse-bearing perikarya enclosed by layers of compact myelin. The identity of these cells, which have myelinated axons and unmyelinated, synapse-bearing dendrites, is unknown.     

Expression of atrial natriuretic factor (ANF) during Xenopus cardiac development

We have isolated the Xenopus orthologue of the atrial natriuretic factor (ANF) gene. Characterization of embryonic expression indicates that the ANF gene is initially expressed throughout the developing myocardium at the late heart tube stage (about stage 32). This is in contrast to all previously characterized Xenopus cardiac differentiation markers that are first expressed in the cardiogenic plate at approximately stage 27. ANF expression becomes restricted exclusively to the atrium at about stage 47, long after the commencement of beating and the original formation of the atrial and ventricular compartments, but shortly after septation of the single atrium into two distinct atria. Received: 5 May 2000 / Accepted: 3 August 2000     

Atrial natriuretic factor (ANF) binding sites in brain and related structures

Visualization of [¹²⁵I]ANF binding sites in rat brain by an autoradiographic technique demonstrated that these sites are highly localized in areas such as the olfactory bulb, subfornical organ, area postrema and nucleus tractus solitarius. This distribution suggests that certain cardiovascular effects of ANF could be centrally mediated and that the existence of brain ANF-related peptides should be considered. Finally, moderate densities of [¹²⁵I]ANF binding sites are found in the rat and guinea pig eye while low densities are seen in pituitary and pineal gland.     

Sexual dimorphisms in the dermal structure of the lesser‐spotted catshark,Scyliorhinus canicula (Linnaeus, 1758)

Sexual dimorphisms in the dermal structures of two elasmobranch species have previously been reported and it has been linked to the use of the mouth by males during copulation. Until relatively recently, the fact, that male Scyliorhinus canicula use their mouths for grasping and biting females during copulation was unknown. This study reveals that not only do adult (M ≥ 525 mm, F ≥ 550 mm) S. canicula show a sexual dimorphism in the epidermis and dermis, but that hatchling S. canicula are born with a sexually dimorphic epidermal layer and this persists into the juvenile stage (M < 525 mm, F < 550 mm). A sexual dimorphism was found in all size classes with both hatchling and juvenile female S. canicula having significantly thicker epidermal layers than hatchling and juvenile male S. canicula. Adult female S. canicula were found to possess both a significantly thicker epidermal and dermal layer than adult male S. canicula. The presence of a sexual dimorphism in the epidermal and dermal layers of adult S. canicula could be directly related to reproductive behaviour in response to the male biting the female prior to copulation.     

Dissociation and identification of intact seminiferous lobules from the testis of the dogfish (Scyliorhinus canicula)

Summary An enzymatic method was developed to obtain intact seminiferous lobules from the testis of the dogfish (Scyliorhinus canicula L.). The freshly isolated lobules were then identified by use of a transillumination technique. Testes from mature dogfish were collected and transverse sections incubated with a mixture of collagenase (0.025%) + pronase (0.08%) at 4° C overnight. Dissociation of the tissue was achieved by mechanical agitation in a calcium and magnesium-free buffer (pH 7.8; 870 mOsm) at room temperature, for 30 min. Based on differences in light absorption and size as well as on comparison with the corresponding histological and ultrastructural cell composition, the seminiferous lobules were identified and classified according to the stages of spermatogenesis of Mellinger (1965). The characteristic changes take place in the size and surface of the lobules and are mainly due to differences in the arrangement of the germ cells, in their number, and in the light absorption characteristics of their nuclei. The combination of the procedures of isolation and transillumination of the dogfish seminiferous lobules, in native condition, offers an original method for the study of germ cell-Sertoli cell interactions in the non-mammalian vertebrate.     

Expression of atrial natriuretic factor gene in heart ventricular tissue

A novel peptide hormone, atrial natriuretic factor (ANF), was recently isolated and characterized in mammalian atria. This hormone has potent natriuretic, diuretic and vasorelaxant activities. Since ANF bioactivity was initially found in atria but not in ventricles, it was assumed that the ANF gene is specifically expressed in atria. We now report that ANF mRNA is present in ventricular tissue as well as in atria. This is clearly demonstrated by in situ hybridization and by Northern blot analysis. Rat ventricular ANF mRNA concentration is a hundred-fold lower than in atria. As in atria, the 126 amino acids precursor form of ANF is predominant in ventricles and it is present at a thousand-fold lower concentration. The ten-fold discrepancy in the ratio of ANF mRNA to immunoreactivity between atria and ventricles could reflect a higher rate of peptide release in the latter. Thus, ventricular ANF production may be physiologically significant in view of the much larger ventricular mass.     

Tachykinin-like immunoreactivity in the sinus venosus of the dogfish (Scyliorhinus canicula)

The sinus venosus of the elasmobranch heart is characterized by the presence of large bundles of unmyelinated nerve fibres that bulge into the cardiac lumen, below the endocardium. In the dogfish (Scyliorhinus canicula), these fibres contain numerous dense-core membrane-bounded granules of about 200 nm in diameter. Most intramural ganglion cells of the sinus venosus also show densely packed granules similar to those found in the subendocardial fibres. We have observed strong substance-P-like immunoreactivity in the large fibre bundles and in the perikarya of the ganglion cells. Preabsorption of the antisera with fragment 7–11 of substance P has shown that the antisera recognize the tachykinin canonic sequence. Our findings suggest that an undetermined tachykinin is secreted in the elasmobranch heart, and that it is probably released into the blood stream in the context of a little-known neuroendocrine system.     

Localization of binding sites for atrial natriuretic factor and angiotensin II in the central nervous system of the clawed toad Xenopus laevis

Summary The distribution of binding sites for atrial natriuretic factor (ANF) and angiotensin II (A II) was investigated in the central nervous system (CNS) of the clawed toad Xenopus laevis by means of in vitro autoradiography using [¹²⁵I]-rat ANF(99–126) or [¹²⁵I] [Val⁵] A II and [¹²⁵I]human A II as labeled ligands. The highest densities of specific ANF-binding were detected in the nucleus habenularis, thalamic regions, hypophyseal pars nervosa and nucleus interpeduncularis. Moderate ANF-binding was found in the bulbus olfactorius, pallium, septum, striatum, lateral forebrain bundle, nucleus infundibularis, hypophyseal pars distalis and tectum. The highest levels of specific A II binding sites were observed in the nucleus praeopticus, nucleus habenularis, hypophyseal pars nervosa and pars distalis, whereas the amygdala contained moderate A II binding. The existence of specific binding sites for ANF and A II in the CNS of Xenopus laevis suggests that both peptides act as neurotransmitters or neuromodulators in the amphibian CNS. The co-localization of dense binding sites for both peptides in the nucleus habenularis, hypophyseal pars nervosa and pars distalis supports the view that ANF and A II have opposite regulatory functions in these regions.     

Distribution patterns and sexual segregations of Scyliorhinus canicula (L.) in the Cantabrian Sea

The influence of factors such as depth, near bottom temperature, near bottom salinity, fish length and diet composition on the aggregation of Scyliorhinus canicula in the Cantabrian Sea was examined. The correlation between these variables and the aggregations of each sex was low. A priori juvenile males and females were not highly discriminated by any of these environmental variables (depth, temperature and salinity) based on a multivariate analysis. The best discriminated groups were the new recruits, and depth and longitude were the variables that contributed most to their segregation, while adult males had some preference for shallower and warm waters. Diet composition varied with body size but it was quite similar in both sexes among which no significant differences were observed. An increase in feeding intensity and abundance of Euphasiacea prey taxa was found in adult females compared to males.     

Occurrence of bombesin-like immunoreactivity in the brain of the cartilaginous fish,Scyliorhinus canicula

Summary The presence and distribution of bombesin-like material were investigated in the brain of the cartilaginous fishScyliorhinus canicula using conventional immunocytochemical techniques. Perikarya containing bombesin-like immunoreactivity were identified in the hypothalamus, within the magnocellular component of the preoptic nucleus. Some immunopositive elements appeared to be of cerebrospinal fluid-contacting type. Beaded immunoreactive fibers were seen crossing the ventral telencephalon and the whole hypothalamus. An important tract of fibers was found in the infundibular floor and in the median eminence, in close contact with the vascular system of the pituitary portal plexus. A moderate number of positive fibers innervated the habenular complex and the dorsal wall of the posterior tuberculum. These findings indicate that a neuropeptide strictly related to amphibian bombesin is located in specific hypothalamic neurons ofS. canicula. The distribution of the immunoreactive fibers and terminals suggests that, in fish, this peptide, may be involved in neuroendocrine and neuromodulator functions.     

Separation of leucocytes in the dogfish (Scyliorhinus canicula) using density gradient centrifugation and differential adhesion to glass coverslips

Summary The blood of the dogfish, S. canicula, contains several types of leucocytes, namely thrombocytes, monocytes, lymphocytes and four populations of granulocytes. Three of these granulocyte types, G1, G3 and G4, are eosinophilic while G2 is heterophilic/neutrophilic. All of the leucocyte types, with the exception of G2 granulocytes and monocytes, can be separated by means of their differential adherent properties to glass and by density gradient centrifugation. Thrombocytes, G3 and G4 granulocytes can be separated in good purity by single-step methods while G1 granulocytes and lymphocytes require a combination of density gradient centrifugation followed by adherence to glass to remove contaminating thrombocytes. Depending on the cell type, between 11–45% of cells with consistently high viability can be recovered after separation. Separated populations of the thrombocytes and granulocytes will be especially useful for studies on the role of such cell types in inflammation.     

Distribution of atrial natriuretic factor in fetal rat atria and ventricles

Summary An immunohistochemical study of rat fetal hearts at 20 days of gestation revealed the presence of immunoreactive atrial natriuretic factor (ANF) in cardiocytes of the left and right atria as well as in certain cells is the left and right ventricles. In the atria, cells of the adluminal pectinate muscles appear more densely labeled than the more peripheral mural cells. In the ventricles, immunoreactive cells were found only in adluminal cardiocytes of the presumptive trabeculae and papillary muscles. The results indicate that ANF is synthesized in the perinatal heart, and that the presence of this hormone in the ventricular cardiocytes may be of only temporary nature during certain stages of pre- and postnatal development.Supported by Miami Valley Chapter of American Heart Association MVH-86-019 and MVH-86-010     

Does specific dynamic action (SDA) regulate return of appetite in the lesser spotted dogfish,Scyliorhinus caniculcla?

Specific dynamic action (SDA) and the return of appetite (RA) were measured in juvenile (weight range 10·9–16·3 g) and adult (weight range 588·8–802·2 g) Scyliorhinus canicula by means of respirometry and re-feeding trials respectively. Juvenile S. canicula consumed relatively more food than the adults at all times indicating faster rates of food processing in juvenile dogfish. At no time during the SDA effect was there any evidence that elevated levels of oxygen consumption completely prevented further food consumption in either adult or juvenile dogfish. The coefficient (Duration_SDA/Duration_RA) was similar for both adult (0·52) and juvenile (0·49) S. canicula suggesting that SDA metabolism was closely associated with the processes involved in appetite return.     

Localization and identification of immunoreactive atrial natriuretic factor (ANF) in the frog ventricle

The localization of ANF-like immunoreactivity in the ventricle of the frog Rana ridibunda was examined by the indirect immunofluorescence and the immunogold techniques, using an antiserum against synthetic ANF (Arg 101-Tyr 126). At the optic level, an appreciable number of positive cardiocytes was observed in the frog ventricle. Electron microscopic studies showed that all secretory granules present in ventricular cardiocytes contain immunoreactive ANF. The immunoreactive material has been characterized by Sephadex G-50 gel chromatography and reverse phase high performance liquid chromatography (RP-HPLC). After gel filtration, ANF-like immunoreactivity eluted in 3 peaks. The major immunoreactive peak corresponded to high molecular weight material, while one peak co-eluted with synthetic ANF (Arg 101-Tyr 126). Further analysis of frog ventricular extracts by RP-HPLC revealed that the low molecular weight material has the same retention time as synthetic ANF, suggesting a high degree of sequence homology between amphibian and mammalian ANF. These results indicate that in amphibians, ventricular cardiocytes synthesize a peptide immunologically and chemically related to mammalian ANF.