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.     

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.     

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.     

Immunocytochemical distribution of FMRFamide-like substance in the brain of the cloudy dogfish,Scyliorhinus torazame

Summary The distribution of the molluscan cardioexcitatory tetrapeptide FMRFamide (Phe-Met-Arg-Phe-NH₂) in the brain of the cloudy dogfish, Scyliorhinus torazame, was examined by immunocytochemistry. FMRFamide-like immunoreactivity was demonstrated to occur extensively in various regions of the dogfish brain, except for the corpus cerebelli. Immunoreactive neuronal perikarya were located in the ganglion of the nervus terminalis, the preoptic area, and the hypothalamic periventricular gray matter consisting of the nucleus medius hypothalamicus, the nucleus lateralis tuberis, and the nucleus lobi lateralis. some of the immunoreactive cells in the hypothalamus were identified as cerebrospinal fluid-contacting neurons. The bulk of the immunostained fibers in the nervus terminalis penetrated into the midventral portion of the telencephalon and ran dorsocaudally toward the basal telencephalon and hypothalamus, showing radial projections or ramifications. The labeled fibers were abundant in the midbasal part of the telencephalon and in the hypothalamus, where some fibers were found in loose networks around the cell bodies of the nucleus septi and hypothalamic periventricular nuclei. The fibers demonstrated in the hypothalamus terminated around the vascular wall of the primary capillary plexus of the median eminence or penetrated deeply into the pars intermedia of the hypophysis. These results suggest that, in the dogfish, an FMRFamide-like substance participates in the regulation of adenohypophysial function. This molecule may have a role as a neurotransmitter and/or neuromodulator in the central nervous system.     

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.     

Effects of some metals on paraoxonase activity from shark Scyliorhinus canicula

Paraoxonase (PON) is an organophosphate hydrolyser enzyme which also has antioxidant properties in metabolism. Due to its crucial functions, the inhibition of the enzyme is undesirable and very dangerous. PON enzyme activity should not be altered in any case. Inhibitory investigations of this enzyme are therefore important and useful. Metal toxicology of enzymes has become popular in the recent years. Here, we report the in vitro inhibitory effects of some metal ions, including Ni²⁺, Cd²⁺, Cu²⁺ and Hg²⁺, on the activity of shark serum PON (SPON). For this purpose, we first purified the enzyme from shark Scyliorhinus canicula (LINNAEUS, 1758) serum and analysed the alterations in the enzyme activity in the presence of metal ions. The K_M and V_max is 0.227?mM and 454.545?U/mL, respectively. The results show that metal ions exhibit inhibitory effects on SPON1 at low concentrations with IC₅₀ values ranging from 0.29 to 2.00?mM. Copper was determined to be the most effective inhibitor with IC₅₀ of 0.29?mM.     

Histochemical localization of FMRFamide-like immunoreactivity in the rat brain

Molluscan cardioexcitatory neuropeptide or FMRFamide is present in the invertebrate central nervous system (CNS) and FMRFamide like peptide has been demonstrated in the mammalian CNS. In this study, the distribution of FMRFamide immunoreactivity was studied in rat brain using the indirect immunofluorescent method. The highest number of FMRFamide staining cell bodies was found in the nucleus (n) arcuatus. N. paraventricularis, n. hypothalamus, n. ventromedialis, n. dorsomedialis and n. tractus solitarii also contained high numbers. FMRFamide positive nerve fibers and terminals were widely distributed. The septal complex contained high densities, especially in n. interstitialis striae terminalis. N. paraventricularis hypothalami, n. paraventricularis, n. hypothalamicus, n. ventromedialis and n. dorsomedialis showed a high to very high degree of immunoreactivity. In myelencephalon, n. tractus solitarii had the densest innervation. Spinal cord had a dense band of FMRFamide positive fibers in lamina I and II of the dorsal horn. The present findings support a neurotransmitter role for a FMRFamide like peptide in the mammalian brain, possibly related to endocrine and autonomic regulation as well as pain modulation.     

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.     

Serum cholesterol and triglyceride levels in Scyliorhinus canicula (L.) during sexual maturation

A study was carried out on the serum levels of cholesterol [total (TC) and that associated with high-density lipoproteins (HDLC)] and triglyceride (TG) in 127 specimens of Scyliorhinus canicula . The values obtained were correlated with sex, size, liver weight and reproductive stage. Results showed that male dogfish have higher levels of TC and HDLC, and lower TG than female. In adult males, TC increased and HDLC decreased with both size and spermatogenesis. Females carrying capsulated eggs showed a noticeable increase in TG together with a decrease of the HDLC levels, which were apparently not related with size.     

Distribution of FMRFamide-like immunoreactivity in the brain and suboesophageal ganglion of the sphinx mothManduca sexta and colocalization with SCPB-, BPP-, and GABA-like immunoreactivity

Summary Using an antiserum against the tetrapeptide FMRFamide, we have studied the distribution of FMRFamide-like substances in the brain and suboesophageal ganglion of the sphinx mothManduca sexta. More than 2000 neurons per hemisphere exhibit FMRFamide-like immunoreactivity. Most of these cells reside within the optic lobe. Particular types of FMRFamide-immunoreactive neurons can be identified. Among these are neurosecretory cells, putatively centrifugal neurons of the optic lobe, local interneurons of the antennal lobe, mushroom-body Kenyon cells, and small-field neurons of the central complex. In the suboesophageal ganglion, groups of ventral midline neurons exhibit FMRFamide-like immunoreactivity. Some of these cells have axons in the maxillary nerves and apparently give rise to FMRFamide-immunoreactive terminals in the sheath of the suboesophageal ganglion and the maxillary nerves. In local interneurons of the antennal lobe and a particular group of protocerebral neurons, FMRFamide-like immunoreactivity is colocalized with GABA-like immunoreactivity. This suggests that FMRFamide-like peptides may be cotransmitters of these putatively GABAergic interneurons. All FMRFamide-immunoreactive neurons are, furthermore, immunoreactive with an antiserum against bovine pancreatic polypeptide, and the vast majority is also immunoreactive with an antibody against the molluscan small cardioactive peptide SCP_B. Therefore, it is possible that more than one peptide is localized within many FMRFamide-immunoreactive neurons. The results suggest that FMRFamide-related peptides are widespread within the nervous system ofM. sexta and might function as neurohormones and neurotransmitters in a variety of neuronal cell types.Abbreviations AL antennal lobe - BPPLI bovine pancreatic polypeptide-like immunoreactivity - FLI FMRFamide-like immunoreactivity - GLI GABA-like immunoreactivity - NSC neurosecretory cell - SCP _B LI small cardioactive peptide_B-like immunoreactivity - SLI serotonin-like immunoreactivity - SOG suboesophageal ganglion     

Distribution of neuropeptide Y-like immunoreactivity in the brain and hypophysis of the cloudy dogfish,Scyliorhinus torazame

Summary Using a specific antiserum raised against synthetic neuropeptide Y, we examined the localization of immunoreactivity in the brain and hypophysis of the cloudy dogfish, Scyliorhinus torazame, by the peroxidase-antiperoxidase method. Immunoreactive perikarya were demonstrated in the ganglion of the nervus terminalis, the dorsocaudal portions of the pallium dorsale, the basal telencephalon, and the nucleus lateralis tuberis and the nucleus lobi lateralis in the hypothalamus. Labeled perikarya were also found in the tegmentum mesencephali, the corpus cerebelli, and the medulla oblongata. Some of the immunoreactive neurons in the hypothalamus were of the CSF-contacting type. The bulk of the labeled fibers in the nervus terminalis ran toward the basal telencephalon, showing radial projections and ramifications. Large numbers of these fibers coursed into the nucleus septi caudoventralis and the nucleus interstitialis commissurae anterioris, where they became varicose and occasionally formed fine networks or invested immunonegative perikarya. In the diencephalon, immunoreactive fibers were observed throughout the hypothalamus, e.g., in the pars neurointermedia of the hypophysis, the subependymal layer of the lobus inferior hypothalami, and in the neuropil of the posterior (mammillary) recess organ. Labeled fibers were scattered throughout the rest of the brain stem and were also seen in the granular layer of the cerebellum. These results suggest that, in the dogfish brain, neuropeptide Y or a related substance is involved in a variety of physiological processes in the brain, including the neuroendocrine control of the hypophysis.     

Mortality estimates of Scyliorhinus canicula in the Cantabrian sea using tag-recapture data

Survival and recovery rates were obtained for small‐spotted catshark Scyliorhinus canicula in the Cantabrian Sea (north of Spain) based on tagging data. A total of 7656 specimens were tagged from 1993 to 2001. Recovery models for multiyear tagging studies were applied using the programme Mark. The best model fitted was achieved considering constant survival and recovery rates following the Brownie parameterization. The outputs were a survival rate ( S ) 0·89 and a recovery rate ( f ) 0·006. The reduced parameterization ( S and r ) provided very similar results, 0·89 and 0·06 respectively. The total finite mortality estimated was therefore Z  = 0·11.     

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.     

On the diel rhythms in metabolism and activity of post-hatching lesser spotted dogfish, Scyliorhinus canicula

The diel rhythms in metabolic rate ( M_R ) and activity level ( A_L ) were measured for single post-hatching dogfish (weight range, 2.76–10.61 g) at 15° C by the indirect calorimetric method of rate of oxygen consumption ( V O₂) and by video-observation respectively, over a period of 72 b. The mean VO ₂ increased from 62.0 (s.e. 2.9) mg O₂ kg⁻¹ h⁻¹ in the daylight hours to 85.5 (s.e. 3.1) mg O₂ kg⁻¹ h⁻¹ during the dark (light regíme, 12 h L: 12 h D). The simultaneous measurement of A _L also showed mean night elevation from 0.6 (s.e. 0.2) min h⁻¹ in the light phase to 14.5 (s.e. 1.6) min h⁻¹ during the darkness. Bimodal nocturnal activity (BNA) was exhibited by the post-hatching dogfish within the 12 h dark period, with V O₂ increasing from 71.4 (s.e. 2.8) mg O₂ kg⁻¹ h⁻¹ before 01.00 hours to 99.5 (s.e. 4.2) mg O₂ kg⁻¹ h⁻¹ after 01.00 hours. Similarly, A _L also increased from 8.9 (s.e. I.7)min h⁻¹ before 01.00 hours to 21.1 (s.e. 2.8) min h⁻¹ after 01.00 hours. The importance of the results presented to the natural behavioural ecology of the hatching dogfish are discussed.     

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.).     

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.     

Immunocytochemical localization of vasotocin-like immunoreactivity in the brain of the cartilaginous fish,Scyliorhinus caniculus

Summary The distribution of vasotocin-like peptides in the central nervous system of the cartilaginous fish Scyliorhinus canicula was determined by indirect immunofluorescence and peroxidase anti-peroxidase techniques, using a specific antiserum raised in rabbits against synthetic vasotocin. Immunoreactive perikarya were mainly detected in the anterior hypothalamus, within the midcaudal part of the preoptic nucleus. The most rostral positive cell bodies were located in the dorso-lateral parts of the preoptic area, whereas at a more caudal level, they took a ventro-medial position within the deepest layers of the nucleus. Throughout the preoptic region these cells varied in shape according to their location. Occasionally, scattered vasotocin-like immunopositive cells were also identified in the nucleus periventricularis hypothalami. Vasotocin immunoreactivity was detected in numerous varicose nerve fibers of the preopticohypophysial tract. These fibers were seen to course through the medio-basal hypothalamus and caudally, after having passed the hypophysial stem, they reached the neurointermediate lobe of the pituitary. Numerous immunoreactive fibers were also observed within the rostro-medial region of the median eminence. At this level the fibers were in close proximity to the capillary loops. In the preoptic region, some stained cells exibited short processes that appeared to contact non-reactive perikarya. By comparing the distribution of vasotocin- and corticotropin-releasing factor immunoreactivity on adjacent then serial sections, it was revealed that these peptides, in S. canicula, do not coexist in the same perikarya. The present results, are compared with those obtained in other vertebrate groups, and their possible functional implications are discussed.     

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.     

Gastric emptying in Scyliorhinus canicula (L.): a comparison of surface-dependent and non-surface dependent models

The usefulness of both surface-dependent and non-surface-dependent models for describing the gastric emptying process in Scyliorhinus canicula (L.) was examined. In most cases the surface-dependent models produced better fits than did the non-surface-dependent models. The exponential model (non-surface-dependent) only adequately described gastric evacuation in the case of small prey items with thin exoskeletons. On the other hand, the exponential model was clearly better than the linear model in all cases. Prey size had little effect on digestion rate. However, the number of prey items ingested was an important factor influencing digestion.