Immune-complex trapping in the splenic ellipsoids of rainbow trout (Oncorhynchus mykiss)

Rainbow trout (Oncorhynchus mykiss), immunised with horseradish peroxidase, were given horseradish peroxidase intravenously, and the trapping of antigen in the spleen was followed 1, 24, and 48 h after injection. After 1 h, the localisation of horseradish peroxidase indicated that the antigen had been extensively trapped in the walls of the splenic ellipsoids. The colocalisation of horseradish peroxidase with rainbow trout immunoglobulin M and complement factor 3 was shown with a double immunofluorescence technique and suggested that horseradish peroxidase was trapped in the form of immune complexes. After 24 and 48 h, very little horseradish peroxidase was detected in the ellipsoids, and horseradish peroxidase was mainly found in association with large cells with prominent cytoplasmic extensions. In nonimmunised fish given horseradish peroxidase intravenously, antigen was not detected in ellipsoids. Thus, the observed difference between immunised and nonimmunised trout suggests a specific role for the splenic ellipsoids in rapid immune-complex trapping and invites speculation on its significance in a secondary immune response.     

Interstitial cells from the testis of the trout (Oncorhynchus mykiss) in vivo and in primary culture

Summary In the testis of the trout, while no changes are apparent in myoid cells at any stage of maturation, Leydig cells display striking structural alterations when observed at different periods of the reproductive cycle. Spermiating testes contain fully differentiated Leydig cells. In regressed testes and those involved in spermatogenesis, poorly differentiated Leydig cells are mixed with cells ranging structurally from normal Leydig cells to fibroblast-like elements. After 3–4 days in culture the myoid cells/fibroblasts progressively acquire the ability to proliferate and then show a positive reaction for 3-hydroxysteroid dehydrogenase. During the same period they undergo structural changes reflecting the emergence of a steroidogenic activity. These changes occur concomitantly with an increase in progestagen secretion. These data suggest that, in vivo, Leydig cells degenerate at the end of a cycle, being then replaced by fibroblastic precursor cells capable of division and differentiation into steroidogenic cells.     

Co-localization of peptides in the Brockmann bodies of the cod (Gadus morhua) and the rainbow trout (Oncorhynchus mykiss)

Endocrine cells exhibiting immunoreactivity to FMRFamide-like, LPLRFamide-like, neuropeptide Y(NPY)-like and peptide YY(PYY)-like peptides were found in the periphery of the Brockmann bodies of the cod, Gadus morhua, and rainbow trout, Oncorhynchus mykiss. No immunoreactivity or very weak labelling was found with antisera to pancreatic polypeptide (PP). Vasoactive intestinal polypeptide (VIP)-like immunoreactivity was found in nerve fibres, whereas labelling with VIP antiserum in endocrine cells disappeared after preincubation with nonimmune serum. There were always more immunoreactive cells in the rainbow trout than in the cod. No immunoreactivity could be seen with antisera to gastrin/cholecystokinin (CCK) or enkephalin. Double-labelling studies were performed to study the colocalization of the peptides in peripheral endocrine cells. Cells immunoreactive to NPY were also labelled with antisera to FMRFamide, LPLRFamide and PYY. The co-localization pattern of NPY varied; in some Brockmann bodies, a population of the immunoreactive cells showed co-localization and others contained NPY-like immunoreactivity only, whereas in other Brockmann bodies, all NPY-labelled cells also contained FMRFamide-like, LPLRFamide-like and PYY-like immunoreactivity. Cells immunoreactive to PYY similarly contained FMRFamide-like, LPLRFamide-like and NPY-like immunoreactivity, comparable to the patterns observed with NPY. Glucagon-like immunoreactivity was found at the periphery of the Brockmann bodies. A subpopulation of the glucagon-containing cells contained NPY-like immunoreactivity. PYY-like immunoreactivity was also found co-localized with glucagon-like immunoreactivity, as were FMRFamide-like and LPLRFamide-like immunoreactivity. Therefore, either NPY-like and PYY-like immunoreactivity together with FMRFamide-like and LPLRFamide-like immunoreactivity occur in the same endocrine cells of the Brockmann body of the cod and rainbow trout, or a hybrid NPY/PYY-like peptide recognized by both NPY and PYY antisera is present in the Brockmann body.     

Degranulation of eosinophilic granule cells induced by capsaicin and substance P in the intestine of the rainbow trout (Oncorhynchus mykiss Walbaum)

Summary Adult rainbow trout (Oncorhynchus mykiss) were injected intraperitoneally with capsaicin, substance P, serotonin, or a control of saline vehicle or bovine serum albumin (0.5 g/g body weight). Fish were sacrificed 30 min and 1,2 and 4 h post-injection, the gut was dissected out, and a small section of the upper intestine was processed for electron microscopy. A significant proportion of eosinophilic granule cells (EGCs) of the intestine were in close association with non-myelinated neuronal bundles in all fish (4 fish per treatment and time period), but there was no significant difference between treatment or time, suggesting that the association was unaffected by these factors. Close examination of EGC ultrastructure showed that fish treated with capsaicin and substance P exhibited limited degranulation of the EGCs in the stratum compactum and extensive crinophagic-like degranulation in the lamina propria. Cells of the lamina propria contained characteristic multivesicular-like bodies. The degranulation was reminiscent of both mast cell degranulation and endocrine cell crinophagy. EGCs of fish treated with serotonin or a control were unaffected, suggesting that the serotoninergic neurons, believed to be involved in gut motility, were not responsible for degranulation. It is apparent that EGCs of the trout intestine may be under nervous control, as has been demonstrated previously for mammalian mast cells.     

Effects of some pesticides on the vital organs of juvenile rainbow trout (Oncorhynchus mykiss)

Gill, trunk kidney, spleen, and liver of rainbow trout (Oncorhynchus mykiss) were examined after exposure to different sublethal concentrations of carbosulfan (25, 50 and 200 μg L⁻¹), propineb (3, 6 and 24 mg L⁻¹), and benomyl (2, 5 and 20 mg L⁻¹) for 14 days. Lesions were observed in gill, trunk kidney, spleen, and liver of rainbow trout exposed to either concentration of pesticides. The most important lesions were determined in the highest concentrations of pesticides. Lamellar fusion, lamellar hyperplasia, epithelial lifting, vacuolization of epithelial tissue, epithelial necrosis, hypertrophy and sloughing of epithelium were observed on fish exposed to carbosulfan, propineb and benomyl. Fish had cell necrosis, degeneration and oedemas in liver, trunk kidney and spleen. None of these lesions were seen in control fish.     

The mucous coat on gill lamellae of rainbow trout (Oncorhynchus mykiss)

The existence of a layer of mucus covering the gill lamellae of healthy rainbow trout (Oncorhynchus mykiss) was investigated. Using cryo-scanning electron microscopy, a smooth, undulating, thin layer was observed which completely covered gill filaments and lamellae, thereby obscuring epithelial microridges. After processing cryopreserved gill arches in glutaraldehyde for conventional scanning electron microscopy, the layer was no longer present and epithelial microridges were clearly visible. The identity of this layer was investigated using cryopreserved gills which were treated in one of two ways. First, gills were incubated with a rabbit antiserum to gill mucus, with normal rabbit serum, or with phosphate-buffered saline. Following fixation in glutaraldehyde and processing, only the gill tissue incubated with the mucus-specific antiserum was still covered with the smooth layer. The layer was also retained on the gills of fish anesthetized in a solution containing mucusspecific antiserum and then processes in glutaraldehyde for conventional scanning electron microscopy. The tenacious nature of the mucous layer was demonstrated by its stability following exposure to formalin and a cationic detergent. Second, the presence of this layer was confirmed on gill tissue which was cryopreserved, followed by freeze-substitution and vapor fixation, and then examined by transmission electron microscopy.     

Calcium signalling in isolated single chromaffin cells of the rainbow trout (Oncorhynchus mykiss)

Chromaffin cells were isolated from the posterior cardinal vein of rainbow trout (Oncorhynchus mykiss) to assess their suitability as a model system for studying mechanisms of catecholamine secretion in fish and to evaluate intracellular calcium changes associated with cholinoreceptor stimulation. Immunocytochemistry in concert with fluorescence microscopy was employed to identify characteristic chromaffin cell proteins and thus to confirm the presence of these specific cells in suspensions and cultures. Dopamine-β-hydroxylase, an enzyme of the catecholamine-synthesising Blaschko pathway, was identified in cytoplasmic vesicles of the isolated chromaffin cells. The actin filament-severing protein, scinderin, was co-localized with actin in the sub-plasmalemmal membrane of these chromaffin cells. Intracellular calcium [Ca²⁺]_i was measured in single chromaffin cells by microspectrofluorometry using the fluorescent dye Fura-2. Significant increases in [Ca²⁺]_i were observed in chromaffin cells in response to depolarisation of the cell membrane by high concentrations of K⁺ or by the stimulation of the cell by the cholinergic receptor agonists, nicotine, acetylcholine or carbachol. The response to the reversible agonist, nicotine, was attenuated following addition of the nicotinic receptor blocker hexamethonium. Such attenuation, however, did not occur when hexamethonium was added after stimulation with the non-specific irreversible cholinergic agonist, carbachol. These results demonstrate the presence of functional cholinoreceptors, linked to intracellular calcium signalling, on isolated trout chromaffin cells and reveal the potential of these cells as a model system for studying aspects of catecholamine secretion in fish.     

Innate immune responses in rainbow trout (Oncorhynchus mykiss, Walbaum) induced by probiotics

Carnobacterium maltaromaticum B26 and Carnobacterium divergens B33, which were isolated from the intestine of healthy rainbow trout (Oncorhynchus mykiss, Walbaum), were selected as being potentially useful as probiotics with effectiveness against Aeromonas salmonicida and Yersinia ruckeri. Thus, rainbow trout administered with feed supplemented with B26 or B33 dosed at >10(7) cells g(-1) feed conferred protection against challenge with virulent cultures of the pathogens. Moreover, both cultures persisted in the gut for up to 3 weeks after administration. The cultures enhanced the cellular and humoral immune responses. Specifically, fish fed with B26 demonstrated significantly increased phagocytic activity of the head kidney macrophages, whereas the use of B33 led to significant increases in respiratory burst and serum lysozyme activity. Also, the gut mucosal lysozyme activity for fish fed with both cultures was statistically higher than the controls.     

The energetic cost of protein synthesis in isolated hepatocytes of rainbow trout (Oncorhynchus mykiss)

Summary To establish the energetic cost of protein synthesis, isolated trout hepatocytes were used to measure protein synthesis and respiration simultaneously at a variety of temperatures. The presence of bovine serum albumin was essential for the viability of isolated hepatocytes during isolation, but, in order to measure protein synthesis rates, oxygen consumption rates and RNA-to-protein ratios, BSA had to be washed from the cells. Isolated hepatocytes were found to be capable of protein synthesis and oxygen consumption at constant rates over a wide range of oxygen tension. Cycloheximide was used to inhibit protein synthesis. Isolated hepatocytes used on average 79.7±9.5% of their total oxygen consumption on cycloheximide-sensitive protein synthesis and 2.8±2.8% on maintaining ouabain-sensitive Na⁺/K⁺-ATPase activity. The energetic cost of protein synthesis in terms of moles of adenosine triphosphate per gram of protein synthesis decreased with increasing rates of protein synthesis at higher temperatures. It is suggested that the energetic cost consists of a fixed (independent of synthesis rate) and a variable component (dependent on synthesis rate).Abbreviations BSA bovine serum albumin - dpm disintegrations per min - k _s fractional rate of protein synthesis - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulphonic acid - PHE phenylalanine; PO₂ oxygen tension - PCA perchloric acid     

Differential responses to copper in rainbow trout (Oncorhynchus mykiss) acclimated to sea water and brackish water

Rainbow trout, Oncorhynchus mykiss, acclimated to 33% sea water (12 mg·ml^-1 salinity) experienced significant (10 meq·1^-1) increases in plasma [Na⁺] and [Cl^-] within 5 h of exposure to 6.3 mol copper·1^-1 indicating severe impairment of branchial ionoregulatory capacity. All plasma ion levels subsequently stabilised once the transbranchial [Na⁺] gradient was reduced to zero. The similar ionic strength of the external medium and their body fluids appeared to protect trout maintained in 33% sea water from further ionoregulatory stress and any secondary physiological disturbances during exposure to copper. Despite three- and fourfold greater transbranchial [Na⁺] and [Cl^-] gradients, trout acclimated to full-strength sea water (35 mg·ml^-1 salinity) suffered no major changes in plasma Na⁺, Cl^-, K⁺, or Ca²⁺, blood gases or haematology during 24 h exposure to 6.3 mol copper·1^-1. This reduction in toxicity in full strength sea water cannot be explained by differences in copper speciation. We suggest that during acute exposure to waterborne copper, active NaCl extrusion is unaffected due to the basolateral location of the gill Na⁺/K⁺-ATPase, but that ionoregulatory disturbances can occur due to gill permeability changes secondary to the displacement of surface-bound Ca²⁺. However, in full strength sea water the three-fold higher ambient [Ca²⁺] and [Mg²⁺] appear to be sufficient to prevent any detrimental permeability changes in the presence of 6.3 mol copper·1^-1. Plasma [NH ₊ ⁴ ] and [HCO _- ³ ] were both significantly elevated during exposure to copper, indicating that some aspects of gill ion transport (specifically the apical Na⁺/NH ₊ ⁴ and Cl^-/HCO _- ³ exchanges involved in acid/base regulation and nitrogenous waste excretion) are vulnerable to inhibition in the presence of waterborne copper.Abbreviations C _aO₂ arterial oxygen content - Hb haemoglobin - Hct haematocrit - MABP mean arterial blood pressure - MCHC mean cell haemoglobin content - MO₂ rate of oxygen consumption - P _a CO₂ arterial carbon dioxide tension - P _aO₂ arterial oxygen partial pressure - S salinity - SW sea water - T _Amm total ammonia (=NH₃+NH ₊ ⁴ ) - T _{CO 2} total carbon dioxide - TEP transepithelial potential - TOC total organic carbon - %Hb-O₂ percentage of haemoglobin saturated with oxygen     

Cellular responses of the skin and changes in plasma cortisol levels of trout (Oncorhynchus mykiss) exposed to acidified water

The skin structure and the plasma cortisol levels of trout, Oncorhynchus mykiss, were examined during 7 days of exposure to water of pH 5. By day-4 and-7, the thickness of the epidermis was significantly (P<0.05) less in acid exposed fish than in controls, and degenerative cells were common in the upper epidermal layers. Many epidermal cells exhibited signs of necrosis, and by day-7 many apoptotic cells were also present. Secretory vesicles of high electron density were abundant in the filament cells of the 3–4 outermost layers of epidermis, and intercellular spaces had increased. Mitotic figures occureed throughout the epidermis, with the exception of the outermost cell layer. Mucous cells became elongated after day-1, and later, newly differentiating mucous cells could be seen close to the skin surface, and many mucocytes contained mucosomes of high electron density. Rodlet cells were occasionally seen. Chloride cells appeared similar to those of control fish. Many leucocytes, mainly macrophages and lymphocytes, had penetrated the epidermis via the highly undulating basal lamina, and at day-7, numerous apoptotic lymphocytes were found. In the dermis, melanosomes became dispersed in the cytoplasmic extensions of melanocytes which were present in the epidermis of all acid-exposed fish. Iridocytes were rate after day-4, while fibroblasts were abundant and secreted large amounts of collagen. After 1 day of exposure to acidified water, a significant (P<0.05) elevation of the plasma cortisol level had occurred, but this subsequently declined, and had returned to control values by day-7. The changes in skin structure, however, remained throughout the whole exposure period.     

Growth hormone stimulates hepatic lipid mobilization in rainbow trout,Oncorhynchus mykiss

Rainbow trout were used to characterize the direct influence of growth hormone on hepatic lipid mobilization in vitro. Liver was removed from fish fasted 72 h, sliced into 1-mm³ pieces and incubated in Hanks' medium containing ovine or salmon growth hormone (0.28 ng·ml^-1–28 g·ml^-1). Lipid mobilization, resulting from triacylglycerol hydrolysis, was evaluated by fatty acid and glycerol release into culture medium. Control rates of fatty acid release and glycerol release were 0.95±0.16 (mean ± SE) and 0.88±0.28 mol·l^-1·mg fresh weight, respectively. Both ovine growth hormone (28 ng·ml^-1) and salmon growth hormone (28 ng·ml^-1) stimulated fatty acid release into culture medium, increasing rates by 112% and 97%, respectively, during the course of a 24-h incubation. Glycerol release was similarly augmented by growth hormone treatment. Growth hormone-stimulated metabolite release became evident within 12 h and persisted for up to 72 h. The presence of amino acids in the culture medium potentiated the lipolytic action of growth hormone. Ovine growth hormone (28 ng·ml^-1) in the presence of amino acids stimulated a 53% increase in fatty acid, and a 108% increase in glycerol, release over rates observed in the absence of amino acids. Salmon growth hormone (28 ng·ml^-1) in the presence of amino acids stimulated a 53% increase in fatty acid, and a 44% increase in glycerol, release over rates observed in the absence of amino acids. Ovine growth hormone (28 ng·ml^-1) also stimulated gluconeogenesis, as indicated by a 75% increase in phosphoenolpyruvate carboxykinase activity in liver pieces incubated in the presence of amino acids. These results indicate that growth hormone directly stimulates lipid breakdown in the liver of trout and that amino acids potentiate growth hormone-stimulated lipolysis.Abbreviations AA amino acid(s) - dGDP deoxy-guanosine diphosphate - ED ₅₀ 50% effective dose - FA fatty acid(s) - fw fesh weight - GH growth hormone - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid - MS-222 tricaine methanesulfonate - MEM minimum essential medium - oGH ovine growth hormone - PEPCK phosphoenolpyruvate carboxykinase - PK_c protein kinase C - rpm revolutions per minute - sGH salmon growth hormone - TG triacylglycerol - w/v weight per volume This paper was presented in abstract form at the Annual Meeting of the American Society of Zoologists, Dec. 26–30, 1991, Atlanta, GA     

Angiotensin II binding to tissues of the rainbow trout,Oncorhynchus mykiss,studied by autoradiography

Summary Tissue slices from seawater-adapted and freshwater-adapted rainbow trout, Oncorhynchus mykiss, were exposed to ¹²⁵I-angiotensin II (1.01·10^-9 M) and binding sites located by light-microscopic autoradiography. Binding/uptake was significantly inhibited by excess (10^-5 M) unlabelled angiotensin II, suggesting specific binding/uptake of angiotensin II to the ventral and dorsal aorta (smooth muscle), urinary bladder (smooth muscle and epithelial lining), glomeruli and proximal tubules, the gill (lamellae and central filament), skin (epithelium), intestine and oesophagus (mucosal epithelium), liver, heart (ventricular myocytes), adrenocortical tissue and brain (cerebellum and medulla oblongata). The specific binding/uptake of angiotensin II to tissues of freshwater- and seawater-adapted animals were generally similar. However, binding/uptake by the proximal tubules was significantly higher in freshwater-adapted trout than seawater-adapted trout. Specific binding/uptake of angiotensin II by the smooth muscle of the bladder was significantly higher in trout adapted to seawater than trout adapted to freshwater.     

Differential production and regulation of gonadotropins (GTH I and GTH II) in the pituitary gland of rainbow trout,Oncorhynchus mykiss,during ovarian development

Summary Biosynthesis of salmon gonadotropins, GTH I and GTH II, during ovarian development, were examined by means of in situ hybridization histochemistry and indirect immunocytochemistry. In rainbow trout pituitary glands, expression of GTH I- and II-subunit genes appeared separately in distinct cells (GTH I- and GTH II-cells), whereas the GTH -subunit gene was expressed in both cell-types. In the GTH I-cells, coordinated increases in GTh, and I messenger ribonucleic acids (mRNAs) occurred coincident with the onset of vitellogenesis, indicating active synthesis of GTH I during vitellogenesis. In contrast, in the GTH II-cells, both GTH -and II-mRNA signals markedly increased from a later stage of vitellogenesis and persisted throughout oocyte maturation and ovulation, supporting the idea that GTH II is actively synthesized as a maturational GTH. GTH -mRNA levels in the GTH I-cells selectively decreased prior to final oocyte maturation, although I-mRNA levels remained elevated, thus suggesting a decline of biosynthesis of GTH I after vitellogenesis. These findings clarify how the synthesis of GTH I and GTH II are coordinated in the piscine pituitary, and indicate that the expression of GTH subunit genes during gametogenesis is regulated differentially in a cell-specific manner, both temporally and spatially.     

Isolation and characterization of the receptor for vitellogenin from follicles of the rainbow trout,Oncorhynchus mykiss

The isolation and characterization of the receptor for vitellogenin from follicle membranes of the rainbow trout, Oncorhynchus mykiss, is described. Follicle membrane proteins subjected to SDS-polyacrylamide gel electrophoresis and subsequently to either protein staining or ligand blotting with radiolabelled vitellogenin (¹²⁵iodine-vitellogenin) demonstrated that the vitellogenin receptor has an apparent molecular mass of 200 kD (probably comprising of two 100-kD subunits) under non-reducing conditions. The vitellogenin binding sites were identified as specific receptors: binding was saturable and the binding sites were both tissue specific to follicle membranes and exhibited ligand specificity. Scatchard analyses of specific binding data revealed a single class of binding sites with a high affinity for rainbow trout vitellogenin (K _d=8.2·10^-9 mol·1^-1). Both brown trout, Salmo trutta, vitellogenin and carp, Cyprinus carpio, vitellogenin were able to displace the radiolabelled rainbow trout vitellogenin from its receptor, although they were less effective than rainbow trout vitellogenin.Abbreviations B _max maximum number of binding sites available - BSA bovine serum albumin - bt-VTG brown trout vitellogenin - c-VTG earp vitellogenin - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - K _d dissociatian constant - NCM nitrocellulose membranes - PMSF phenylmethylsulphonylfluoride - rt-VTG rainbow trout vitellogenin - VTG vitellogenin     

Modulation of catecholamine storage and release by the pituitary-interrenal axis in the rainbow trout,Oncorhynchus mykiss

This study examined the effects of pituitary-interrenal hormones on catecholamine storage and release in the rainbow trout Oncorhynchus mykiss. An extract of trout pituitary elicited the release of adrenaline, but not noradrenaline, using an in situ perfusion preparation. A variety of doses of adrenocorticotropic hormone (2–2000 mU) caused the release of both catecholamines in situ which was unaffected by pre-treatment with the ganglion blocker, hexamethonium, or the serotonergic receptor antagonist, methysergide, but was abolished in calcium-free media. Intra-arterial injections of adrenocorticotrophic hormone in vivo caused an elevation of plasma adrenaline but not noradrenaline levels. Injections of cortisol in situ did not elicit catecholamine release. Trout given an intraperitoneal implant of cortisol (50 mg·kg^-1 body weight) had significantly higher plasma cortisol concentrations when compared to controls after 7 days of implantation. Increases in the levels of stored catecholamines were observed in various regions of the kidney and posterior cardinal vein following 3 and 7 days of cortisol treatment. The ability of the chromaffin cells to release catecholamines in response to cholinergic stimulation was assessed in situ after 7 days of treatment. Basal (non-stimulated) adrenaline outflowing perfusate levels were greater in the cortisol-treated fish. Cortisol treatment increased the responsiveness of the catecholamine release process to low doses of the cholinoceptor agonist carbachol. Three or 7 days of cortisol treatment did not alter the in vitro activity of the enzyme phenylethanolamine-N-methyl-transferase. The results of this study demonstrate that interactions within the pituitary-adrenal axis can influence both catecholamine storage and release in the rainbow trout.Abbreviations ACTH adrenocorticotropic hormone - AK anterior third of the kidney - APCV anterior third of the PCV - HPLC high performance liquid chromatography - MK middle third of the kidney - M1 maximum value - MPCV middle third of the PCV - MS222 ethyl-aminobenzoate - P1 pre value - PCA perchloric acid - PCV posterior cardinal vein - PK posterior third of the kidney - PNMT phenylethanolamine-N-methyltransferase - PPCV posterior third of the PCV - rbc red blood cells - SEM standard error of the mean - TK total kidney (i.e. the sum of the AK, MK, and PK) - TPCV total PCV (i.e. the sum of the APCV, MPCV and PPCV)     

The effect of exogenous catecholamines on the ventilatory and cardiac responses of normoxic and hyperoxic rainbow trout,Oncorhynchus mykiss

Ventilation frequency, opercular pressure amplitude, heart rate, dorsal aortic pressure, arterial pH, arterial O₂ tension, and plasma catecholamine levels were recorded in rainbow trout, Oncorhynchus mykiss, during normoxia (19.7 kPa, 148 mmHg) or hyperoxia (51.2 kPa, 384 mmHg) after injection of various concentrations of catecholamines. In normoxic fish, adrenaline injection resulted in a depression of arterial O₂ tension, hypoventilation due to a drop in ventilation frequency, and a drop in heart rate, while dorsal aortic pressure increased. Noradrenaline depressed ventilation frequency, but opercular pressure amplitude increased to a far greater extent, and dorsal aortic pressure increased. During hyperoxia, adrenaline injection lowered ventilation frequency, opercular amplitude and heart rate, but dorsal aortic pressure increased. The stimulatory effects of noradrenaline on ventilation were abolished during hyperoxia, but the cardiac responses were similar to those seen during normoxia. These results indicate that catecholamines can modify the ventilatory output from the respiratory centre, and modification of ventilation frequency can occur independently of opercular pressure amplitude.Abbreviations f _g ventilation frequency - HPLC high performance liquid chromatography - P _op opercular pressure amplitude - f _h heart rate - P _DA dorsal aortic pressure - pH_a arterial pH - P _aO₂ arterial oxygen tension - PO₂ oxygen tension     

Localization and diurnal variations of carbonic anhydrase mRNA expression in the inner ear of the rainbow trout Oncorhynchus mykiss

Physiological studies have suggested that carbonic anhydrase (CA) plays a central role in otolith biomineralization via ion transport. However, the presence and exact function of CA in the inner ear have not been determined. In the present study, to investigate the localization of CA and its involvement in otolith calcification, we cloned two cDNAs encoding CAs from the rainbow trout sacculus. These two cDNAs, designated rainbow trout CAa (rtCAa) and rtCAb, both had an open reading frame encoding 260 amino acids with a sequence identity of 78%. Remarkably, rtCAb has a high degree of homology (82%) with “high activity CA” in the zebrafish, and its mRNA expression showed variation in the range 1.9–11.4 × 10⁴ copies/ng total RNA in the sacculus. In contrast, rtCAa mRNA was constantly expressed at approximately 3 × 10⁴ copies/ng total RNA. In situ hybridization revealed that rtCAb mRNA was strongly expressed in the distal squamous epithelial cells and transitional epithelial cells, except the mitochondria-rich cells, whereas, rtCAa was localized in extrasaccular tissue. These results suggest that the rtCAb isozyme is involved in the daily increment formation and calcification of otoliths via phase and spatial differences of the bicarbonate supply to the endolymph.     

Developmental sequence of bone-resorbing cells induced by intramuscular implantation of mineral-containing bone particles into rainbow trout,Oncorhynchus mykiss

Mineral-containing bone particles (BPs) were implanted intramuscularly into rainbow trout (Oncorhynchus mykiss) to investigate the sequence of appearance of bone-resorbing cells. A fibrous substance first surrounded the implanted BPs and was gradually replaced by connective tissue containing capillaries. Two weeks after BP implantation, relatively small multinucleated cells (type-1 cells), whose cytoplasm stained deeply with hematoxylin, appeared along the surfaces of the BPs. At later stages (after 4–8 weeks), the majority of cells which appeared to be resorbing the BPs were multinucleated cells whose cytoplasm stained deeply with eosin (type-2 cells). Type-2 cells contained more nuclei than type-1 cells. Electron-microscopical observations revealed that type-2 cells had the characteristic features of osteoclasts: the presence of numerous mitochondria, vacuoles and granules, and a differentiation of the cell membrane and cytoplasm into a ruffled border and clear zone, respectively. A tartrate-resistant acid phosphatase activity, which is an established characteristic of osteoclasts in terrestrial vertebrates, but which had not previously been examined in teleosts, was demonstrated histochemically in the type-2 cells. Development of type-2 cells was closely correlated with the development of connective tissue. These findings suggest that the development of a capillary network around the implanted BPs enables circulating osteoclast-progenitors to reach the surface of the BPs.