Acetylcholinesterase-containing nerve cells and their distribution in the pineal organ of the goldfish,Carassius auratus

Summary Histochemically, an intense acetylcholinesterase (AChE) reaction has been observed in the perikarya of the nerve cells and in the neuropil formations of the pineal organ in the goldfish, Carassius auratus. A group of AChE-rich nerve cells has also been observed between the caudal end of the pineal stalk and the habenular ganglion. No component of the complex revealed butyrylcholinesterase (BuChE) activity.Two different types of nerve cells were recognized on the basis of their size, AChE activity and distribution. Type I cells are characterized by large perikarya possessing a moderate AChE activity and by the presence of an extensive AChE-rich neuropil formation in their vicinity; they are restricted to the rostro-lateral regions of the pineal vesicle. Type II cells are situated in the medio-rostral area of the pineal vesicle and along the entire length of the stalk, and are smaller than Type I cells; they show an intense AChE activity in their perikarya.The neuropil formations in the medio-rostral area of the pineal vesicle are almost as large as those in the vicinity of the Type I cells; they exhibit a strong AChE activity. In the rostral half of the vesicle several sensory cells are associated with each nerve cell, while in the caudal portion only a few cells are apposed to each nerve cell. Thus, the ratio of the number of sensory cells to that of AChE-containing nerve cells in the anterior half of the pineal vesicle is high when compared with the remaining area. In the anterior half of the vesicle the outer segments of the sensory cells are more distinct and their inner segments possess a higher AChE activity than those in the posterior region and the stalk. A gradation in the degree of development of neuropil formations along the pineal axis is remarkable; their size and AChE activity gradually diminish in a caudal direction. In view of the structural specialization of the rostral region of the pineal organ, it has been argued that its terminal portion is more photosensitive.This work was supported by a fellowship from the Alexander von Humboldt Foundation, Federal Republic of Germany.     

Distribution of GABA-immunoreactive neurons in the forebrain of the goldfish,Carassius auratus

Summary The distribution of gamma-aminobutyric acid (GABA) immunoreactivity was studied in the forebrain (tel-and diencephalon) of the goldfish by means of immunocytochemistry on Vibratome sections using antibodies against GABA. Positive perikarya were detected in the olfactory bulbs and in all divisions of the telencephalon, the highest density being found along the midline. In the diencephalon, GABA-containing cell bodies were found in the hypothalamus, in particular in the preoptic and tuberal regions. The inferior lobes, the nucleus recessus lateralis, and more laterodorsal regions, such as the nucleus glomerulosus and surrounding structures, also exhibited numerous GABA-positive perikarya. Cell bodies were also noted in the thalamus, in particular in the dorsomedial, dorsolateral and ventromedial nuclei. The relative density of immunoreactive fibers was evaluated for each brain nucleus and classified into five categories. This ubiquitous distribution indicates that, as in higher vertebrates, GABA most probably represents one of the major neurotransmitters in the brain of teleosts.     

Effects of salmon calcitonin and anti-salmon calcitonin antibody on plasma calcium concentrations in the goldfish,Carassius auratus

The role of calcitonin (CT) in plasma calcium regulation was studied by the administration of exogenous CT and anti-salmon(s) CT antibody using goldfish,Carasius auratus, loaded or otherwise with calcium. CT elicited a decrease in plasma calcium concentrations at a dose of 10 ng/g body weight 1 h after administration. However, no effects were observed following doses of 30 ng and 50 ng/g 1 h, nor for the three doses 3 h after administration. In calcium-loaded fish, the effect of CT was different depending on the dosage of CT. Ten ng and 50 ng/g induced a decrease and an increase in plasma calcium concentrations, respectively, 3 h after administration. Anti-sCT antibody (0.02 μg or 0.1 μg/g) did not affect plasma calcium concentrations. In calcium-loaded fish, neither dose of anti-sCT antibody changed plasma calcium concentrations 1 h after administration. However, following a dose of 0.1 μg/g, plasma calcium concentrations decreased after 3 h. A positive correlation between plasma calcium concentrations and the gonad somatic index (GSI) in females was no longer apparent after administration of anti-sCT antibody. There was no relationship between plasma calcium concentrations and GSI in control and anti-sCT antibody-treated males. These results suggested that CT regulates plasma calcium concentrations in different ways depending on the dosage with CT having a role in calcium physiology during vitellogenesis.     

The 65-kDa cytosolic protein associated with warm temperature acclimation in goldfish,Carassius auratus

Goldfish Carassius auratus were acclimated to either 10 or 30°C for a minimum of 5 weeks. A 65-kDa protein specific to warm-temperature-acclimated fish was extracted from the gel with 70% formic acid after two-dimensional electrophoresis of the muscle cytoplasmic protein fraction. The 65-kDa protein thus prepared to homogeneity was used to raise specific antibodies in rabbit by conventional methods. The antibody produced exhibited specific reaction with a protein having the same molecular weight from brain and liver tissue, suggesting that the 65-kDa protein is a ubiquitous cytosolic component in warm-acclimated goldfish. When water temperature was increased from 20 to 30°C over a 20-h period, a prominent amount of the 65-kDa protein was observed in muscle tissue extracts within 5 days of additional rearing; this was demonstrated by immunoblotting with the specific antibody. The N-terminal amino acid sequence of the 65-kDa protein was determined as Asp-Glu-Pro-Gln-Gly-His-Gln-His (or Asp)-Glu-Leu, differing from that of a family of known heat-shock proteins having about 70 kDa in molecular mass (hsp 70). No interaction between ATP and the 65-kDa protein revealed by ATP-agarose affinity chromatography further confirmed the different properties of the 65-kDa protein from those of hsp 70.Abbreviations ATP adenosine 5-triphosphate - hsp heat-shock protein(s) - IgG immunoglobulin G - mRNA messenger ribonucleic acid - PMSF phenylmethylsulphonyl fluoride - PVDF polyvinylidene difluoride - SDS sodium dodecyl sulphate - SDS-PAGE SDS-polyacrylamide gel electrophoresis     

Synapse-like contacts between axons of the pineal tract and the subcommissural organ in Rana perezi (Anra) and their absence in Carassius auratus (Teleostei): ultrastructural tracer studies

The present study was designed to investigate the controversial subject of the existence of a neural input from the pineal organ via the pineal tract to the subcommissural organ (SCO) in teleosts and anurans. Horseradish peroxidase was injected into the pineal organ and pineal tract of Carassius auratus and Rana perezi. Within the pinealofugal fibers the tracer was visualized at the light-and electron-microscopic levels either by immunocytochemistry using an anti-peroxidase serum, or by revealing the enzymatic activity of peroxidase. In both species, labeled myelinated and unmyelinated fibers of the pineal tract were readily traced by means of electron microscopy. In R. perezi, numerous terminals contacting the SCO cells in a synapse-like (synaptoid, hemisynaptic) manner bore the label, whereas a different population of endings was devoid of the tracer, indicating that in this species the SCO receives a dual neural input, one of pineal origin, the other of unknown source and nature. In the SCO of C. auratus, neither labeled nor unlabeled synapse-like contacts were found. Thus, in this latter species, a direct neural input to the SCO is missing. It is concluded that the secretory activity of the SCO can be controlled by different mechanisms in different species, and that more than one neural input mechanism may operate in the same species.     

The effects of dopamine on temperature regulation in goldfish

Summary Microinjections of dopamine (DA) were made into specific forebrain loci in goldfish (Carassius auratus: 40–85 g) to study the involvement of DA in behavioral thermoregulation. Injections of 25, 50, 100 and 250 ng DA into the anterior aspect of the nucleus preopticus periventricularis (NPP) led to consistent, dose-dependent decreases in selected temperature was observed following injections of 5 or 10 ng DA. Injections of the control solution were without effect.Injections of DA into other forebrain loci, including the posterior half of the NPP, either had no thermoregulatory effect or had minor thermoregulatory effects which, in comparison to injections into the most effective sites, were inconsistent and required larger doses to obtain. The decrease in selected temperature following injections of 100 ng DA into the anterior NPP was blocked by haloperidol, a dopaminergic antagonist, but not by phentolamine, a noradrenergic antagonist. Injections of haloperidol alone resulted in a minor, but statistically significant, increase in selected temperature.The most sensitive DA sites lie caudal to the sites most sensitive to norepinephrine within the anterior NPP. DA acts on the dopaminergic receptors of central thermoregulatory neurons in the anterior NPP of goldfish. These receptors appear to mediate behavioral responses to excessively warm environments.Abbreviations DA dopamine - NE norepinephrine - NPP nucleus preopticus periventricularis - PBS phosphate buffer solution     

Tyrosinase positive oculocutaneous albinism in the goldfish,Carassius auratus L., an ultrastructural and biochemical study of the eye

Summary Ultrastructural studies, and cytochemical and biochemical determinations of tyrosinase activity were conducted on the pigment epithelium of albino and xanthic goldfish eyes. In eyes of xanthic goldfish, two types of melanosomes are present, spherical and elongated. Melanized melanosomes are absent in the eyes of the albino goldfish, but elongated lamellar premelanosomes are observed. Internal vesicles are present in both melanosome types in the pigment epithelium of the xanthic goldfish but are absent in premelanosomes of the albino. There are also differences in the distribution of lipid droplets, smooth endoplasmic reticulum and Golgi complexes with the latter two being more abundant in the albino. Tyrosinase was not identified cytochemically; however, the enzyme was demonstrated biochemically in the pigment epithelia of both albino and xanthic goldfish. The enzyme is associated with the particulate and soluble fractions of both types of eyes. Particulate albino tyrosinase may be solubilized by triton X-100 treatment. Tyrosinase inhibitors are present in the particulate fractions of both albino and xanthic goldfish eyes. Thus, in the goldfish, ocular albinism appears to be a multiple defect at the molecular and ultrastructural levels.Contribution Number 362, Department of Biology     

Gonadotropin production and release in female goldfish (Carassius auratus) after administration of pimozide and an LHRH analogue as studied by electron microscopy and radioimmunoassay

Summary The effect of pimozide and an LHRH-analogue (LHRH-A) on gonadotropic cells of the goldfish pituitary gland were described qualitatively and quantitatively. A scale of four categories was devised to reflect various ultrastructural appearances of the cells. Experimental animals were divided into a control group, a group injected with LHRH-A alone, pimozide alone, and groups receiving these two substances in combination. Fish injected with the single substance were killed 12 h after injection while the groups receiving the combined treatments were killed at 4, 12 and 48 h. Serum levels of gonadotropin measured by radioimmunoassay were used to indicate whether an increase in hormone release had occurred. An immunocytochemical technique, the protein A-gold procedure, assured that the cells studied were gonadotropes. The control group showed variation in the profiles of gonadotropic cells. The single treatment groups showed some increase in secretory inclusions. At 4 h after injection the combined treatment caused a significant increase in hormone granules; at 12 and 48 h there was a gradual decrease in content of secretory products, and an increase in vacuolization. The results indicate that the combined pimozide and LHRH-A treatment stimulated gonadotropin production as well as release.     

Intermediate filaments reminiscent of immature cells expressed by goldfish (Carassius auratus) astrocytes and oligodendrocytes in vitro

The expression of intermediate filaments is developmentally regulated. In the mammalian embryo keratins are the first to appear, followed by vimentin, while the principal intermediate filament of the adult brain is glial fibrillary acidic protein. The intermediate filaments expressed by a cell thus reflect its state of differentiation. The differentiation state of cells, and especially of glial cells, in turn determines their ability to support axonal growth. In this study we used three new antibodies directed against three fish intermediate filaments (glial fibrillary acidic protein, keratin 8 and vimentin), in order to determine the identity and level of expression of intermediate filaments present in fish glial cells in culture. We found that fish astrocytes and oligodendrocytes are both able to express keratin 8 and vimentin. We further demonstrate that under proliferative conditions astrocytes express high keratin 8 levels and most oligodendrocytes also express keratin 8, whereas under nonproliferative conditions the astrocytes express only low keratin 8 levels and most oligodendrocytes do not express keratin 8 at all. These results suggest that the fish glial cells retain characteristics of immature cells. The findings are also discussed in relation to the fish glial lineage.     

A reinvestigation of the Gn-RH (gonadotrophin-releasing hormone) systems in the goldfish brain using antibodies to salmon Gn-RH

Summary The organization of Gn-RH systems in the brain of teleosts has been investigated previously by immunohistochemistry using antibodies against the mammalian decapeptide which differs from the teleostean factor. Here, we report the distribution of immunoreactive Gn-RH in the brain of goldfish using antibodies against synthetic teleost peptide.Immunoreactive structures are found along a column extending from the rostral olfactory bulbs to the pituitary stalk. Cell bodies are observed within the olfactory nerves and bulbs, along the ventromedial telencephalon, the ventrolateral preoptic area and the latero-basal hypothalamus. Large perikarya are detected in the dorsal midbrain tegmentum, immediately caudal to the posterior commissure. A prominent pathway was traced from the cells located in the olfactory nerves through the medial olfactory tract and along all the perikarya described above to the pituitary stalk. In the pituitary, projections are restricted to the proximal pars distalis. A second immunoreactive pathway ascends more dorsally in the telencephalon and arches to the periventricular regions of the diencephalon. Part of this pathway forms a periventricular network in the dorsal and posterior hypothalamus, whereas other projections continue caudally to the medulla oblongata and the spinal cord. Lesions of the ventral preoptic area demonstrate that most of the fibers detected in the pituitary originate from the preoptic region.     

The outer segments of photoreceptive pinealocytes in the pineal organ of the funa,Carassius gibelio langsdorfi

Summary The form and size of the outer segments of photoreceptive pinealocytes in the pineal organ of the funa, Carassius gibelio langsdorfi, were observed with the scanning electron microscope. The height of the outer segments measures between 1 and 3 m and the diameter varies widely from 1.5 to 8 m. Various forms of outer segments, i.e. a slender type, a dome-like type, a cap-like type and a helical type, were demonstrated. The parallel-oriented filamentous processes of the inner segments have the same length as the outer segments and a diameter of approximately 100 nm; they are projections from the apical border of the inner segment and surround the cone-like outer segments. The processes make a right angle with the lamellar disks. The distance between two processes averages 100 nm. The lamellar disks of the outer segments are oriented at right angles to the modified cilium in the basal part, but the angle often changes in the peripheral part, where the lamellar disks are raised and become parallel to the cilium.Supported by a fellowship from the Japan Society for the Promotion of Science and a grant from the Deutsche Forschungsgemeinschaft to M. UeckSupported by a grant from the Ministry of Education of Japan to K.Wake     

Studies on resistance characteristic and cDNA sequence conservation of transferrin from crucian carp, Carassius auratus

Transferrin (Tf) is a kind of non-heme β-globulin with two iron ions (Fe³⁺)-binding sites. To prove Tf’s physiological functions, Fe³⁺-proteins, serum iron contents, and total iron-binding capabilities were tested for Tfs of crucian carps (Carassius auratus) and sliver carps (Hypophthalmichthys molitrix). The above results demonstrated that sliver carps shared 1/3 Tf alleles with crucian carps; Tf of crucian carps had stronger Fe³⁺-binding ability and transportation ability in plasma than that of sliver carps. In addition, the results of oxygen consumption experiments indicated that crucian carps had the higher oxygen utility rate than sliver carps. For acute hypoxia exposure assay, normoxic gas mixture, hypoxic gas mixture A, and hypoxic gas mixture B were used to induce oxygen-regulated gene expression of crucian carps in acute hypoxia. The results of quantitative real-time PCR revealed that mRNA levels of Tf gene, Tfr gene and ATPase gene were down-regulated in acute hypoxia but mRNA level of LDHa gene was up-regulated in acute hypoxia. The results of crucian carp Tf-cDNA sequence analysis showed that cDNA regions of two Fe³⁺-binding sites were T₇₄₇–T₁₀₂₆ and T₁₇₃₇–A₁₈₈₄ based on the principle of bioinformatics. The sequence conservation of two Fe³⁺-binding sites was higher than that of the other five regions, which were confirmed according to the subregion model of Tf-cDNA sequence.     

Neuropeptides in the intestine of two teleost species (Oreochromis mossambicus,Carassius auratus): Localization and electrophysiological effects on the epithelium

Summary The presence of bioactive peptides in the gut and their possible electrophysiological effects on the intestinal epithelium were studied in two teleost species, the tilapia (Oreochromis mossambicus) and the goldfish (Carassius auratus). Vasoactive intestinal polypeptide-like immunoreactive nerve fibres were found beneath the intestinal epithelium of both species. Galanin-, metenkephalin-and calcitonin gene-related peptide-like immunoreactive nerve fibres were found exclusively in the mucosa of the tilapia. Both species had vasoactive intestinal polypeptide-, enkephalin- or neuropeptide Y-like immunoreactive endocrine cells; calcitonin gene-related peptide-like immunoreactive endocrine cells were additionally found in the tilapia. Somatostatin- and dopamine--hydroxylase-like immunoreactivities were not observed. Nerve cell bodies in the myenteric plexus of both species showed immunoreactivity for calcitonin gene-related peptide-, vasoactive intestinal polypeptide-, and galanin-like peptide. Enkephalin-like immunoreactive nerve cell bodies were present in the tilapia only. None of the peptides had a pronounced electrogenic effect. However, calcitonin gene-related peptide added to stripped intestinal epithelium of the tilapia, reduced the ion selectivity, and addition of galanin increased the ion selectivity. In goldfish intestine, both galanin and calcitonin gene-related peptide were without effect. Enkephalin counteracted the serotonin-induced reduction of the ion selectivity of the goldfish intestinal epithelium, but had no effect on the tilapia epithelium. In both species, vasoactive intestinal polypeptide reduced the ion selectivity of the intestinal epithelium, and neuropeptide Y induced an increase of the ion selectivity. Somatostatin showed no effect on the epithelial ion selectivity of either species. Tetrodotoxin did not inhibit the effects of the peptides studied. The changes in ion selectivity suggest that the enterocytes may be under the regulatory control of these peptides.     

The fine structure of the fin musculature in two teleost species with different swimming modes,the puffer,Tetraodon steindachneri,and the goldfish,Carassius auratus

Summary Puffer fish (Tetraodon steindachneri) can execute precise maneuvers due to their highly specialized mode of propulsion. In the conventional locomotion exemplified by the goldfish (Carassius auratus), the fish thrusts are generated by lateral beating of the caudal fin. In contrast, the puffer generates its propulsive force by very rapid undulating movements of the pectoral, dorsal and anal fins. The fine structure of the fin muscles is identical in the two species of fishes, despite the differences in fin movement; cytologically, the fibers are intermediate between those of red and of white muscle. On the other hand, both the fusion frequency and the number of motor endplates are considerably higher in the fin muscles of the puffer than in those of the goldfish.     

Distribution and characterization of neuropeptide Y-like immunoreactivity in the brain and pituitary of the goldfish

Summary The distribution of neuropeptide Y (NPY) immunoreactivity has been studied by means of immunocytochemistry and radioimmunoassay in the brain of the goldfish. It was found that NPY had a widespread distribution in the entire brain in particular in the telencephalon, diencephalon, optic tectum and rhombencephalon. In the pituitary gland, positive type-B fibers were observed in the various lobes frequently in direct contact with secretory cells, in particular the gonadotrophs, somatotrophs and MSH (melanocyte-stimulating hormone) secreting cells. When measured by radioimmunoassay, the highest NPY concentrations were found in the pituitary and telencephalon, confirming the results of immunocytochemistry. The displacement curves obtained with serial dilutions of brain extracts were parallel to that of synthetic porcine NPY. Following high performance liquid chromatography, the NPY-like material extracted from goldfish brain co-eluted as a single peak with synthetic porcine NPY. These data demonstrate the presence of an NPY-like substance widely distributed in the goldfish brain. The observation of NPY-immunoreactive fibers in the pituitary gland suggests that, among its other functions, NPY may play a role in the neuroendocrine regulation of pituitary function.     

Neural connections between the brain and the pineal gland of the golden hamster (Mesocricetus auratus)

Summary Neurons projecting from the brain to the pineal gland via the pineal stalk were investigated in the golden hamster with the use of the retrograde horseradish-peroxidase tracing method both in vivo and in vitro. Labelled perikarya were observed in the medial and lateral habenular nuclei as well as in the posterior commissure. Single cells located in the ependymal lining of the pineal- and suprapineal recesses were also retrogradely labelled. These results show that a distinct central innervation of the pineal gland exists in the golden hamster, in agreement with findings in other mammalian species investigated by means of a similar methodology. In addition, also direct signals from the cerebrospinal fluid to the parenchyma might be conducted via cells located within the ependymal layer of the pineal- and suprapineal recesses.This study was supported by grants from the Deutscher Akademischer Austauschdienst to M.M. (312/dk-4-is), the Deutsche Forschungsgemeinschaft to H.W.K. (Ko 758/2-2, 2-3), and the Carlsberg Foundation     

Generalization and categorization of spectral colors in goldfish. II. Experiments with two and six training wavelengths

In part I of this study (Kitschmann and Neumeyer 2005), goldfish categorized spectral colors only in the sense that wavelengths in a range of about twice as large as the just noticeable difference were treated as similar to a given training wavelength. Now, we trained goldfish on more than one wavelength to prevent very accurate learning. In one experiment goldfish were trained on six adjacent wavelengths with equal numbers of rewards, and, thus, equal numbers of learning events. Generalization tests showed that some wavelengths were chosen more often than others. This indicated that certain spectral ranges are either more attractive or more easily remembered than others. As this is a characteristic of the “focal” colors or centers of color categories in human color vision, we interpret the findings in goldfish accordingly. We conclude (Figs. 5 and 6) that there are four categories in spectral ranges approximately coinciding with the maximal sensitivities of the four cone types, and three categories in-between. Experiments with two training colors indicate that there is no direct transition between categories analogous to human “green” and “red”, but that there is a color analogous to human “yellow” in-between (Figs. 2, 3; Table 1).     

Immunocytochemical localization of hypocalcin in the endocrine cells of the corpuscles of Stannius in three teleost species (trout,flounder and goldfish)

Summary In order to identify the cell-type responsible for the production of hypocalcin (the recently isolated hypocalcemic hormone of teleost fish), the corpuscles of Stannius (CS) of trout, flounder and goldfish, were immunocytochemically stained with antisera raised against trout hypocalcin. The secretory granules of the type-1 cells of the CS, considered to be the hypocalcin-producing cells, showed intense immunoreactivity in all species examined. However, in trout and flounder, the secretory granules produced by the type-2 cells, which have been suggested to represent a functionally different cell-type, also showed an intense immunoreactivity. In goldfish, no type-2 cells were observed. We tentatively conclude that type-1 and type-2 cells represent structurally different forms of the same functional cell-type.     

苯并(a)芘对鲫鱼肝脏EROD活性的影响

研究了典型多环芳烃类有机污染物苯并(a)芘(BaP)对鲫鱼(Carassius auratus)肝脏7-乙氧基-3-异吩唑酮-脱乙基酶(EROD)活性的影响。结果表明,注射后96 h,10和100 mg.kg-1处理组肝脏EROD活性被明显诱导,分别为对照组的2.3(P<0.05)和3.1倍(P<0.01)。肝脏EROD活性随着时间延长继续升高,至注射后14 d,1 mg.kg-1处理组鲫鱼肝脏EROD活性为对照的3.0倍(P<0.001),而100 mg.kg-1处理组则高达5.8倍(P<0.001)。鲫鱼肝脏EROD活性可作为反映BaP暴露水平的生物标志物。BaP对鲫鱼的最低效应浓度为1 mg.kg-1(鱼体重)。     

Generalization and categorization of spectral colors in goldfish I. Experiments with one training wavelength

Goldfish have a tetrachromatic color vision with a high discrimination ability for spectral colors as well as for object colors. We investigate the question whether goldfish organize the high number of discriminable colors in terms of color categories, i.e. in a few larger groups of colors independent of wavelength discrimination. Twenty-four goldfish were trained with food reward, each fish on one out of 13 wavelengths between 371 nm and 630 nm. In transfer tests two different wavelengths were presented, one shorter and one longer than the training wavelength, and the choice behavior was determined. Choice frequencies of ≥50% were assumed to indicate similarity to the training color. The wavelength ranges ≥50% were about 100 nm and twice as large as the just noticeable differences measured in wavelength discrimination tests (Fig. 7). The ranges were surprisingly about the same for all training wavelengths, provided the data were plotted on a wavelength scale weighted according to discrimination ability (Fig. 4). Thus, with the training method chosen goldfish showed a kind of categorization which, however, depends on training wavelength and discrimination ability. Generalization tests in which training wavelength and test wavelengths were shown separately for 2 min each gave the same results as wavelength discrimination tests (Figs. 5 and 6) and are, therefore, not indicative for color categories.