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.     

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.     

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     

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     

The effects of blinding,pinealectomy and exposure to constant dark conditions on gonadal activity in the female goldfish,Carassius auratus

Synopsis The effects of pinealectomy, blinding, and exposure to constant darkness were examined in female goldfish during different seasons. Neither blinding nor pinealectomy under short or decreasing photoperiod conditions, nor exposure to constant darkness had an effect on ovarian activity in goldfish during fall and winter. In spring, constant darkness has an inhibitory effect on ovarian activity. Pinealectomy under increasing photoperiod conditions partially inhibits reproductive activity, but not to the extent of constant darkness. Blinding, under the same regime also inhibited to some extent the ovarian response to increasing photoperiod. Our data suggest that retinal pathways and the pineal organ are involved in the photosexual response to increasing day lengths.Address correspondence to: Dr. V. L. de Vlaming, Marquette University     

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.     

Otolith resorption induced by anaerobic stress in the goldfish, Carassius auratus

To examine otolith resorption induced by anaerobic stress, ⁴⁵Ca-prelabelled goldfish, Carassius auratus , were kept in oxygen-deficient ambient water (O₂ < 0.5 .1 1⁻¹, 26° C) for 2 days and otoliths (asterisci) were analysed for ⁴⁵Ca retention. In a second experiment, fish were anaerobically stressed for 2 days, and then received a single intraperitoneal injection of ⁴⁵Ca. They were maintained under stress for one more day and killed to examine ⁴⁵Ca deposition in otoliths. Plasma was analysed for total and radioactive calcium. Otoliths (lapilli) were examined for stress-induced check formation by scanning electron microscopy (SEM).
Stress significantly reduced plasma calcium levels and the rate of calcium retention in otoliths, which was calculated from the ⁴⁵Ca specific activity of the plasma. On the other hand, the rate of calcium deposition in otoliths was the same in the stressed as in the control fish. SEM observation revealed that the applied stress resulted in a check formation in otoliths. These results indicate that a 48-h stress of oxygen deficiency induces calcium resorption in otoliths.     

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.     

Fine-structural study of leucocytes in the goldfish, Carassius auratus

An electron microscopic study was performed on leucocytes from circulating blood of the goldfish. The leucocytes were divided into eight types: neutrophil, eosinophil, large granular leucocyte (LGL), medium-sized granular leucocyte (MGL), small granular leucocyte (SGL), fine granular leucocyte (FGL), lymphocyte, and monocyte. In this report the thrombocyte was excluded from leucocytes, and LGL, MGL, SGL and FGL were tentatively classified based on the size of intracytoplasmic granules possessed by each cell. The existence of goldfish monocytes was electron microscopically demonstrated for the first time in the present report.     

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.     

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 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     

The effects of calcitonin on plasma calcium levels and bone metabolism in the fresh water teleost Channa punctatus

Administration of salmon calcitonin (sCT) caused significant reduction in total and ultrafiltrable plasma calcium content in the plasma of a fresh water female teleost Channa punctatus. A time-bound analysis on the effect of sCT showed a highly significant short duration reduction in total and ultrafiltrable plasma calcium content in fish kept in normal tap water and low-calcium water and a moderate hypocalcemia in fish kept in high-calcium water. Sexually immature adult fish showed a greater response than the sexually mature ones. Using tartrate-resistant acid phosphatase (TRACP) and alkaline phosphatase (ALP) activities in plasma and hydroxyproline (HYP) excretion in urine, the effect of sCT on the inhibition of bone calcium resorption were examined. In both sexually mature and immature adult fish, kept in normal tap water, sCT significantly suppressed TRACP and ALP activities in plasma and excretion of HYP in urine within 2-6 h with a maximum at 4 h after injection. Salmon CT treatment to sexually immature adult fish caused significant increase in skeletal bone calcium concentration. Taken together, all this information indicates that CT in a fresh water female teleost is an effective regulator of plasma calcium levels, and its action, at least in part, operates through inhibition of bone calcium resorption.     

The dopaminergic innervation of the goldfish pituitary

Summary The dopaminergic innervation of the goldfish pituitary gland was studied by immunocytochemistry at the electron-microscope level using highly specific antibodies against dopamine coupled to bovine serum albumin with glutaraldehyde. A satisfactory preservation of the tissue was achieved after immersion in 5% glutaraldehyde in phosphate buffer containing sodium metabisulfite to prevent oxidation of the endogenous dopamine. The immunocyto-chemical procedure was performed on Vibratome sections using the preembedding method. Immunoreactivity was restricted to part of the neurosecretory type-B fibers (diameter of the secretory vesicles lower than 100 nm) in which it was found to occupy the whole cytoplasm. Labeled fibers were observed within the neurohypophysis in the different parts of the gland and in the adenohypophyseal tissue where immunoreactive profiles were detected in close apposition to the different cell types. These data are in agreement with previous results obtained by means of radioautography and further support a role for dopamine in the neuroendocrine regulation of pituitary functions in teleosts.     

Diurnal variations in the physiology of the goldfish,Carassius auratus

Abstract

Liver glycogen, liver lipid, liver triglycerides, plasma glucose, plasma total lipid, plasma cholesterol, plasma corticoids, hypothalamic serotonin and pituitary pro‐lactin levels were assayed at five times over a 24‐h period in Carassius auratus maintained under a specific photoperiod regime at various times throughout the year. Diurnal variations were observed in all parameters monitored. Daily variations of liver glycogen, plasma glucose, plasma lipid, plasma corticoids and hypothalamic serotonin were affected by time of feeding. Liver glycogen, plasma lipid and plasma corticoid levels were also affected by time of feeding. Diurnal variations of liver glycogen, plasma glucose and plasma lipid were influenced by light‐dark cycles. These data illustrate that feeding time, photoperiod and time of sacrifice are important considerations in the study of metabolic and hormonal parameters in fishes.     

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.     

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.     

Segregation of FMRF amide-immunoreactive efferent fibers from NPY-immunoreactive amacrine cells in goldfish retina

Summary Immunocytochemical studies were conducted on goldfish to determine whether a retinal efferent fiber system, immunoreactive to the tetrapeptide Phe-Met-Arg-Phe-NH₂ (FMRFamide), might contain instead a substance similar to one of the 36-amino acid pancreatic polypeptides, the C-terminus of which is similar to FMRFamide.Our results demonstrate the presence of two separate peptidergic systems, one containing FMRFamide-like, and the other pancreatic polypeptide-like peptides. Antisera to FMRFamide reveal the efferent fibers, whose axons exit the optic nerve and terminate in layer 1 of the inner plexiform layer, as previously described. Antisera to porcine neuropeptide Y, and to avian and bovine pancreatic polypeptides label a sparse population of putative amacrine cell bodies and a dense fiber plexus in layers 1, 3, and 5 of the inner plexiform layer. Based on intensity of staining, this amacrine cell peptide appears to be most similar to neuropeptide-Y.Radioimmunoassay and immunocytochemical staining of retinas in which the efferent fiber peptide was depleted by optic nerve crush confirm in large part the observation that the two peptide systems are distinct. However, there is some cross-recognition of the FMRFamide-like tissue antigen by pancreatic polypeptide antibodies.Double-label studies with antisera to tyrosine hydroxylase and neuropeptide-Y indicate that the pancreatic polypeptide antigen is not co-localized with catecholamines.     

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.     

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.