The welfare of captive animals is influenced by their ability to express natural behaviours. Foraging is one behaviour that may be particularly important in this respect; many species will continue to work for food even when it is freely available. The role of substrate, and in particular particle size, on the foraging behaviour of goldfish (Carassius auratus) was examined through three repeated measures experiments. In the first, tanks were set up with five uniform substrates: plastic grid, coarse sand, fine gravel, pebbles, and cobbles. In the second, fish were provided with a choice between coarse sand and fine gravel, fine gravel and pebbles, and pebbles and cobbles. In the third, they were provided with two choices between coarse sand and cobbles, one where the sand contained more food and one where the cobbles did. Our results show that particle size significantly affected the amount of time goldfish spent foraging, and that goldfish exhibited foraging behaviour even in the absence of a substrate they can manipulate. Goldfish foraged longest when provided with coarse sand. Fish foraged significantly longer over smaller particle size substrates when given a choice, although they did not distinguish between the two finest substrates, coarse sand and gravel. Increases in total time spent foraging were achieved through more, rather than longer, bouts. Food density did not significantly alter preference for smaller particle substrates. In general, coarse sand (1.5 mm) was found to be the most appropriate substrate in terms of facilitating natural foraging behaviours. These findings are discussed with respect to the welfare and husbandry of goldfish and aquarium fish in general.

A moving stimulus paradigm was designed to investigate color contrast encoding in the retina. Recently, this paradigm yielded suggestive evidence for color contrast encoding in zebrafish but the significance and generality remain uncertain since the properties of color coding in the zebrafish inner retina are largely unknown. Here, the question of color contrast is pursued in the goldfish retina where there is much accumulated evidence for retinal mechanisms of color vision and opponent color-coding, in particular. Recordings of a sensitive local field potential of the inner retina, the proximal negative response, were made in the intact, superfused retina in the light-adapted state. Responses to color contrast and achromatic contrast were analyzed by comparing responses to a green moving bar on green versus red backgrounds. The quantitative form of the irradiance/response curves was distinctly different under a range of conditions in 32 retinas, thereby providing robust evidence for red–green color contrast. The color contrast is based on successive contrast, occurs in the absence of overt color opponency, and clearly differs from previous findings in the goldfish retina for simultaneous color contrast mediated by color-opponent neurons. The form of the irradiance/response curves suggests that successive color contrast is particularly important when achromatic contrast is low, as often occurs in natural environments. The present results provide a parallel with the well-known principle of human color vision, first proposed by Kirschmann as the third law of color contrast, and may also have implications for the evolution of vertebrate color vision.

Aqua feeds should be formulated to provide complete and balanced nutrition to achieve optimal health and growth in fish, including adequate levels of essential amino acids (EAA). There are few or no data relating to the EAA requirements for ornamental fish species, with the majority of quantitative data for these nutrients being available for commercially farmed fish. The determination of EAA requirements is usually established through dose–response studies, which can be costly and time consuming, especially if determining the requirement for many amino acids (AA). An alternative method for predicting the EAA of fish, which is also relatively fast and inexpensive, is the assessment of whole-body AA composition. A total of eight goldfish with a mean wet weight of 34·2 (sem 1·4) g were obtained as a result of a routine cull by breeders. The fish were freeze-dried and AA was content analysed by hydrolysis or performic ‘acid’ oxidation. EAA values ranged between 0·97 (sem 0·02) for tryptophan and 7·9 (sem 0·14) for lysine (g/100 g AA). Compositional data were also used to estimate the essential amino acid ratios of these fish. The findings are in agreement with those for juvenile common goldfish, suggesting that there are no differences in whole-body AA composition between juvenile and adult, or fancy and common goldfish. However, these indices do not provide a quantitative total amount of each AA required by the fish, but can be used proportionally to provide guidelines to formulate diets for ornamental species.

Vision is used in nearly all aspects of animal behavior, from prey and predator detection to mate selection and parental care. However, the light environment typically is not uniform in every direction, and visual tasks may be specific to particular parts of an animal’s field of view. These spatial differences may explain the presence of several adaptations in the eyes of vertebrates that alter spectral sensitivity of the eye in different directions. Mechanisms that alter spectral sensitivity across the retina include (but are not limited to) variations in: corneal filters, oil droplets, macula lutea, tapeta, chromophore ratios, photoreceptor classes, and opsin expression. The resultant variations in spectral sensitivity across the retina are referred to as intraretinal variability in spectral sensitivity (IVSS). At first considered an obscure and rare phenomenon, it is becoming clear that IVSS is widespread among all vertebrates, and examples have been found from every major group. This review will describe the mechanisms mediating differences in spectral sensitivity, which are in general well understood, as well as explore the functional significance of intraretinal variability, which for the most part is unclear at best.

The viability of ornamental fish culture relies on the maintenance of high-quality breeds. To improve the profitability of culture operations we attempted to produce cloned fish from the somatic nucleus of the high-quality Japanese goldfish (Carassius auratus auratus) breed ‘Ranchu’. We transplanted the nucleus of a cultured fin-cell from an adult Ranchu into the non-enucleated egg of the original goldfish breed ‘Wakin’. Of the 2323 eggs we treated, 802 underwent cleavage, 321 reached the blastula stage, and 51 reached the gastrula stage. Two of the gastrulas developed until the hatching stage. A considerable number of nuclear transplants retained only the donor nucleus. Some of these had only a 2n nucleus derived from the same donor fish. Our results provide insights into the process of somatic cell nuclear transplantation in teleosts, and the cloning of Ranchu.

The role of dopamine as the endogenous signal-initiating light-dependent changes in the distribution of pigment granules in goldfish retinal pigment epithelium was investigated. In normal retinas, light adaptation resulted in the dispersion of pigment granules. This effect of light was mimicked by the intraocular injection of dopamine or serotonin, which is thought to increase endogenous dopamine release, into dark-adapted eyes. The effect of light, dopamine, or serotonin on dark-adapted retinas was blocked by the dopamine receptor antagonists haloperidol and sulpiride. However, lesioning the endogenous source of retinal dopamine, by prior intraocular injection of 6-hydroxydopamine (6-OHDA), did not block the dispersion of pigment granules in light-adapted retinas. No significant differences in pigment dispersion were noted between unlesioned and lesioned light- or dark-adapted retinas. However, the effect of light on pigment dispersion was no longer blocked by haloperidol or sulpiride in 6-OHDA lesioned animals. Dopamine and serotonin mimicked the effect of light when injected into lesioned dark-adapted eyes, but their effects were also not blocked by haloperidol or sulpiride. These results suggest that dopamine, acting on D2 receptors, is sufficient to induce pigment migration in unlesioned animals. In 6-OHDA-lesioned animals, however, pigment migration is mediated by a receptor mechanism other than D2.

Increment threshold functions of the electroretinogram (ERG) b–wave were obtained from goldfish using an in vivo preparation to study intraretinal mechanisms underlying the increase in perceived brightness induced by depletion of retinal dopamine by 6–hydroxydopamine (6–OHDA). Goldfish received unilateral intraocular injections of 6–OHDA plus pargyline on successive days. Depletion of retinal dopamine was confirmed by the absence of tyrosine-hydroxylase immunoreactivity at 2 to 3 weeks postinjection as compared to sham-injected eyes from the same fish. There was no difference among normal, sham-injected or 6–OHDA-injected eyes with regard to ERG waveform, intensity-response functions or increment threshold functions. Dopamine-depleted eyes showed a Purkinje shift, that is, a transition from rod-to-cone dominated vision with increasing levels of adaptation. We conclude (1) dopamine-depleted eyes are capable of photopic vision; and (2) the ERG b–wave is not diagnostic for luminosity coding at photopic backgrounds. We also predict that (1) dopamine is not required for the transition from scotopic to photopic vision in goldfish; (2) the ERG b–wave in goldfish is influenced by chromatic interactions; (3) horizontal cell spinules, though correlated with photopic mechanisms in the fish retina, are not necessary for the transition from scotopic to photopic vision; and (4) the OFF pathway, not the ON pathway, is involved in the action of dopamine on luminosity coding in the retina.

We have produced and characterized a monoclonal antibody, MH1, which selectively labels rod horizontal cells and Miiller cells in the goldfish retina. Biochemical and tissue distribution studies indicate that MH1 may recognize four out of five classes of intermediate filament proteins in goldfish: vimentin, desmin, glial fibrillary acidic protein (GFAP), and keratin, but not neurofilament. The intermediate filament which is labeled strongest in the retina is vimentin. In the goldfish retina, the only type of horizontal cells recognized by MH1 appear to be rod horizontal cells. This result suggests that the rod horizontal cell, an interneuron, and Miiller (glial) cells share a common antigen: vimentin, which is usually only expressed in mesenchymal origin cells.

The development of rod horizontal cells in the goldfish retina was also studied using MH1. The cells were not labeled by MH1 until 4–6 weeks posthatching, a stage in which the animals are already visually active. MH1 also did not label any horizontal cell in the region close to the ora terminalis in the goldfish retina. These results suggest that either the emergence and maturation of rod horizontal cells occur late during goldfish retinal development or the expression of vimentin itself occurs late in the development of rod horizontal cells.

Extracellular recordings were made from the isolated goldfish retina during superfusion with various serotonergic agonists and antagonists to determine the effects of these drugs on the maintained activity and response properties of the ganglion cells. Superfusion of the retina with serotonin (25–500 μM) increased the maintained activity of OFF-center ganglion cells and decreased the maintained activity of ON-center ganglion cells. In addition, serotonin also attenuated the excitatory responses to annular stimuli, suggesting a decrease in the strength of surround input to the ganglion cells. The effects of serotonin on OFF-center ganglion cells were mimicked by the nonselective 5-HT1, agonist 5-MeOT and the 5-HT1 receptor agonist 8-OH-DPAT, while only 5-MeOT mimicked the action of serotonin on ON-center ganglion cells. The effects of exogenously applied serotonin on the ganglion cells could be blocked by the mixed 5-HT1/5-HT2 receptor antagonist methysergide but not by the 5-HT2 receptor antagonist mianserin or the dopamine receptor antagonist haloperidol.

These results support previous anatomical and biochemical evidence that serotonin functions in a neurotransmitter or neuromodulatory role in the teleost retina and suggest that serotonin may be involved in modulating the maintained activity and surround input to the ganglion cells. The results also indicate that two different types of receptors may mediate the actions of serotonin in the ON and OFF pathways, respectively.

Recordings of field potentials in nucleus isthmi (NI) were obtained in an in vitro preparation of goldfish brain using a lateral approach. Horseradish peroxidase (HRP) was injected from recording electrodes to verify recordings within the nucleus and to label axonal pathways and cell bodies. Activity in NI was repetitive and could be elicited by stimulation of the optic nerve, tectum, pretectum, or tectobulbar tract. Spontaneous activity was present in some preparations and consisted of bursts with intervening silent periods. Anatomical and electrophysiological evidence indicated that the primary isthmotectal pathway is composed of fine fibers that exit NI rostrally and pass through pretectum to enter tectum rostrally. An afferent pathway consisting of both fine- and large-diameter fibers entered NI ventromedially; the large diameter axons have been previously reported in percomorph fishes, but were not thought to be present in cyprinids such as goldfish. The large diameter axons arise from labeled cell bodies in the region of the lateral thalamic nucleus. No labeled cell bodies were seen in ipsilateral nucleus pretectalis superficialis, pars magnocellularis, where they are seen in percomorphs. The fine axons, which have not been reported in percomorph fishes, were shown to arise from tectal bipolar (type VI) neurons. As in percomorphs, tectal type XIV neurons were also labeled. This and corroborating recordings from nucleus isthmi constitute the first demonstration of a tectoisthmic projection in a cyprinid fish.

The effect of unilateral depletion of retinal dopamine on goldfish visual behavior was studied using a behavioral reflex, the dorsal light reaction (DLR). Retinal dopamine was depleted by intraocular injections of 6–hydroxydopamine (6–OHDA) on two successive days. By 2 weeks postinjection, dopamine interplexiform cells (DA-IPC) were not detected using tyrosine-hydroxylase immunoreactivity (TH-IR). By 6 weeks postinjection, generation of DA-IPC was observed at the marginal zone and by 9 months postinjection, 2–3 rows of DA-IPC were present at the marginal zone. Neurites extended several hundred micrometers toward the central retina. By 2 weeks postinjection, all 6–OHDA lesioned fish tilted 7–15 deg toward the injected eye under uniform overhead illumination. The tilting did not occur under scotopic illumination and reappeared within 1 min of light adaptation. Quantitation of the DLR showed that 6–OHDA lesioned fish behaved as if light were 2.4 log units more intense to the injected eye. Partial recovery was observed by 9 months postinjection, paralleling the reappearance of DA-IPC at the marginal zone. Tilting also was induced by unilateral intraocular injection with Dl and D2 dopamine receptor antagonists, SCH 23390 and S(—)-sulpiride, respectively. Fish did not tilt if they were light adapted at the time of injection. Tilting was observed if the animals were dark-adapted for 3 h and left in the dark for 1 h postinjection. Fish tilted toward the drug-injected eye within 2 min of light adaptation and gradually returned to vertical within 2 h. The tilting response to S(—)-sulpiride was stronger (˜20 deg vs. ˜5 deg) and occurred at lower concentration (1 μM vs. 10 μM) compared to SCH 23390. We conclude that dopamine depletion mimics the dorsal light reaction by increasing the luminosity output of the eye and that dopamine is directly involved in photopic luminosity function.

The effect of background illumination on horizontal cell receptive-field size and dye coupling was investigated in isolated superfused goldfish retinas. Background illumination reduced both horizontal cell receptive-field size and dye coupling. The effect of light on horizontal cell receptive-field size was mimicked by treating the retina with 20 μM dopamine. To test the hypothesis that the effects of light were due to endogenous dopamine release, the effect of light was studied in goldfish retinas in which dopaminergic interplexiform cells were lesioned using 6-hydroxydopamine treatment. In lesioned retinas, background illumination reduced both horizontal cell receptive-field size and dye coupling. Furthermore, the effect of background illumination on unlesioned animals could not be blocked by prior treatment with the D1 dopamine receptor antagonist SCH-23390. These results suggest that, in goldfish retina, dopamine release is not the only mechanism by which horizontal cell receptive-field size could be reduced by light.

The synaptic organization of dopaminergic interplexiform cells (DA-IPC) in the goldfish retina was studied by a combined double-label electron-microscopical (EM) immunocytochemical/autoradiographical study. DA-IPCs were labeled with antisera against tyrosine hydroxylase. The possibility of synaptic contact with GABAergic amacrine cells in the proximal inner plexiform layer (IPL) was studied by using 3H-GABA uptake. Most synaptic input and output from DA-IPC processes involved amacrine cell processes. In addition, synaptic interactions were observed between DA-IPC processes and bipolar cell terminals, other DA-IPC processes, very small dendrites in the IPL, ganglion cell and optic fiber layers (OFL), and cell bodies in the ganglion cell layer (GCL). Input and output synapses with GABAergic amacrine processes also were observed. Two-thirds of the DA-IPC boutons in the proximal IPL were involved in “junctional appositions,” that is, the junctions appeared to be specialized but they were different than classical chemical synapses. The synaptic organization of DA-IPCs in the goldfish IPL appears to be far more complex than previously thought. Although earlier studies have attempted to explain the action of dopamine in terms of interaction only with amacrine cells, the present study shows that effects involving bipolar cells, other DA-IPCs, unidentified processes and cell bodies in the GCL and OFL must be considered as well.

The vertebrate retina processes visual information in parallel neural pathways known as the ON and OFF pathways. These pathways encode increments and decrements of light independently as excitatory responses. We examined the photopic spectral response of ON and OFF mechanisms in goldfish by measuring the sensitivity of optic nerve responses to the onset and termination of stimuli of various wavelengths. Using various adapting backgrounds, we found that the ON and OFF responses have different spectral sensitivities. The weighting of the cone inputs to the responses was estimated by an algebraic summation model. This model suggests that for the ON response, input from S-cones is stronger and more independent than for the OFF response, and M- and L-cones show stronger antagonism in the ON response than in the OFF response. The OFF response probably receives input from all cone types, but spectral antagonism is weak and its dominant input is from L-cones.

The glycinergic system in goldfish retina was studied by immunocytochemical localization of glycine antiserum at the light-microscopical level. Numerous amacrine cells, a type of interplexiform cell, interstitial cell, and displaced amacrine cell were glycine-immunoreactive (IR). Amacrine cells, accounting for 97% of the glycine-IR neurons, were of four types based solely on their level of dendritic stratification: stratified amacrine cells of the first, third, and fifth sublayers and bistratified amacrine cells of the first and fifth sublayers. Double-labeling experiments were carried out to determine possible co-localization of glycine-IR with GABA-IR, serotonin-IR, substance P-IR and somatostatin-IR. No evidence for co-localization of glycine-IR with these other transmitter substances was found, despite reports of co-localization of these substances in retinas of other species. Glycinergic neurons in goldfish retina appear to consist of a heterogeneous population of at least seven morphologically distinct subtypes that are also neurochemically distinct in regard to GABA, serotonin, substance P, and somatostatin. Since dendritic stratification in the inner plexiform layer is correlated with ON-, OFF-response types, we suggest that the subtypes of glycine-IR amacrine cells play different roles in the encoding of visual information.

We have recorded whole-cell membrane currents in response to exogenously applied acetylcholine (ACh), nicotine, and 1,1 dimethyl-4-phenyl piperazinium iodide on retinal ganglion cells enzymatically dissociated from goldfish retina. Agonist applications induced nicotinic-type responses in a majority of cells when cells were isolated under optimal conditions. Currents were reminiscent of nicotinic-type ganglionic responses. Dose-response measurements of ACh-induced currents indicated an EC50 of 52 μM and a Hill coefficient of 0.6. Currents were selective for Na+ over Cl− and were highly inwardly rectifying. Responses were blocked reversibly by d-tubocurarine, hexamethonium chloride, and N-methyl-D-glucamine. In 50% of the cases, α-bungarotoxin reversibly blocked the current induced by ACh application. The blocking action of mecamylamine was irreversible and independent of the presence of agonist but was more effective in the presence of ACh. We conclude that functional nicotinic ACh receptors exist on most goldfish retinal ganglion cells.

Visual responses of goldfish to rotating square-wave gratings were recorded before and after intraocular injection of 2-amino-4-phosphonobutyric acid (APB). High doses of APB reduced the rate of optokinetic nystagmus (OKN) to a relatively high spatial frequency grating moving at a high temporal frequency. Responses to a low spatial frequency grating were not altered, nor were responses to the higher spatial frequency when it rotated slowly. The effects of APB were transient and lasted no longer than 3 d. We conclude that APB reduces OKN to high spatiotemporal frequencies in goldfish.

There are four types of horizontal cell in the goldfish retina, three cone- and one rod-type. The neurotransmitter of only one type, the H1 (cone) horizontal cell, has been identified as GABA. 3H-adenosine uptake was examined as a possible marker for the other classes of horizontal cell. Isolated goldfish retinae were incubated in 3H-adenosine (10–40 μCi) in HEPES-buffered saline for 30 min, then fixed, embedded in plastic, and processed for light-microscopic autoradiography (ARG). For double-label immuno/ARG studies, l-μm-thick sections were processed for GABA postembed immunocytochemistry, then for ARG. 3H-adenosine uptake was localized to cone photoreceptors, presumed precursor cells in the proximal outer nuclear layer, and to a single, continuous row of horizontal cell bodies in the inner nuclear layer. No uptake was localized to the region of horizontal cell axon terminals. 3H-adenosine uptake did not colocalize with GABA-IR in H1 horizontal cells, but it did colocalize with adenosine deaminase immunoreactivity. It is concluded that 3H-adenosine uptake selectively labels rod horizontal cells in the goldfish retina based on position and staining pattern, which are similar to rod horizontal cells stained by Golgi or HRP injection methods. The use of 3H-adenosine uptake may provide a useful tool to study other properties of rod horizontal cells (i.e. development) as well as provide clues as to the transmitter used by these interneurons.

We have produced and characterized a monoclonal antibody, AT101, which selectively labels both viable and formaldehyde-fixed horizontal cell axon terminals, but not their somas or axons, of the goldfish (Carassius auratus) retina. The antigen recognized by AT101 appears to be a cell surface glycoprotein with a molecular weight of about 35,000 Daltons, and is present exclusively or predominantly in nervous tissues of all vertebrate species examined. We have used AT101 as a probe to analyze immunocytochemically the organization of horizontal cell axon terminals (HCATs) in the adult goldfish retina, and the emergence and maturation of these terminals during retinal development. Because of continued growth at the retinal margin in adult goldfish, there is a peripheral-to-central gradient in the age of cells, with the most mature in the center and the youngest in the periphery. In the center and near periphery of the adult retina, HCATs have a fusiform morphology and form a dense network in the middle and proximal part of the inner nuclear layer. In the far peripheral retina, the axon terminals appear round or ellipsoid. The retina closest to the retinal margin is devoid of AT101 staining, indicating that either HCATs are absent or the antigen recognized by AT101 is not present on HCATs at this stage. A similar sequence of changes in staining pattern is seen during development. Although AT101 staining can first be demonstrated in the larval retina at 1 month after hatching, it appears mostly as punctate structures. At a later stage, there are round or ellipsoid structures that resemble in morphology and location (in the inner nuclear layer) those found in the far peripheral adult retina. Double-labeling experiments with AT101 and antiserum against tubulin also indicate that AT101 labels the HCATs when they first appear during development. These data suggest that the emergence and maturation of HCAT is a late event in retinal development.

Selective labeling of intravitreal Procion yellow dye by presumed blue-sensitive cone photoreceptors has been demonstrated in primate retina. To determine whether Procion yellow is selective for this cone type in an unrelated vertebrate species, labeling by this dye was studied in goldfish retina, where cone pigment type can be directly inferred from photoreceptor morphology. At low vitreal concentrations of the dye (<0.4%), only cone outer segments were labeled. At vitreal concentrations of 0.4–0.5%, the inner segments of short-double cones and a subset of long single cones (presumed green-sensitive cones) were selectively stained. At still higher vitreal concentrations (0.6–0.7%), the inner segments of short-single cones and miniature short-single cones (presumed blue-sensitive cones) showed evidence of Procion label, but were not as heavily labeled. The inner segments of long-double cones and a subset of long-single cones (presumed red-sensitive cones) did not label at any of these concentrations. These results show that Procion yellow is not a selective marker for blue-sensitive cones in the goldfish retina. In addition, stained rod and cone nuclei were observed at each dye concentration, including those concentrations at which no inner segments were labeled.