Retina Of Rainbow Trout Research Articles

Near absence of differential gene expression in the retina of rainbow trout after exposure to a magnetic pulse: implications for magnetoreception.

The ability to perceive the Earth's magnetic field, or magnetoreception, exists in numerous animals. Although the mechanism underlying magnetoreception has not been clearly established in any species, in salmonid fish, it is hypothesized to occur by means of crystals of magnetite associated with nervous tissue such as the brain, olfactory organ or retina. In this study, rainbow trout (Oncorhynchus mykiss) were exposed to a brief magnetic pulse known to disrupt magnetic orientation behaviour in several animals. Changes in gene expression induced by the pulse were then examined in the retina. Analyses indicated that the pulse elicited differential expression of only a single gene, gamma-crystallin M3-like (crygm3). The near absence of an effect of the magnetic pulse on gene expression in the retina stands in sharp contrast to a recent study in which 181 genes were differentially expressed in brain tissue of O. mykiss after exposure to the same pulse. Overall, our results suggest either that magnetite-based magnetoreceptors in trout are not located in the retina, or else that they are unaffected by magnetic pulses that can disrupt magnetic orientation behaviour in animals.

Aquatic Environments and Spectral Sensitivity of Rod Visual Pigments in the Retina of Rainbow Trout (Oncorhynchus mykiss): A Microspectrophotometric Study

The vertebrate retina is a part of the central nervous system (CNS) that can be visualized in a non-invasive manner. The retina is a thin multi-layered sensory tissue of neural cells that contains millions of photoreceptors. There are two physiologically distinct types of photoreceptors across the retina, the cones and the rods. The light sensitivity of the photoreceptors results from the presence of visual pigment molecules in the outer segment of the cone and rod cells. All visual pigments are composed of two components: retinal (vitamin A aldehyde), termed a chromophore, bound to a protein called opsin. The spectral absorption properties of a visual pigment are mainly determined by the amino acid sequence of the opsin protein and the type of chromophore (A1 or A2 or mixed) bound to the opsin. There is a shifting from A1- to A2-based chromophores in the same opsin due to different environmental factors, e.g. aquatic environments. A1/A2 visual pigment ratios play a dominant role in determining rod λmax in rainbow trout (Oncorhynchus mykiss), shifting the λmax of the visual pigment and spectral bandwidth of absorbance (HBW, half-band width). The spectral absorption characteristics of the retinal rod photoreceptors in the Rainbow Trout (Oncorhynchus mykiss) were measured using the charge-coupled device (CCD)- based microspectrophotometer (MSP) system. We investigated retina preparations of juvenile rainbow trout (Oncorhynchus mykiss) using 54 rod photoreceptors. The spectral sensitivity of each rod photoreceptor was calculated using λmax (wavelength of maximum absorbance) and HBW. The mean wavelength of maximum absorbance values for the alpha-bands were 503.5±2.5nm for rhodopsin- dominated rods, 523.1±7.9nm for porphyropsin-dominated rods, and 509.6±1.9nm for rhodopsin porphyropsin mixed rods. The half-band width (HBW) of the main absorption bands was 4567±771 cm-1 for vitamin A1-based visual pigments and 4697±487 cm-1 for vitamin A2-based visual pigments. Our results provide the evidence of shifting the λmax of the visual pigment to a longer wavelength, broadening spectral bandwidth of absorbance, and presence three fundamentally different classes of rod photoreceptors in the retina of the rainbow trout depending on A1/A2 visual pigment ratios due to different environmental variables, e.g. aquatic environments.

In ovo thyroxine exposure alters later UVS cone loss in juvenile rainbow trout

Thyroid hormones (THs) play a vital role in vertebrate neural development, and, together with the beta isoform of the thyroid hormone receptor (TRβ), the development and differentiation of cone photoreceptors in the vertebrate retina. Rainbow trout undergo a natural process of cone cell degeneration during development and this change in photoreceptor distribution is regulated by thyroxine (T4; a thyroid hormone). In an effort to further understand the role of T4 in photoreceptor ontogeny and later developmental changes in photoreceptor subtype distribution, the influence of enhanced in ovo T4 content on the onset of opsin expression and cone development was examined. Juvenile trout reared from the initial in ovo-treated embryos were challenged with exogenous T4 at the parr stage of development to determine if altered embryonic exposure to yolk THs would affect later T4-induced short-wavelength-sensitive (SWS1) opsin transcript downregulation and ultraviolet-sensitive (UVS) cone loss. In ovo TH manipulation led to upregulation of both SWS1 and long-wavelength-sensitive (LWS) opsin transcripts in the pre-hatch rainbow trout retina and to changes in the relative expression of TRβ. After 7 days of exposure to T4, juveniles that were also treated with T4 in ovo had greatly reduced SWS1 expression levels and premature loss of UVS cones relative to T4-treated juveniles raised from untreated eggs. These results suggest that changes in egg TH levels can have significant consequences much later in development, particularly in the retina.

Daily Rhythmic Expression Patterns of Clock1a, Bmal1, and Per1 Genes in Retina and Hypothalamus of the Rainbow Trout, Oncorhynchus Mykiss

Living organisms show daily rhythms in physiology, behavior, and gene expression, which are due to the presence of endogenous clocks that synchronize biological processes to the 24-h light/dark cycle. In metazoans, generation of circadian rhythmicity is a consequence of specialized tissues known as “master clocks,” having different locations among species. A few studies have described clock-gene expression in fish neural tissues, but none of them assessed clock-gene expression in different discrete regions. The present study was designed to explore the presence/absence of circadian clock-gene expression in the rainbow trout (Oncorhynchus mykiss) retina and hypothalamus. Juvenile fish were acclimated to a 12:12 light (L)-dark (D) cycle. Then, retina and hypothalamus were collected from animals kept under LD conditions or constant darkness (DD) for 24 h. Real-time quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) assays were performed to quantify expression of the core circadian genes Clock1a, Bmal1, and Per1 as representative members of the circadian oscillator. All clock genes analyzed in the retina and hypothalamus showed circadian fluctuations. However, gene expression peaked in the rainbow trout hypothalamus with a 3-h (Clock1a and Bmal1) or 6-h (Per1) delay relative to that observed in the retina, the latter showing highest expression levels at zeitgeber times 9 (ZT9) for Clock1a and Bmal1, and at ZT21 for Per1. When exposed to DD, the rhythmic gene expression pattern was maintained for all genes in the rainbow trout retina, but only for Clock1a and Per1 in the hypothalamus. Bmal1 failed to cycle under DD, suggesting that hypothalamic clock function might depend on either several clock-gene isoforms or regulation from external inputs. Overall, these data indicate that representative molecular members of the core circadian clock are present in both the retina and hypothalamus of rainbow trout. (Author correspondence: jmmiguez@uvigo.es)

Systemic thyroid hormone is necessary and sufficient to induce ultraviolet-sensitive cone loss in the juvenile rainbow trout retina

Rainbow trout possess ultraviolet-sensitive (UVS) cones in their retina that degenerate naturally during development. This phenomenon can be induced with exogenous thyroxine [T4, a thyroid hormone (TH)] treatment. However, the previous T4 exposure experiments employed static water immersion; a method that could introduce confounding stress effects on the fish. Because of this, it was uncertain if T4 alone was sufficient to induce retinal changes or if stress-related hormones were also necessary to initiate this process. Furthermore, it was unclear whether endogenous T4 was the factor responsible for initiating natural UVS cone loss during development. The current study examined the role of systemic T4 on the juvenile rainbow trout retina using a slow-release implant. Exogenous T4 treatment resulted in SWS1 opsin downregulation and UVS cone loss after four weeks of exposure, signifying that T4 is sufficient to induce this process. Blocking endogenous T4 production with propylthiouracil (PTU, an anti-thyroid agent) attenuated SWS1 downregulation and UVS cone loss in the retina of naturally developing rainbow trout, suggesting that endogenous T4 is necessary to initiate retinal remodelling during development. Quantitative real-time RT-PCR analysis demonstrated that several TH-regulating components are expressed in the trout retina, and that expression levels of the TH receptor isoform TRbeta and the type 2 deiodinase (D2) change with T4 treatment. This suggests that T4 may act directly on the retina to induce UVS cone loss. Taken together, these results demonstrate that systemic TH is necessary and sufficient to induce SWS1 opsin downregulation and UVS cone loss in the retina of juvenile rainbow trout.

Chromatic organization of cone photoreceptors in the retina of rainbow trout: single cones irreversibly switch from UV (SWS1) to blue (SWS2) light sensitive opsin during natural development

The retinas of salmonid fishes have single and double cones arranged in square to row formations termed mosaics. The square mosaic unit is formed by four double cones that make the sides of the square with a single (centre) cone in the middle, and a single (corner) cone at each corner of the square when present. Previous research using coho salmon-derived riboprobes on four species of anadromous Pacific salmon has shown that all single cones express a SWS1 (UV sensitive) visual pigment protein (opsin) at hatching, and that these cones switch to a SWS2 (blue light sensitive) opsin during the juvenile period. Whether this opsin switch applies to non-anadromous species, like the rainbow trout, is under debate as species-specific riboprobes have not been used to study opsin expression during development of a trout. As well, a postulated recovery of SWS1 opsin expression in the retina of adult rainbow trout, perhaps via a reverse process to that occurring in the juvenile, has not been investigated. Here, we used in situ hybridization with species-specific riboprobes and microspectrophotometry on rainbow trout retina to show that: (1) single cones in the juvenile switch opsin expression from SWS1 to SWS2, (2) this switch is not reversed in the adult, i.e. all single cones in the main retina continue to express SWS2 opsin, and (3) opsin switches do not occur in double cones: each member expresses one opsin, maximally sensitive to green (RH2) or red (LWS) light. The opsin switch in the single cones of salmonid fishes may be a general process of chromatic organization that occurs during retinal development of most vertebrates.

Spatio-temporal characterization of retinal opsin gene expression during thyroid hormone-induced and natural development of rainbow trout

The abundance and spatial distribution of retinal cone photoreceptors change during thyroid hormone (TH)-induced and natural development of rainbow trout (Oncorhynchus mykiss). These changes are thought to allow the fish to adapt to different photic environments throughout its life history. To date, the ontogeny of rainbow trout cone photoreceptors has been examined using physiological and morphological approaches. In this study, we extended these observations by measuring opsin gene expression in retinal quadrants during natural and TH-induced development. Gene expression during natural development was investigated in retinae from fish at both parr and smolt stages. The role of TH in modulating opsin gene expression was determined in TH-treated parr and control fish sampled after two, nine, and 22 days of treatment. Total RNA was isolated from each retinal quadrant and steady-state opsin mRNA levels were measured using reverse transcriptase real-time quantitative polymerase chain reaction (QPCR) analysis. Expression of ultraviolet-sensitive opsin (SWS1), rod opsin (RH1), middle wavelength-sensitive opsin (RH2), and long wavelength-sensitive opsin (LWS) transcripts vary spatially in the parr retina. Smolts, compared to parr, had downregulated SWS1 expression in all quadrants, lower LWS expression dorsally, higher RH1 expression nasally, and higher RH2 expression dorsally. In TH-treated parr, SWS1 opsin expression was downregulated in the nasal quadrants by two days. SWS1 displayed the greatest degree of downregulation in all quadrants after nine days of treatment, with an increase in short wavelength-sensitive (SWS2) and RH2 opsin mRNA expression in the temporal quadrants. This study reveals that opsin genes display spatially significant differences within rainbow trout retina in their level of mRNA expression, and that regulation of opsin expression is a dynamic process that is influenced by TH. This is particularly evident for SWS1 gene expression in parr following TH-induced and natural development.

Identification of a unique transcript down-regulated in the retina of rainbow trout ( Oncorhynchus mykiss) at smoltification

Developmental and physiological changes in the retina of salmonid fishes occur during smoltification, a metamorphic event associated with thyroid hormone that prepares salmon for oceanic migration. These changes include loss of ultraviolet-sensitive (UVS) cone photoreceptors, switching of visual pigments, alterations in thyroid hormone regulation, and associated changes in behavior. This model provides an opportunity to study substantial neuronal development within an established retina. Little is known, however, about how higher order neurons are altered or how retinal gene expression changes during this transition. Here, we have used differential display RT-PCR and RACE-PCR to identify a previously uncharacterized gene transcript in Oncorhynchus mykiss under developmental regulation in the retina during smoltification, rtp12.5. This unique cDNA encodes a putative protein 112 amino acids long similar to a hypothetical human open reading frame located on chromosome 14.Q24.2. Differential expression was confirmed by RNA dot blot and in situ hybridization. We also present O. mykiss sep15 cDNA sequence and describe its expression in the vertebrate retina. Considering the expression pattern within retinal tissue observed by in situ hybridization, rtp12.5 may be under TH regulation and involved in neuronal remodeling of the retina during loss of UVS cones.

Gradual and partial loss of corner cone-occupied area in the retina of rainbow trout

Several studies have indicated that the rainbow trout ( Oncorhynchus mykiss) loses ultraviolet (UV) sensitivity and the associated UV-sensitive corner cones when the animal transforms from a small (parr) juvenile to a larger, silver-coloured, smolt. Similar changes supposedly take place when parr juveniles are treated with thyroid hormone (T 4) or retinoic acid. In contrast to previous investigations, this study shows that parr juveniles lack corner cones throughout the lower half of the ventral retina, suggesting that corner cones cease to be incorporated into the ventral retina some time after hatching. This uneven incorporation of corner cones across the retina, when combined with retinal growth, creates a progressively smaller area of lower retina occupied by corner cones. Because in previous studies, the stimulating illumination was directed primarily at the ventral retina, the reported age-dependent changes in UV or polarization sensitivities can be explained by differences in the area of corner cones that was illuminated, and not necessarily by a loss of corner cones. This study also shows: (1) that the double cones from non-ventral mosaics of parr rainbow trout may change in cross-sectional shape, altering the mosaic formation from a square to a row, (2) the existence of a ‘pure’ (non-changing) square mosaic in the ventral retina, and (3) a potential method, based on differential staining of cone nuclei, to classify paired cones into double or twin cones.

Developmental switch in excitability, Ca(2+) and K(+) currents of retinal ganglion cells and their dendritic structure.

In the retina of teleost fish, continuous neuronal development occurs at the margin, in the peripheral growth zone (PGZ). We prepared tissue slices from the retina of rainbow trout that include the PGZ and that comprise a time line of retinal development, in which cells at progressive stages of differentiation are present side by side. We studied the changes in dendritic structure and voltage-dependent Ca(2+), Na(+), and K(+) currents that occur as ganglion cells mature. The youngest ganglion cells form a distinct bulge. Cells in the bulge have spare and short dendritic trees. Only half express Ca(2+) currents and then only high-voltage-activated currents with slow inactivation (HVAslow). Bulge cells are rarely electrically excitable. They express a mixture of rapidly inactivating and noninactivating K(+) currents (IKA and IKdr). The ganglion cells next organize into a transition zone, consisting of a layered structure two to three nuclei thick, before forming the single layered structure characteristic of the mature retina. In the transition zone, the dendritic arbor is elaborately branched and extends over multiple laminae in the inner plexiform layer, without apparent stratification. The arbor of the mature cells is stratified, and the span of the dendritic arbor is well over five times the cell body's diameter. The electrical properties of cells in the transition and mature zones differ significantly from those in the bulge cells. Correlated with the more elaborate dendritic structures are the expression of both rapidly inactivating HVA (HVAfast) and of low-voltage-activated (LVA) Ca(2+) currents and of a high density of Na(+) currents that renders the cells electrically excitable. The older ganglion cells also express a slowly activating K(+) current (IKsa).

Molecular Cloning and Characterization of a cDNA Encoding the Retinal ArylalkylamineN-Acetyltransferase of the Rainbow Trout, Oncorhynchus mykiss

Melatonin synthesis in the retina as well as in the pineal gland exhibits daily variations with higher levels during the dark phase of light-dark cycles. To analyze the molecular mechanism of melatonin synthesis in the retina, we have cloned, sequenced and characterized a putative cDNA for arylalkylamine N-acetyltransferase (AANAT; EC 2.3.1.87), a rate-limiting enzyme in melatonin production, from the retina of the rainbow trout (Oncorhynchus mykiss). The trout AANAT cDNA (1,585 bp) contains an open reading frame encoding 240 amino acid protein (predicted molecular weight, 27,420) that is 51-65% identical to avian and mammalian AANAT. The trout retinal AANAT protein contains motifs A and B that are conserved among the N-acetyltransferase superfamily and eight potential phosphorylation sites. Southern blot analysis demonstrated that the protein is expressed by a single copy gene. A single AANAT transcript (1.6 kb) was detected in the retina but not in the liver by Northern blot analysis. The levels of AANAT mRNA in the retina exhibited day-night changes with 3.3-fold increase at night. These results indicate that in the rainbow trout retina, the activity of AANAT and thus melatonin synthesis are regulated at least in part at the transcriptional level.

Evidence for an endogenous clock in the retina of rainbow trout: II. Circadian rhythmicity of serotonin metabolism.

The purpose of the present study was to investigate patterns of circadian rhythmicity in the retina of a salmonid fish, the rainbow trout (Oncorhynchus mykiss). Our data present the first demonstration of intraretinal variations of serotonergic substances under light/dark-conditions (LD) and during continuous darkness (DD). All substances examined (serotonin, N-acetyl serotonin, 5-hydroxytryptophol, 5-hydroxyindole acetic acid) were rhythmic in LD. Serotonin, N-acetyl-serotonin, and 5-hydroxyindole acetic acid showed a preservation of specific features of rhythmicity in DD indicating the involvement of an endogenous pacemaker in the regulation of serotonin metabolism in the rainbow trout eye.

Evidence for an endogenous clock in the retina of rainbow trout: I. Retinomotor movements, dopamine and melatonin.

The purpose of the present study was to investigate patterns of circadian rhythmicity in the retina of a salmonid fish, the rainbow trout (Oncorhynchus mykiss). In a first attempt to show both morphological and neurochemical variations during light/dark conditions (LD) and during continuous darkness (DD), we investigated retinomotor movements and the associated regulatory transmitters, dopamine and melatonin. All parameters studied showed patterns of rhythmicity in LD and DD, clearly indicating the presence of an endogenous clock in the rainbow trout eye. The most salient variations of all parameters studied were found at the transitions from light to dark and vice versa in LD and from subjective day to subjective night and vice versa in DD. The amplitudes of rhythms compared between LD and DD were similar in pigment index, whereas a clear reduction was found for cone index, dopamine and melatonin.

Microanatomy of the dopaminergic system in the rainbow trout retina

We have investigated the morphology of dopaminergic interplexiform cells as well as the distribution of two classes of dopamine receptors in the retina of the rainbow trout. Interplexiform cells were visualized using an antiserum against tyrosine hydroxylase and PAP immunocytochemistry. In whole amounts, these cells have a density of between 91 and 182 cells per mm2 with highest values in the lower temporal quadrant. Their cell bodies lie at the inner margin of the inner nuclear layer with only 12-17 cells per retina displaced to the ganglion cell layer. There are three levels of stratification in the inner plexiform layer, one at the distal and proximal borders respectively, and one in the middle. They arise mostly from a radially oriented, stout primary dendrite. Tangential processes are about 1 micron in diameter and show a number of varicosities. The density of processes is greatest in sublayer 5, but no major difference in the general organization is apparent between the three sublayers. In the outer retina, there are two levels of dense ramification confined to the layer of horizontal cells. Light and electron microscopic analysis shows synaptic input to horizontal cells, but not to photoreceptors. The distribution of D1 receptors was assessed by studying the binding pattern of a specific, fluorescent-labelled antagonist, SCH 23390, in unfixed frozen sections. We found displaceable binding in the inner and outer plexiform layers and in the region of horizontal cell perikarya. We used an anti-peptide antibody directed to an extracellular domain of the rat D2 receptor and a fluorescent secondary antiserum to study the localization of D2 receptors. In addition to marked label in both plexiform layers, the outer, and especially the inner segments of rods and cones show specific immunoreactivity. In addition, there is distinct label at the level of the horizontal cell bodies; in the inner retina, specific fluorescence is found in somata of some amacrine cells. The significance of the connectivity pattern and the distribution of the two receptor types is discussed with respect to the role of dopamine in controlling adaptational processes in the outer retina, such as retinomotor movements and changes in horizontal cell morphology and physiology.

Oxygen diffusion in the trout retina

Oxygen tensions were measured in vivo within the different layers of the rainbow trout retina. Oxygen microelectrodes were advanced in 10 μm increments through the retinas and the P O 2 measured at each location. Mean retinal P O 2 ranged from 124 mm Hg at the retinal-vitreal interface to 281 mm Hg at the choriocapillaris. These data reaffirm that the cellular layers of the normal trout retina are continually exposed to supra-arterial oxygen tensions that are known to cause toxicity in other species. The intraretinal P O 2 gradient was mathematically characerized and results indicate that the in vivo oxygen profile can be described by a two-component exponential function. In order to gain a better understanding of oxygen delivery to the retina, the data were also subjected to an analysis based upon the classical equation for planar diffusion. The trout retina is an excellent model for studying oxygen diffusion in vivo since this tissue is supplied with oxygen from a single source, thereby simplifying the mathematical analysis. Calculations yielded values of 1·86 × 10 −5 and 0·58 × 10 −5 ml O 2 min −1 cm −1 atm −1 (at 9°C) for the Krogh permeation coefficient ( DS) for the photoreceptor region and the remaining neural retina, respectively.

Superoxide dismutase provides protection against the hyperoxia in the retina of the rainbow trout ( Salmo gairdneri)

1. 1. A modified hydroxylamine oxidation assay method coupled with a parallel line analysis to detect endogenous inferfering substances, was used to determine superoxide dismutase (SOD). 2. 2. Retinal SOD activities in trout, frog and rat were compared. SOD activity in liver and brain tissues was also determined. 3. 3. Based on estimates of superoxide anion production there appears to be excess SOD protection in the rainbow trout retina, which could, at least in part, account for its resistance to oxygen toxicity.

The Function of Photomechanical Movements in the Retina of the Rainbow Trout (Salmo Gairdneri)

ABSTRACT The function of photomechanical movements in the retina of rainbow trout (Salmo gairdneri) was investigated by determining both the effect of light on the level of extractable visual pigment, and the electroretinographic b-wave sensitivity, during various stages of photomechanical light and dark adaptation. Dark-adapted fish, light-adapted fish, and dark-adapted fish exposed to ten minutes direct sunlight had on average visual pigment concentrations of 100, 82 and 36% respectively. The intensity of illumination required to bleach a specified amount of visual pigment in the light-adapted retina was found to be 1·29 log units higher than that needed to bleach the same amount of visual pigment in a dark-adapted eye. The level of extractable visual pigment was observed to be relatively constant over natural twilight periods. A close temporal correlation was observed between the time course of electroretinographic adaptation, measured by the b-wave sensitivity, and photomechanical changes. All these observations tend to support the hypothesis that photo-mechanical movements serve, at least in part, to protect the rod visual pigment from overstimulation in the light-adapted retina.

Visual pigment changes in rainbow trout in response to temperature.

Lower water temperature (6 degrees C in comparison to 16 degrees C) favors a higher proportion of porphyropsin in the retina of rainbow trout (Salmo gairdneri), regardless of the light conditions (constant darkness or 12 hours of light and 12 of darkness). This response to temperature does not follow a Q10 relation, namely an increase in the rate of a reaction produced by raising the temperature 10 degrees C.

Comparison of direct and indirect calorimetry as a method for determining total energy produced by the isolated retina of rainbow trout ( Salmo gairdneri)

A method for determining heat generation in isolated retinas by direct calorimetry is described and comparisons are made between the values thus determined and values obtained by indirect calorimetry. The heat production figures obtained by these two methods were significantly different and they correspond closely to values calculated from previously published biochemical data. Even though indirect calorimetry does provide a good estimated of heat produced from aerobic reactions it cannot be used to estimate the total heat production in isolated tissues such as the teleost retina which relies on anaerobic biochemical reactions to provide some 50–60% of its total energy needs. It is concluded that in future determinations of metabolic rates on isolated tissues, consideration of non-oxidative pathways be made before indirect calorimetry is pursued.