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Purpose: Although zebrafish rods begin to develop as early as 2 days postfertilization (dpf), they are not deemed anatomically mature and functional until 15 to 21 dpf. A recent study detected a small electroretinogram (ERG) from rods in a cone mutant called no optokinetic response f (nof) at 5 dpf, suggesting that young rods are functional. Whether they can mediate behavioral responses in larvae is unknown., Methods: We first confirmed rod function by measuring nof ERGs under photopic and scotopic illumination at 6 dpf. We evaluated the role of rods in visual behaviors using two different assays: the visual-motor response (VMR) and optokinetic response (OKR). We measured responses from wild-type (WT) larvae and nof mutants under photopic and scotopic illuminations at 6 dpf., Results: Nof mutants lacked a photopic ERG. However, after prolonged dark adaptation, they displayed scotopic ERGs. Compared with WT larvae, the nof mutants displayed reduced VMRs. The VMR difference during light onset gradually diminished with decreased illumination and became nearly identical at lower light intensities. Additionally, light-adapted nof mutants did not display an OKR, whereas dark-adapted nof mutants displayed scotopic OKRs., Conclusions: Because the nof mutants lacked a photopic ERG but displayed scotopic ERGs after dark adaptation, the mutants clearly had functional rods. WT larvae and the nof mutants displayed comparable scotopic light-On VMRs and scotopic OKRs after dark adaptation, suggesting that these responses were driven primarily by rods. Together, these observations indicate that rods contribute to zebrafish visual behaviors as early as 6 dpf.

Mutations in myosin VIIa (MYO7A) cause Usher Syndrome 1B (USH1B), a disease characterized by the combination of sensorineural hearing loss and visual impairment termed retinitis pigmentosa (RP). Although the shaker-1 mouse model of USH1B exists, only minor defects in the retina have been observed during its lifespan. Previous studies of the zebrafish mariner mutant, which also carries a mutation in myo7aa, revealed balance and hearing defects in the mutants but the retinal phenotype has not been described. We found elevated cell death in the outer nuclear layer (ONL) of myo7aa(-/-) mutants. While myo7aa(-/-) mutants retained visual behaviors in the optokinetic reflex (OKR) assay, electroretinogram (ERG) recordings revealed a significant decrease in both a- and b-wave amplitudes in mutant animals, but not a change in ERG threshold sensitivity. Immunohistochemistry showed mislocalization of rod and blue cone opsins and reduced expression of rod-specific markers in the myo7aa(-/-) ONL, providing further evidence that the photoreceptor degeneration observed represents the initial stages of the RP. Further, constant light exposure resulted in widespread photoreceptor degeneration and the appearance of large holes in the retinal pigment epithelium (RPE). No differences were observed in the retinomotor movements of the photoreceptors or in melanosome migration within the RPE, suggesting that myo7aa(-/-) does not function in these processes in teleosts. These results indicate that the zebrafish myo7aa(-/-) mutant is a useful animal model for the RP seen in humans with USH1B., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

In fish, axons that originate in the olfactory bulb innervate the retina and increase luminance sensitivity. In this issue of Neuron, Esposti et al. (2013) investigate the mechanisms underlying this interaction to report modulation of synaptic gain and sensitivity in OFF bipolar cells but rarely in ON bipolar cells., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Acetylcholine is present in and released from starburst amacrine cells in the inner plexiform layer (IPL), but its role in retinal function except, perhaps, in early development, is unclear. Nicotinic acetylcholine receptors are thought to be present on ganglion, amacrine, and bipolar cell processes in the IPL, and it is known that acetylcholine increases the spontaneous and light-evoked responses of retinal ganglion cells. The effects of acetylcholine on bipolar cells are not known, and here we report the effects of nicotine on the b-wave of the electroretinogram in larval zebrafish. The b-wave originates mainly from ON-bipolar cells, and the larval zebrafish retina is cone-dominated. Only small rod responses can be elicited with dim lights in wild-type larval zebrafish retinas, but rod responses can be recorded over a range of intensities in a mutant ( n o optokinetic response f ) fi sh that has no cone function. We fi nd that nicotine strongly enhances cone-driven b-wave response amplitudes but depresses rod driven b-wave response amplitudes without, however, affecting rod- or cone-driven b-wave light sensitivity.

Bipolar cells convey luminance, spatial, and color information from photoreceptors to amacrine and ganglion cells. We studied the photoreceptor connectivity of 321 bipolar cells in the adult zebrafish retina. 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) was inserted into whole-mounted transgenic zebrafish retinas to label bipolar cells. The photoreceptors that connect to these DiI-labeled cells were identified by transgenic fluorescence or their positions relative to the fluorescent cones, as cones are arranged in a highly ordered mosaic: rows of alternating blue- (B) and ultraviolet-sensitive (UV) single cones alternate with rows of red-(R) and green-sensitive (G) double cones. Rod terminals intersperse among cone terminals. As many as 18 connectivity subtypes were observed, 9 of which-G, GBUV, RG, RGB, RGBUV, RGRod, RGBRod, RGBUVRod, and RRod bipolar cells-accounted for 96% of the population. Based on their axon terminal stratification, these bipolar cells could be further subdivided into ON, OFF, and ON-OFF cells. The dendritic spread size, soma depth and size, and photoreceptor connections of the 308 bipolar cells within the nine common connectivity subtypes were determined, and their dendritic tree morphologies and axonal stratification patterns compared. We found that bipolar cells with the same axonal stratification patterns could have heterogeneous photoreceptor connectivity whereas bipolar cells with the same dendritic tree morphology usually had the same photoreceptor connectivity, although their axons might stratify on different levels., (Copyright © 2012 Wiley Periodicals, Inc.)

Vitamin A deficiency causes impaired vision and blindness in millions of children around the world. Previous studies in zebrafish have demonstrated that retinoic acid (RA), the acid form of vitamin A, plays a vital role in early eye development. The objective of this study was to describe the effects of early RA deficiency by treating zebrafish with diethylaminobenzaldehyde (DEAB), a potent inhibitor of the enzyme retinaldehyde dehydrogenase (RALDH) that converts retinal to RA. Zebrafish embryos were treated for 2 h beginning at 9 h postfertilization. Gross morphology and retinal development were examined at regular intervals for 5 days after treatment. The optokinetic reflex (OKR) test, visual background adaptation (VBA) test, and the electroretinogram (ERG) were performed to assess visual function and behavior. Early treatment of zebrafish embryos with 100 μM DEAB (9 h) resulted in reduced eye size, and this microphthalmia persisted through larval development. Retinal histology revealed that DEAB eyes had significant developmental abnormalities but had relatively normal retinal lamination by 5.5 days postfertilization. However, the fish showed neither an OKR nor a VBA response. Further, the retina did not respond to light as measured by the ERG. We conclude that early deficiency of RA during eye development causes microphthalmia as well as other visual defects, and that timing of the RA deficiency is critical to the developmental outcome.

Background: In a recent genomic study, Leung et al. used a factorial microarray analysis to identify Smarca4 (Brg1)-regulated genes in micro-dissected zebrafish retinas. Two hundred and fifty nine genes were grouped in three-way ANOVA models which carried the most specific retinal change. To validate the microarray results and to elucidate cellular expression patterns of the significant genes for further characterization, 32 known genes were randomly selected from this group. In situ hybridization of these genes was performed on the same types of samples (wild-type (WT) and smarca4a50/a50 (yng) mutant) at the same stages (36 and 52 hours post-fertilization (hpf)) as in the microarray study., Results: Thirty out of 32 riboprobes showed a positive in situ staining signal. Twenty seven out of these 30 genes were originally further classified as Smarca4-regulated retinal genes, while the remaining three as retinal-specific expression independent of Smarca4 regulation. It was found that 90.32% of the significant microarray comparisons that were used to identify Smarca4-regulated retinal genes had a corresponding qualitative expression change in the in situ hybridization comparisons. This is highly concordant with the theoretical true discovery rate of 95%. Hierarchical clustering was used to investigate the similarity of the cellular expression patterns of 25 out of the 27 Smarca4-regulated retinal genes that had a sufficiently high expression signal for an unambiguous identification of retinal expression domains. Three broad groups of expression pattern were identified; including 1) photoreceptor layer/outer nuclear layer specific expression at 52 hpf, 2) ganglion cell layer (GCL) and/or inner nuclear layer (INL) specific expression at both 36 & 52 hpf, and 3) GCL and/or INL specific expression at 52 hpf only. Some of these genes have recently been demonstrated to play key roles in retinal cell-type specification, differentiation and lamination. For the remaining three retinal-specific genes that are independent of Smarca4 regulation, they all had a subtle expression difference between WT and smarca4a50/a50 retinas as detected by in situ hybridization. This subtle expression difference was also detected by the original microarray analysis. However, the difference was lower than the fold change cut-off used in that study and hence these genes were not inferred as Smarca4-regulated retinal genes., Conclusions: This study has successfully investigated the expression pattern of 32 genes identified from the original factorial microarray analysis. The results have demonstrated that the true discovery rate for identifying Smarca4-regulated retinal genes is 90.3%. Hence, the significant genes from the microarray study are good candidates for cell-type specific markers and will aid further investigation of retinal differentiation.

Purpose: The bugeye mutant has an enlarged eye phenotype, presumably because of elevated intraocular pressure. Since elevated intraocular pressure is a significant risk factor for glaucoma, the bugeye zebrafish mutant may be a model organism for the disease., Methods: The optomotor response (OMR) was used to assess visual responsiveness in both larval and adult zebrafish. Electroretinograms (ERGs) were recorded to measure outer retinal function, and histologic analyses were performed on WT and mutant eyes., Results: At 5 days old, bugeye mutants have an OMR, ERGs, and retinal morphology indistinguishable from those of wild-type (WT) animals. By 2 months of age, bugeye mutants begin to develop an enlarged eye phenotype. At 3 months, some mutants show deficits in the OMR assay, including lower contrast sensitivity. The data suggest that there is a correlation between the size of the enlarged eye and the degree of OMR deficit. Histologic analysis of the bugeye mutant retina revealed decreases in retinal ganglion cell densities by 3 months. By 5 months, the mutant's ERG b-wave had smaller amplitudes and longer latencies at brighter light intensities than those of the WT fish., Conclusion: After phenotypic onset at 3 months, the bugeye mutants begin to develop visual deficits. At 3 months, bugeye mutants exhibit a decrease in retinal cell densities and by 5 months, they show diminished outer retinal function. In summary, the bugeye mutant provides a means of studying glaucoma-associated phenotypes in the zebrafish.

Darkness serves as a stimulus for vertebrate photoreceptors; they are actively depolarized in the dark and hyperpolarize in the light. Here, we show that larval zebrafish essentially turn off their visual system at night when they are not active. Electroretinograms recorded from larval zebrafish show large differences between day and night; the responses are normal in amplitude throughout the day but are almost absent after several hours of darkness at night. Behavioral testing also shows that larval zebrafish become unresponsive to visual stimuli at night. This phenomenon is largely circadian driven as fish show similar dramatic changes in visual responsiveness when maintained in continuous darkness, although light exposure at night partially restores the responses. Visual responsiveness is decreased at night by at least two mechanisms: photoreceptor outer segment activity decreases and synaptic ribbons in cone pedicles disassemble.

Horizontal cells (HCs) are involved in establishing the center-surround receptive field organization of photoreceptor and bipolar cells. In many species, HCs respond differentially to colors and may play a role in color vision. An earlier study from our laboratory suggested that four types of HCs exist in the zebrafish retina: three cone HCs (H1, H2 and H3) and one rod HC. In this study, we describe their photoreceptor connections. Cones are arranged in a mosaic in which rows of alternating blue (B)- and ultraviolet (UV)-sensitive single cones alternate with rows of red (R)- and green (G)-sensitive double cones; the G cones are adjacent to UV cones and B cones adjacent to R cones. Two small-field (H1 and H2) and two large-field (H3 and rod HC) cells were observed. The cone HC dendritic terminals connected to cones with single boutons, doublets, or rosettes, whereas the rod HCs connected to rods with single boutons. The single boutons/doublets/rosettes of cone HCs were arranged in double rows separated by single rows for H1 cells, in pairs and singles for H2 cells, and in a rectilinear pattern for H3 cells. These connectivity patterns suggest that H1 cells contact R, G, and B cones, H2 cells G, B, and UV cones, and H3 cells B and UV cones. These predictions were confirmed by applying the DiI method to SWS1-GFP retinas whose UV cones express green fluorescent protein. Each rod HC was adjacent to the soma or axon of a DiI-labeled cone HC and connected to 50-200 rods.

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the visual scene. However, the OKR requires that an animal detect moving stripes and it is possible that fish that fail to exhibit an OKR may not be completely blind. One zebrafish mutant, the no optokinetic response c (nrc) has no OKR under any light conditions tested and was reported to be completely blind. Previously, we have shown that OFF-ganglion cell activity can be recorded in these mutants. To determine whether mutant fish with no OKR such as the nrc mutant can detect simple light increments and decrements we developed the visual motor behavioral assay (VMR). In this assay, single zebrafish larvae are placed in each well of a 96-well plate allowing the simultaneous monitoring of larvae using an automated video-tracking system. The locomotor responses of each larva to 30 minutes light ON and 30 minutes light OFF were recorded and quantified. WT fish have a brief spike of motor activity upon lights ON, known as the startle response, followed by return to lower-than baseline activity, called a freeze. WT fish also sharply increase their locomotor activity immediately following lights OFF and only gradually (over several minutes) return to baseline locomotor activity. The nrc mutants respond similarly to light OFF as WT fish, but exhibit a slight reduction in their average activity as compared to WT fish. Motor activity in response to light ON in nrc mutants is delayed and sluggish. There is a slow rise time of the nrc mutant response to light ON as compared to WT light ON response. The results indicate that nrc fish are not completely blind. Because teleosts can detect light through non-retinal tissues, we confirmed that the immediate behavioral responses to light-intensity changes require intact eyes by using the chokh (chk) mutants, which completely lack eyes from the earliest stages of development. In our VMR assay, the chk mutants exhibit no startle response to either light ON or OFF, showing that the lateral eyes mediate this behavior. The VMR assay described here complements the well-established OKR assay, which does not test the ability of zebrafish larvae to respond to changes in light intensities. Additionally, the automation of the VMR assay lends itself to high-throughput screening for defects in light-intensity driven visual responses.

In a zebrafish recessive mutant young (yng), retinal cells are specified to distinct cell classes, but they fail to morphologically differentiate. A null mutation in a brahma-related gene 1 (brg1) is responsible for this phenotype. To identify retina-specific Brg1-regulated genes that control cellular differentiation, we conducted a factorial microarray analysis. Gene expression profiles were compared from wild-type and yng retinas and stage-matched whole embryos at 36 and 52 hours postfertilization (hpf). From our analysis, three categories of genes were identified: (i) Brg1-regulated retinal differentiation genes (731 probesets), (ii) retina-specific genes independent of Brg1 regulation (3,038 probesets), and (iii) Brg1-regulated genes outside the retina (107 probesets). Biological significance was confirmed by further analysis of components of the Cdk5 signaling pathway and Irx transcription factor family, representing genes identified in category 1. This study highlights the utility of factorial microarray analysis to efficiently identify relevant regulatory pathways influenced by both specific gene products and normal developmental events.

The pineal gland of zebrafish (Danio rerio) contains light-sensitive photoreceptor cells and plays an important role in the neuroendocrine system. The zebrafish exorhodopsin gene encodes a pineal-specific photoreceptive protein, whose promoter region harbors a cis-acting element, pineal expression-promoting element (PIPE), directing pineal-specific gene expression. For in vivo genetic studies on PIPE-binding proteins and their regulatory mechanisms, we generated a transgenic zebrafish line, Tg(P(20)-rh/P:gfp), that expresses green fluorescent protein (GFP) under the control of the zebrafish rhodopsin promoter fused with 20 PIPE repeats. In Tg(P(20)-rh/P:gfp) fish, PIPE-dependent gene expression is visualized by GFP fluorescence in the pineal gland along with PIPE-independent GFP signals in the retinal rod photoreceptors. The transgenic fish exhibit detectable and reproducible GFP fluorescence in the larval pineal gland by 5 days postfertilization. Antisense morpholino-mediated knock-down of a pineal transcription factor gene, otx5, suppresses pineal GFP expression in the transgenic line. In a pilot screen of N-ethyl-N-nitrosourea-treated fish of the GFP transgenic line, we isolated potential dominant mutations that cause attenuation of pineal GFP fluorescence with a marginal effect on the retinal GFP signal. The results suggest that the Tg(P(20)-rh/P:gfp) line will be useful for detecting deficits in PIPE-dependent gene expression in the pineal gland.

We describe here different types of horizontal cells in the zebrafish retina and how they connect to photoreceptors. To label horizontal cells, crystals of DiI were placed onto the tips of pulled glass pipettes and inserted into the inner nuclear layer of fixed whole-mount retinas. The DiI-labeled horizontal cells were imaged by confocal microscopy and analyzed according to dendritic arborization, cell depth, dendritic terminal morphology, and connectivity with photoreceptors. Three types of horizontal cells were unequivocally identified: two cone-connecting (H1/2 and H3) and one rod-related cell. H1/2 cells have dendritic terminals that are arranged in "rosette" clusters and that connect to cone photoreceptors without any apparent specificity. H3 cells are larger and have dendritic terminal doublets arranged in a rectilinear pattern. This pattern corresponds to the mosaic of the single cones in the zebrafish photoreceptor mosaic and indicates that H3 cells connect specifically to either the blue-sensitive (long-single) or ultraviolet-sensitive (short-single) cones. Thus, H3 cells are likely to be chromaticity-type cells that process specific color information, whereas H1/2 cells are probably luminosity-type cells that process luminance information. Rod horizontal cells were identified by their shape and dendritic pattern, and they connect with numerous rod photoreceptors via small spherical terminals., ((c) 2007 Wiley-Liss, Inc.)

Over the last decade, the use of the zebrafish as a genetic model has moved beyond the proof-of-concept for the analysis of vertebrate embryonic development to demonstrated utility as a mainstream model organism for the understanding of human disease. The initial identification of a variety of zebrafish mutations affecting the eye and retina, and the subsequent cloning of mutated genes have revealed cellular, molecular and physiological processes fundamental to visual system development. With the increasing development of genetic manipulations, sophisticated techniques for phenotypic characterization, behavioral approaches and screening strategies, the identification of novel genes or novel gene functions will have important implications for our understanding of human eye diseases, pathogenesis, and treatment.

Whereas the zebrafish retina has long been an important model system for developmental and genetic studies, little is known about the responses of the inner retinal neurons. Here we report single-unit ganglion cell recordings from 5- to 6-day-old zebrafish larvae. In wild-type larvae we identify at least five subtypes of ganglion cell responses to full-field illumination, with ON-OFF and ON-type cells predominating. In the nrc mutant retina, in which the photoreceptor terminals develop abnormally, we observe normal OFF responses but abnormal ON-OFF responses and no ON responses. Previously characterized as blind, these mutants lack an optokinetic reflex (OKR), but in another behavioral assay nrc mutant fish have near-normal responses to the offset of light and slow and sluggish responses to the onset of light. Pharmacological block of the ON pathway mimics most of the nrc visual defects. We conclude that the abnormal photoreceptor terminals in nrc mutants predominantly perturb the ON pathway and that the ON pathway is necessary to drive the OKR in larval zebrafish.

Purpose: Eye development and photoreceptor maintenance is dependent on the retinal pigment epithelium (RPE), a thin layer of cells that underlies the neural retina. Despite its importance, development of RPE has not been studied by a genomic approach. In this study, a microarray expression-profiling methodology was established for studying RPE development., Methods: The intact retina with RPE attached was dissected from developing embryos, and differentially expressed genes in RPE were inferred by comparing the dissected tissues with retinas without RPE, in microarray and statistical analyses., Results: Of the probesets used, 8810 were significantly expressed in RPE at 52 hours postfertilization (hpf), of which 1443 may have biologically meaningful expression levels. Further, 78 and 988 probesets were found to be significantly over- or underexpressed in RPE, respectively, compared with retina. Also, 79.2% (38/48) of the known overexpressed probesets were independently validated as RPE-related transcripts., Conclusions: The results strongly suggest that this methodology can obtain in vivo RPE-specific gene expression from the zebrafish embryos and identify novel RPE markers.

Purpose: Children born to mothers who have consumed alcohol during pregnancy have an array of retinal abnormalities and visual dysfunctions. In the past, rodent systems have been used to study the teratogenic effects of ethanol on vertebrate embryonic development. The exact developmental windows in which ethanol causes specific developmental defects have been difficult to determine because rodents and other mammals develop in utero. In this study, we characterized how ethanol affects the function and development of the visual system in an ex utero embryonic system, the zebrafish., Methods: Zebrafish embryos were raised in fish water containing various concentrations of ethanol from 2 to 5 days after fertilization. The effects of ethanol on retinal morphology were assessed by histologic and immunohistochemical analyses and those on retinal function were analyzed by optokinetic response (OKR) and electroretinography (ERG)., Results: Zebrafish embryos exposed to moderate and high levels of ethanol during early embryonic development had morphological abnormalities of the eye characterized by hypoplasia of the optic nerve and inhibition of photoreceptor outer segment growth. Ethanol treatment also caused an increased visual threshold as measured by the OKR. Analysis with the ERG indicated that there was a severe reduction of both the a- and b-waves, suggesting that ethanol affects the function of the photoreceptors. Indeed, low levels of ethanol that did not cause obvious morphologic changes in either the body or retina did affect both the OKR visual threshold and the a- and b-wave amplitudes., Conclusions: Ethanol affects photoreceptor function at low concentrations that do not disturb retinal morphology. Higher levels of ethanol inhibit photoreceptor development and cause hypoplasia of the optic nerve.

Whole cell patch recording was performed from morphologically identified cone-driven on-off bipolar cells (Cabs) in giant danio retinal slices to study their glutamate receptors and light-evoked responses. Specific agonists were puffed in the presence of cobalt, picrotoxin, and strychnine to identify glutamate receptors on these cells. Most Cabs responded to both the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptor agonist kainate and the excitatory amino acid transporter (EAAT) substrate D-aspartate, and both responses were localized to the dendrites. Kainate generated depolarizations whereas D-aspartate had E(rev) close to E(Cl) and generated hyperpolarizations, indicating that the AMPA/kainate receptors are sign-preserving, whereas the EAATs are sign-inverting. In response to white light, some Cabs gave on bipolar cell-like responses whereas others gave off bipolar cell-like ones, but many cells' responses had both on and off bipolar cell components. In response to appropriately colored center-selective stimuli, many Cabs responded to short and long wavelengths with opposite polarities and were thus double color-opponent. The depolarizing components of the responses to white or colored stimuli were suppressed by the EAAT blocker DL-threo-beta-benzyloxyaspartate (TBOA), whereas the hyperpolarizing components were reduced by the AMPA/kainate receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX). These results are consistent with the hypothesis that both EAATs and AMPA/kainate receptors are involved in the generation of light-evoked responses in Cabs and that they confer these cells with on and off bipolar cell properties, respectively. Cabs can generate double color-opponent center responses by receiving inputs from certain cones through EAATs and from other cones through AMPA/kainate receptors.

Several molecules, such as growth factors and neurotrophic factors, are required both for the differentiation of specific retinal cell types and the long-term cell survival of all retinal neurons. As diffusible factors, these molecules act non-cell-autonomously. Here, we describe the loss of function phenotype for dazed (dzd), a gene that acts cell-autonomously for retinal cell survival and affects the differentiation of rod photoreceptors and the Muller glia. By 3 days after fertilization, dazed mutant embryos have small eyes and slight heart edema. Acridine orange staining indicated a significant degree of retinal cell death occurring by 48 hr after fertilization, and histological analysis revealed that dying cells were found in the inner and outer nuclear layers and near the marginal zones. Although molecular and morphological differentiation of the inner retina and cone photoreceptors occurred, rod photoreceptors failed to differentiate beyond a small patch in the ventral retina and rod precursors failed to respond to exogenously added retinoic acid, which normally potentiated rod differentiation. Mosaic analysis indicated that the dazed gene acts cell-autonomously for rod production and cell survival, as dazed clones failed to produce rods outside the ventral patch and dazed cells were not maintained in wild-type hosts. Raising mutants under constant light resulted in severe retinal degeneration, whereas raising embryos under constant darkness did not provide any additional protection from cell death. Behavioral analysis showed that a subpopulation of adult fish that were heterozygous for the dazed mutation had elevated visual thresholds and were night blind, suggesting that dazed may also be required for long-term dim-light vision. Taken together, our studies suggest a role for the dazed gene in rod and Muller cell development and overall retinal cell survival and maintenance., (Copyright 2005 Wiley-Liss, Inc)

Genetic analysis in zebrafish has been instrumental in identifying genes necessary for visual system development and function. Recently, a large-scale retroviral insertional mutagenesis screen, in which 315 different genes were mutated, that resulted in obvious phenotypic defects by 5 days postfertilization was completed. That the disrupted gene has been identified in each of these mutants provides unique resource through which the formation, function, or physiology of individual organ systems can be studied. To that end, a screen for visual system mutants was performed on 250 of the mutants in this collection, examining each of them histologically for morphological defects in the eye and behaviorally for overall visual system function. Forty loci whose disruption resulted in defects in eye development and/or visual function were identified. The mutants have been divided into the following phenotypic classes that show defects in: (1) morphogenesis, (2) growth and central retinal development, (3) the peripheral marginal zone, (4) retinal lamination, (5) the photoreceptor cell layer, (6) the retinal pigment epithelium, (7) the lens, (8) retinal containment, and (9) behavior. The affected genes in these mutants highlight a diverse set of proteins necessary for the development, maintenance, and function of the vertebrate visual system.

The mechanisms by which retinal neurons are patterned along the dorsal/ventral axis remain largely unknown, yet this patterning is integral for the topographic mapping of visual space. With an interest in elucidating the mechanisms that regulate the development of this retinal axis, we have characterized a T-box family transcription factor, Tbx2b, during zebrafish retinogenesis. Tbx2b is expressed throughout all phases of retinal development with a striking asymmetry of distribution highest dorsally to lowest ventrally. To examine Tbx2b function during retinal development, two morpholino antisense oligonucleotides were created; one blocking the translational start site of Tbx2b and the other interfering with Tbx2b mRNA splicing. Injection of either of these morpholinos resulted in profound defects in the development of the dorsal retina. By using molecular markers for neuronal subtypes, the ventral retina contained all cell types, whereas in the dorsal retina, only retinal ganglion cells expressed markers of differentiation. The cells of the dorsal retina were postmitotic, however, as demonstrated by a lack of BrdUrd incorporation during the normal periods of retinal differentiation. Markers for dorsal and ventral retinal compartments were also expressed normally in Tbx2b morphants. Combined, these observations suggest that the cellular mechanisms regulating neuronal differentiation within the retina are asymmetric about the dorsal/ventral axis and that Tbx2b mediates this process within the dorsal retina.

A behavioral assay based on the optokinetic reflex was used to screen chemically mutagenized zebrafish larvae for deficits in visual function. A homozygous recessive mutation, lazy eyes (lze), was isolated based on the observation that 5-day postfertilization (dpf) mutants displayed weaker and less frequent eye movements than wild-type fish in response to moving stripes. Electroretinographic (ERG) recordings revealed that mutants had severely reduced a- and b-wave amplitudes relative to wild-type fish, indicating outer retinal dysfunction. Retinal lamination and cellular differentiation were normal in the lze retina; however, mutant photoreceptor cells had small outer segments and pyknotic nuclei were occasionally observed in the outer retina and the marginal zone of lze. Cone, rod, amacrine, bipolar, and Müller cell marker analyses indicated that the typical lze retina contained fewer rod photoreceptors and fewer Müller cells than wild-type fish at 5 dpf. At 3 dpf, however, mutant retinas had normal numbers of rod photoreceptors and Müller cells, suggesting that the initial differentiation of these cell types occurred normally. Rod photoreceptor histology was normal at this early stage, but Müller cells were often hypertrophied, suggesting that they were unhealthy. Constant light rearing of mutant animals accelerated the Müller cell degeneration, severely worsened the visual deficit, but had no obvious affect on the photoreceptors. When ERG responses and Müller cell degeneration from the same mutant animals were analyzed, the extent of the Müller cell loss matched closely the degree to which ERG responses were reduced. In summary, the lze gene appears to be required for Müller cell viability and normal visual function. The lze mutant may be a model for the study of the involvement of Müller cells in photoreceptor development and function., (Copyright 2003 Wiley-Liss, Inc.)

Non-visual opsins mediate various light-dependent physiological events. Our previous search for non-visual opsin genes in zebrafish led to the discovery of VAL-opsin (VAL-opsinA) in deep brain cells and retinal horizontal cells of the adult fish. In this study, we report the identification and characterization of its duplicated gene, VAL-opsinB, in zebrafish. A molecular phylogenetic analysis indicates that VAL-opsinB is orthologous to a previously reported salmon gene and that the duplication of the VAL-opsin gene occurred in the teleost lineage. The recombinant protein of zebrafish VAL-opsinB forms a green-sensitive photopigment when reconstituted with 11- cis-retinal. VAL-opsinB expression was detected in a limited number of cells of the brain and the eye, and the expression pattern is distinct from that of the VAL-opsinA gene. Such a differential expression pattern suggests that VAL-opsinA and VAL-opsinB are involved in different physiological events in zebrafish. [ABSTRACT FROM AUTHOR]

Abstract: Finding genetic polymorphisms and mutations linked to addictive behavior can provide important targets for pharmaceutical and therapeutic interventions. Forward genetic approaches in model organisms such as zebrafish provide a potentially powerful avenue for finding new target genes. In order to validate this use of zebrafish, the molecular nature of its reward system must be characterized. We have previously reported the use of cocaine-induced conditioned place preference (CPP) as a reliable method for screening mutagenized fish for defects in the reward pathway. Here we test if CPP in zebrafish involves the dopaminergic system by co-treating fish with cocaine and dopaminergic antagonists. Sulpiride, a potent D2 receptor (DR2) antagonist, blocked cocaine-induced CPP, while the D1 receptor (DR1) antagonist SCH23390 had no effect. Acute cocaine exposure also induced a rise in the expression of tyrosine hydroxylase (TH), an important enzyme in dopamine synthesis, and a significant decrease in the expression of elongation factor 1α (EF1α), a housekeeping gene that regulates protein synthesis. Cocaine selectively increased the ratio of TH/EF1α in the telencephalon, but not in other brain regions. The cocaine-induced change in TH/EF1α was blocked by co-treatment with sulpiride, but not SCH23390, correlating closely with the action of these drugs on the CPP behavioral response. Immunohistochemical analysis revealed that the drop in EF1α was selective for the dorsal nucleus of the ventral telencephalic area (Vd), a region believed to be the teleost equivalent of the striatum. Examination of TH mRNA and EF1α transcripts suggests that regulation of expression is post-transcriptional, but this requires further examination. These results highlight important similarities and differences between zebrafish and more traditional mammalian model organisms. [Copyright &y& Elsevier]