Farida Emran

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities

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.

OFF ganglion cells cannot drive the optokinetic reflex in zebrafish

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.

Zebrafish larvae lose vision at night.

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.

The bugeye mutant zebrafish exhibits visual deficits that arise with the onset of an enlarged eye phenotype.

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 mutants 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.

Cellular Expression of Smarca4 (Brg1)-regulated Genes in Zebrafish Retinas

BackgroundIn 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.ResultsThirty 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.ConclusionsThis 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.

Larval zebrafish turn off their photoreceptors at night.

Studies in several vertebrate species have shown that visual sensitivity and a number of other retinal phenomena are regulated by circadian mechanisms. For example, ultra-structural studies of 5 day old zebrafish larvae have shown that synaptic ribbons in photoreceptor terminals undergo dramatic diurnal alterations. These synaptic ribbons are very prominent during the day, but are almost completely absent at night. The implications of this circadian driven process on visual function are not well understood. We recently showed that larval zebrafish essentially lose visual responsiveness at night. This shut-down of retinal function at night is regulated by at least two mechanisms: the disassembly of synaptic ribbons in cone pedicles and a decrease of outer segment activity. Here, we summarize our recently reported observations and further discuss our hypothesis on how this phenomenon of shutting-down retinal function at night may provide a means for zebrafish larvae to conserve energy.