A connectomics approach to understanding a retinal disease.

Macular telangiectasia type 2 (MacTel), a late-onset macular degeneration, has been linked to a loss in the retina of Müller glial cells and the amino acid serine, synthesized by the Müller cells. The disease is confined mainly to a central retinal region called the MacTel zone. We have used electron microscopic connectomics techniques, optimized for disease analysis, to study the retina from a 48-y-old woman suffering from MacTel. The major observations made were specific changes in mitochondrial structure within and outside the MacTel zone that were present in all retinal cell types. We also identified an abrupt boundary of the MacTel zone that coincides with the loss of Müller cells and macular pigment. Since Müller cells synthesize retinal serine, we propose that a deficiency of serine, required for mitochondrial maintenance, causes mitochondrial changes that underlie MacTel development.

Vitamin A: its many roles-from vision and synaptic plasticity to infant mortality.

The recognition that a dietary factor is essential to maintain good and sensitive vision as well as overall health goes back over 3,000 years to the ancient Egyptians. With the discovery of the vitamins at the turn of the twentieth century, fat-soluble vitamin A was soon shown to be the essential factor. In the first half of the twentieth century, the role vitamin A plays in vision, as precursor to the light-sensitive visual pigment molecules in the photoreceptors was elegantly worked out, especially by George Wald and his colleagues. Beginning in the 1960s, with the recognition of the active metabolite of vitamin A, its acid form now called retinoic acid, the roles of vitamin A in maintaining overall health of an organism began to be explored, and this research continues to this day. Receptors activated by retinoic acid, the RARs and RXRs have been shown to regulate gene transcription in a surprisingly wide variety of biological processes from early growth and development to the maintenance of epithelial tissues in many organs, the regulation of the immune system, and even the modulation of synaptic function in the brain involved in mechanisms underlying memory and learning. Therapeutic uses for retinoic acid have been developed, including one for a specific form of leukemia. The story is by no means complete and it is likely more surprises await with regard to this remarkable molecule.

myosin 7aa(-/-) mutant zebrafish show mild photoreceptor degeneration and reduced electroretinographic responses.

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.

The effects of nicotine on cone and rod b-wave responses in larval zebrafish.

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.

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.

Neuromodulation: Actions of Dopamine, Retinoic Acid, Nitric Oxide, and Other Substances on Retinal Horizontal Cells.

Whereas excitation and inhibition of neurons are well understood, it is clear that neuromodulatory influences on neurons and their synapses play a major role in shaping neural activity in the brain. Memory and learning, emotional and other complex behaviors, as well as cognitive disorders have all been related to neuromodulatory mechanisms. A number of neuroactive substances including monoamines such as dopamine and neuropeptides have been shown to act as neuromodulators, but other substances thought to play very different roles in the body and brain act as neuromodulators, such as retinoic acid. We still understand little about how neuromodulatory substances exert their effects, and the present review focuses on how two such substances, dopamine and retinoic acid, exert their effects. The emphasis is on the underlying neuromodulatory mechanisms down to the molecular level that allow the second order bipolar cells and the output neurons of the retina, the ganglion cells, to respond to different environmental (ie lighting) conditions. The modulation described affects a simple circuit in the outer retina, involves several neuroactive substances and is surprisingly complex and not fully understood.

Early retinoic acid deprivation in developing zebrafish results in microphthalmia.

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.

Restoring partial vision to a blind patient.

This paper reports an important breakthrough in partially restoring sight to a man who had lost his vision due to retinitis pigmentosa (RP), a heritable retinal degenerative disease that affects approximately 1 in 4000 people. Long considered an insurmountable challenge, a stellar team of vision scientists, engineers, basic biologists, and others, working together for many years, has enabled a man who had been legally blind for decades to begin distinguishing objects and navigating his environment1.

Cellular expression of Smarca4 (Brg1)-regulated genes in zebrafish retinas.

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.

Factorial microarray analysis of zebrafish retinal development.

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.

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.

Rods Contribute to Visual Behavior in Larval Zebrafish.

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.

Zebrafish: a model system for the study of eye genetics.

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.

Differential expression of duplicated VAL-opsin genes in the developing zebrafish.

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.

Morphological types and connectivity of horizontal cells found in the adult zebrafish (Danio rerio) retina.

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.

Probing pineal-specific gene expression with transgenic zebrafish.

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.

A Life in Vision.

I was drawn into research in George Wald's laboratory at Harvard, where as an undergraduate and graduate student, I studied vitamin A deficiency and dark adaptation. A chance observation while an assistant professor at Harvard led to the major research of my career-to understand the functional organization of vertebrate retinas. I started with a retinal circuit analysis of the primate retina with Brian Boycott and intracellular retinal cell recordings in mudpuppies with Frank Werblin. Subsequent pharmacology studies with Berndt Ehinger primarily with fish focused on dopamine and neuromodulation. Using zebrafish, we studied retinal development, neuronal connectivity, and the effects of genetic mutations on retinal structure and function. Now semi-retired, I have returned to primate retinal circuitry, undertaking a connectomic analysis of the human fovea in Jeffrey Lichtman's laboratory.