Adult Zebrafish Retina Research Articles

3571 Expression profile of the ribbon synapse protein Ribeye in the zebrafish

OBJECTIVES/SPECIFIC AIMS: Although two Ribeye protein isoforms have been identified in zebrafish, information about the identities of their variants is incomplete. This study aims to identify and characterize both of the Ribeye isoforms and their splice variants. METHODS/STUDY POPULATION: Immunohistochemistry was performed on the retina and neuromasts of zebrafish larva and adults.Ribeye expression was analyzed by western blot. Ribeye proteins will be separated, isolated and identified by mass spectrophotometry. RESULTS/ANTICIPATED RESULTS: Immunohistochemistry performed on larval and adult zebrafish retinas revealed the expression of Ribeye A in the inner and outer plexiform layers. Ribeye B was likewise expressed in both plexiform layers in larval zebrafish, but more pronounced expression in the outer plexiform layer in the adult zebrafish retina. Immunohistochemical experiments also demonstrated the co-expression of both Ribeye isoforms in the hair cells of both larval and adult neuromasts. Analysis of Ribeye expression by western blot showed the presence of more than the three previously identified variants. Current experiments are being conducted to characterize the additional Ribeye variants. We expect to identify the residual Ribeye protein as a result of this analysis. DISCUSSION/SIGNIFICANCE OF IMPACT: This study is necessary in order to gain a clear understanding of Ribeye expression in zebrafish tissues. Doing so will enable us to target this protein for gene editing to address outstanding questions about the mechanisms that govern ribbon synapse function. Synapse and synapse-associated proteins are involved in a wide-array of diseases that arise as a result of their dysfunction (e.g. blindness, deafness, bradycardia, autism spectrum disorders, and schizophrenia). Thus, it is important for us to identify the shared and distinct mechanisms that give rise to diseases associated with synaptic dysregulation. Such information could provide the basis for novel therapeutic interventions for synaptic disorders.

Differential expression of PKCα and -β in the zebrafish retina.

The retina is a complex neural circuit, which processes and transmits visual information from light perceiving photoreceptors to projecting retinal ganglion cells. Much of the computational power of the retina rests on signal integrating interneurons, such as bipolar cells. Commercially available antibodies against bovine and human conventional protein kinase C (PKC) α and -β are frequently used as markers for retinal ON-bipolar cells in different species, despite the fact that it is not known which bipolar cell subtype(s) they actually label. In zebrafish (Danio rerio) five prkc genes (coding for PKC proteins) have been identified. Their expression has not been systematically determined. While prkcg is not expressed in retinal tissue, the other four prkc (prkcaa, prkcab, prkcba, prkcbb) transcripts were found in different parts of the inner nuclear layer and some as well in the retinal ganglion cell layer. Immunohistochemical analysis in adult zebrafish retina using fluorescent in situ hybridization and PKC antibodies showed an overlapping immunolabeling of ON-bipolar cells that are most likely of the BON s6 and BON s6L or RRod type. However, comparison of transcript expression with immunolabeling, implies that these antibodies are not specific for one single zebrafish conventional PKC, but rather detect a combination of PKC -α and -β variants.

Extensive growth is followed by neurodegenerative pathology in the continuously expanding adult zebrafish retina.

The development of effective treatments for age-related neurodegenerative diseases remains one of the biggest medical challenges today, underscoring the high need for suitable animal model systems to improve our understanding of aging and age-associated neuropathology. Zebrafish have become an indispensable complementary model organism in gerontology research, yet their growth-control properties significantly differ from those in mammals. Here, we took advantage of the clearly defined and highly conserved structure of the fish retina to study the relationship between the processes of growth and aging in the adult zebrafish central nervous system (CNS). Detailed morphological measurements reveal an early phase of extensive retinal growth, where both the addition of new cells and stretching of existent tissue drive the increase in retinal surface. Thereafter, and coinciding with a significant decline in retinal growth rate, a neurodegenerative phenotype becomes apparent,-characterized by a loss of synaptic integrity, an age-related decrease in cell density and the onset of cellular senescence. Altogether, these findings support the adult zebrafish retina as a valuable model for gerontology research and CNS disease modeling and will hopefully stimulate further research into the mechanisms of aging and age-related pathology.

Poor Splice-Site Recognition in a Humanized Zebrafish Knockin Model for the Recurrent Deep-Intronic c.7595-2144A>G Mutation in USH2A.

The frequent deep-intronic c.7595-2144A>G mutation in intron 40 of USH2A generates a high-quality splice donor site, resulting in the incorporation of a pseudoexon (PE40) into the mature transcript that is predicted to prematurely terminate usherin translation. Aberrant USH2A pre-mRNA splicing could be corrected in patient-derived fibroblasts using antisense oligonucleotides. With the aim to study the effect of the c.7595-2144A>G mutation and USH2A splice redirection on retinal function, a humanized zebrafish knockin model was generated, in which 670 basepairs of ush2a intron 40 were exchanged for 557 basepairs of the corresponding human sequence using an optimized CRISPR/Cas9-based protocol. However, in the retina of adult homozygous humanized zebrafish, only 7.4% ± 3.9% of ush2a transcripts contained the human PE40 sequence and immunohistochemical analyses revealed no differences in the usherin expression and localization between the retina of humanized and wild-type zebrafish larvae. Nevertheless, we were able to partially correct aberrant ush2a splicing using a PE40-targeting antisense morpholino. Our results indicate a clear difference in splice-site recognition by the human and zebrafish splicing machinery. Therefore, we propose a protocol in which the effect of human splice-modulating mutations is studied in a zebrafish-specific cell-based splice assay before the generation of a humanized zebrafish knockin model.

Characterization of proliferative, glial and angiogenic responses after a CoCl2 -induced injury of photoreceptor cells in the adult zebrafish retina.

The adult zebrafish is considered a useful model for studying mechanisms involved in tissue growth and regeneration. We have characterized cytotoxic damage to the retina of adult zebrafish caused by the injection of cobalt chloride (CoCl2 ) into the vitreous cavity. The CoCl2 concentration we used primarily caused injury to photoreceptors. We observed the complete disappearance of cones, followed by rods, across the retina surface from 28 to 96hr after CoCl2 injury. The loss of 30% of bipolar cells was also observed by 50hr after lesion (hpl). CoCl2 injury provoked a strong induction of the proliferative activity of multipotent Müller glia and derived progenitors. The effect of CoCl2 on retina cells was significantly reduced by treatment with glutamate ionotropic receptor antagonists. Cone photoreceptor regeneration occurred 25days after injury. Moreover, a single dose of CoCl2 induced vascular damage and regeneration, whereas three injections of CoCl2 administered weekly provoked neovascular-like changes 20days after injury. CoCl2 injury also caused microglial reactivity in the optic disc, retina periphery and fibre layer. CoCl2 -induced damage enhanced pluripotency and proneural transcription factor gene expression in the mature retina 72 hpl. Tumour necrosis factor alpha, vascular endothelial growth factor (VEGF) and VEGF receptor mRNA levels were also significantly enhanced by 72 hpl. The injury paradigm we have described in this work may be useful for the discovery of signalling molecules and pathways that participate in the regenerative response and it may serve as a model to screen for compounds that could potentially treat aberrant angiogenesis.

Effect of Resveratrol on Sirtuins, OPA1, and Fis1 Expression in Adult Zebrafish Retina.

We determined whether sirtuins (SIRT1-SIRT7) are expressed in the zebrafish retina, evaluated the modulatory effect of resveratrol in the normal retina, and examined N-Methyl-D-aspartic acid (NMDA)-induced zebrafish retinal damage associated with mitochondrial sirtuins and mitochondrial fusion and fission mediators, OPA1 and Fis1. Sirtuins, OPA1, and Fis1 mRNA expression was analyzed by RT-PCR and quantitative real time PCR (qPCR) in adult zebrafish (AB type) retina and liver. qPCR showed an effect of resveratrol on SIRTs (SIRT1, 3, 4, 5) and OPA1 and Fis1 in low and high concentrations (5 and 50 mg/L) at different time points (0, 1, 24, and 48 hours) in the retina. Western blots were performed to examine the expression of SIRTs and OPA1 proteins under high concentrations of resveratrol for 24 hours. Hematoxylin and eosin staining, qPCR and mitochondrial copy number, and DNA damage assays then were used to confirm the protective effects of resveratrol on NMDA-induced retinal damage. The seven sirtuins and OPA1 were highly expressed in zebrafish retina compared to the liver. Treatment with resveratrol promoted SIRT1, mitochondrial sirtuins, and OPA1 gene and protein expression, and improved mitochondrial DNA repair in adult zebrafish retina. Interestingly, the effect of resveratrol on SIRT4 gene and protein expression was significantly higher in the zebrafish retina. Importantly, resveratrol offered protection against NMDA-induced retinal damage by activating the SIRT1 gene and subsequent protein expression. Mitochondrial sirtuins and OPA1 genes likely had a role in regulating mitochondrial dynamics. To our knowledge, our study is the first composite analysis of sirtuins in adult zebrafish retina and provides sufficient evidence that resveratrol, as an activator of SIRT1, protects NMDA-induced zebrafish retinal damage by potentially mediating mitochondrial sirtuins and OPA1 genes.

Transient acceleration of autophagic degradation by pharmacological Nrf2 activation is important for retinal pigment epithelium cell survival

Non-exudative age-related macular degeneration (AMD) is mainly caused by the accumulation of lipofuscin and drusen on the retinal pigment epithelium (RPE). Both oxidative stress and autophagic dysfunction accelerate the deposition of lipofuscin at the RPE. One of the key regulators in the response against oxidative stress is the NF-E2-Related Factor 2 (Nrf2)-kelch like ECH associated protein 1 (Keap1) axis, which is also closely associated with the autophagy pathway. Nrf2 activation upregulates the expression levels of certain anti-oxidative enzymes [e.g. Heme oxygenase-1 (HO-1)], which attenuates oxidative damage. However, until now, the relationship between cytoprotective effects of Nrf2 activation and autophagic degradation remain unclear. To address these questions, we investigated the effects of a novel Nrf2 activator, RS9, on RPE damage. We found that RS9 protected ARPE-19 cells against NaIO3-induced oxidative damage, and that the protective effects of RS9 were inhibited by co-treatment with zinc protoporphyrin, an HO-1 inhibitor. Next, we examined the involvement of autophagic degradation in the protective effects of RS9. Co-treatment with RS9 and chloroquine, a lysosomal acidification inhibitor, inhibited the protective effect. Furthermore, western blotting and immunostaining showed that RS9 accelerated autophagy flux and induced transient upregulation of p62 [also known as sequestosome 1 (SQSTM1)]. Co-treatment with chloroquine and RS9 also inhibited the degradation of autophagosomes. Transient upregulation of SQSTM1 by RS9 was unaltered by HO-1 knockdown using siRNA. RS9 and chloroquine had the same actions in light damaged adult zebrafish retina as those in vitro. In conclusion, we clarified the relationship between acceleration of the autophagy pathway and the cytoprotective effects of Nrf2 activation in RPE cells and zebrafish retina. These findings indicated that Nrf2 activation could be a promising therapeutic approach for non-exudative AMD by supporting RPE maintenance.

Capn5 Expression in the Healthy and Regenerating Zebrafish Retina.

PurposeAutosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV) is a devastating inherited autoimmune disease of the eye that displays features commonly seen in other eye diseases, such as retinitis pigmentosa and diabetic retinopathy. ADNIV is caused by a gain-of-function mutation in Calpain-5 (CAPN5), a calcium-dependent cysteine protease. Very little is known about the normal function of CAPN5 in the adult retina, and there are conflicting results regarding its role during mammalian embryonic development. The zebrafish (Danio rerio) is an excellent animal model for studying vertebrate development and tissue regeneration, and represents a novel model to explore the function of Capn5 in the eye.MethodsWe characterized the expression of Capn5 in the developing zebrafish central nervous system (CNS) and retina, in the adult zebrafish retina, and in response to photoreceptor degeneration and regeneration using whole-mount in situ hybridization, FISH, and immunohistochemistry.ResultsIn zebrafish, capn5 is strongly expressed in the developing embryonic brain, early optic vesicles, and in newly differentiated retinal photoreceptors. We found that expression of capn5 colocalized with cone-specific markers in the adult zebrafish retina. We observed an increase in expression of Capn5 in a zebrafish model of chronic rod photoreceptor degeneration and regeneration. Acute light damage to the zebrafish retina was accompanied by an increase in expression of Capn5 in the surviving cones and in a subset of Müller glia.ConclusionsThese studies suggest that Capn5 may play a role in CNS development, photoreceptor maintenance, and photoreceptor regeneration.

Evidence of Oxidative Phosphorylation in Zebrafish Photoreceptor Outer Segments at Different Larval Stages.

Previous studies on purified bovine rod outer segments (OS) disks pointed to Oxidative Phosphorylation (OXPHOS) as being the most likely mechanism involved in ATP production, as yet not fully understood, to support the first phototransduction steps. Bovine and murine rod OS disks, devoid of mitochondria, would house respiratory chain complexes I to IV and ATP synthase, similar to mitochondria. Zebrafish ( Danio rerio) is a well-suited animal model to study vertebrate embryogenesis as well as the retina, morphologically and functionally similar to its human counterpart. The present article reports fluorescence and Transmission Electron Microscopy colocalization analyses of respiratory complexes I and IV and ATP synthase with zpr3, the rod OS marker, in adult and larval zebrafish retinas. MitoTracker Deep Red 633 staining and assays of complexes I and III-IV activity suggest that those proteins are active in OS. Results show that an extramitochondrial aerobic metabolism is active in the zebrafish OS at 4 and 10 days of larval development, as well as in adults, suggesting that it is probably maintained during embryogenesis. Data support the hypothesis of an extramitochondrial aerobic metabolism in the OS of zebrafish.

Absence of histopathological changes in the retina of zebrafish treated with sodium iodate

In ophthalmological research, the use of zebrafish to investigate visual behaviors has been increasing, but can produce misleading, false-positive results if compounds adversely affect their motor functions or central nervous system. Therefore, histological analysis to identify a target organ is important in zebrafish toxicity assay. We investigated the retinal degeneration in zebrafish, using typical retinal toxicants, mainly sodium iodate and N-methyl-N-nitrosourea (MNU). No histopathological changes were found after sodium iodate exposure at 1.0 mM for 5 or 7 days in the retina of larval, juvenile, and adult zebrafish. There were also no obvious histopathological changes in the retina of adult zebrafish at 0.1 mM, even after 30 days treatment with sodium iodate. In addition, many proliferating cell nuclear antigen-positive cells were found not only in the ciliary marginal zone, but also in the outer nuclear layer, especially in larval and juvenile zebrafish with or without sodium iodate exposure. However, the concentrations of iodine in the blood and the eyeballs of adult zebrafish increased remarkably after the treatment. General retinal damage emerged after MNU exposure at 150 mg/l for 60 min in adult zebrafish, but first pyknotic cells appeared in the inner nuclear layer and the ganglion cell layer. Our findings indicate that zebrafish retina have a different reactivity pattern from mammalian animals against some retinal toxicants, and in them it is difficult to detect histopathological changes.

Repeatability and Reproducibility of In Vivo Cone Density Measurements in the Adult Zebrafish Retina.

Zebrafish (Danio rerio) are widely used as an experimental model for a wide range of retinal diseases. Previously, optical coherence tomography (OCT) was introduced for quantitative analysis of the zebrafish cone photoreceptor cell mosaic; however no data exists on the intersession reproducibility or intrasession repeatability of such measurements. We imaged 14 wild-type (WT) fish three times each, with 48h between each time point. En face images of the UV cone mosaic were generated from the OCT volume scans at each time point. These images were then aligned and the overlapping area cropped for analysis. Using a semiautomated cone-counting algorithm, a single observer identified each cone to calculate the cone density for every image, counting each image twice (84 total counts). The OCT cone density measurements were found to have an intersession reproducibility of 0.9988 (95% CI=0.9978-0.9999) and an intrasession repeatability of 136.0±10.5 cones/mm2 (about 0.7%). Factors affecting image quality include gill movement during acquisition of the OCT volume and variable inclusion of non-UV cone mosaics in the contours used to generate the en face images.

Connectivity of cone photoreceptor telodendria in the zebrafish retina.

The connectivity amongst photoreceptors is critical to their function, as it underpins lateral inhibition and effective translation of stimuli into neural signals. Despite much work characterizing second-order interneurons in the outer retina, the synapses directly connecting photoreceptors have often been overlooked. Telodendria are fine processes that connect photoreceptor pedicles. They have been observed in diverse vertebrate groups, yet their roles in vision remain speculative. Here, we visualize telodendria via fluorescent protein expression in photoreceptor subtypes. We characterized short wavelength cone telodendria in adult and larval zebrafish retina. Additionally, in the larval retina, we investigated rod telodendria and UV cone telodendria in mutant and transgenic retinas with altered complements of cone types. In the adult retina, telodendria are twice as abundant and branch almost twice as often on blue cones compared to UV cones. Pedicles of neighboring UV and blue cones typically converge into contiguous pairs, despite the regular spacing of their cell bodies. In contrast to adults, larval UV cone telodendria are more numerous (1.3 times) than blue cone telodendria. UV cone telodendria are not detectably affected by ablation of blue cones, and are reduced twofold in mutant larval retina with few UV cones. We thus saw no evidence that telodendria increase in number in the absence of their typical cellular neighbors. We also found that larval rod telodendria are less abundant than short wavelength cone telodendria. In summary, we describe the development and morphology of zebrafish photoreceptor synaptic connectivity toward appreciating the function of telodendria in visual signal processing.

Restoration of Dendritic Complexity, Functional Connectivity, and Diversity of Regenerated Retinal Bipolar Neurons in Adult Zebrafish.

Adult zebrafish (Danio rerio) are capable of regenerating retinal neurons that have been lost due to mechanical, chemical, or light damage. In the case of chemical damage, there is evidence that visually mediated behaviors are restored after regeneration, consistent with recovery of retinal function. However, the extent to which regenerated retinal neurons attain appropriate morphologies and circuitry after such tissue-disrupting lesions has not been investigated. Adult zebrafish of both sexes were subjected to intravitreal injections of ouabain, which destroys the inner retina. After retinal regeneration, cell-selective markers, confocal microscopy, morphometrics, and electrophysiology were used to examine dendritic and axonal morphologies, connectivities, and the diversities of each, as well as retinal function, for a subpopulation of regenerated bipolar neurons (BPs). Although regenerated BPs were reduced in numbers, BP dendritic spreads, dendritic tree morphologies, and cone-bipolar connectivity patterns were restored in regenerated retinas, suggesting that regenerated BPs recover accurate input pathways from surviving cone photoreceptors. Morphological measurements of bipolar axons found that numbers and types of stratifications were also restored; however, the thickness of the inner plexiform layer and one measure of axon branching were slightly reduced after regeneration, suggesting some minor differences in the recovery of output pathways to downstream partners. Furthermore, ERG traces from regenerated retinas displayed waveforms matching those of controls, but with reduced b-wave amplitudes. These results support the hypothesis that regenerated neurons of the adult zebrafish retina are capable of restoring complex morphologies and circuitry, suggesting that complex visual functions may also be restored.SIGNIFICANCE STATEMENT Adult zebrafish generate new retinal neurons after a tissue-disrupting lesion. Existing research does not address whether regenerated neurons of adults successfully reconnect with surrounding neurons and establish complex morphologies and functions. We report that, after a chemical lesion that ablates inner retinal neurons, regenerated retinal bipolar neurons (BPs), although reduced in numbers, reconnected to undamaged cone photoreceptors with correct wiring patterns. Regenerated BPs had complex morphologies similar to those within undamaged retina and a physiological measure of photoreceptor-BP connectivity, the ERG, was restored to a normal waveform. This new understanding of neural connectivity, morphology, and physiology suggests that complex functional processing is possible within regenerated adult retina and offers a system for the future study of synaptogenesis during adult retinal regeneration.

Characterization of the pleiotropic roles of Sonic Hedgehog during retinal regeneration in adult zebrafish

In contrast to the mammalian retina, the zebrafish retina possesses the ability to regenerate. This is primarily accomplished through Müller glial cells, which, upon damage, re-enter the cell cycle to form retinal progenitors. The progenitors continue to proliferate as they migrate to the area of damage and ultimately differentiate into new neurons. The purpose of this study was to characterize the expression and function of Sonic Hedgehog (Shh) during regeneration of the adult zebrafish retina. Expression profiling of Shh pathway genes showed a significant upregulation of expression associated with stages of progenitor proliferation and neuronal differentiation. Activation of Shh signaling during early stages of retinal regeneration using intraocular injections of the recombinant human SHH (SHH-N) resulted in increased Müller cell gliosis, proliferation, and neuroprotection of damaged retinal neurons. Continued activation of Shh resulted in a greater number of differentiated amacrine and ganglion cells in the fully regenerated retina. Conversely, inhibition of Shh signaling using intraocular injections of cyclopamine resulted in decreased Müller glial cell proliferation and a fewer number of regenerated amacrine and ganglion cells. These data suggest that Shh signaling plays pleiotropic roles in proliferation and differentiation during adult zebrafish retinal regeneration.

Injury-induced purinergic signalling molecules upregulate pluripotency gene expression and mitotic activity of progenitor cells in the zebrafish retina.

Damage in fish activates retina repair that restores sight. The purinergic signalling system serves multiple homeostatic functions and has been implicated in cell cycle control of progenitor cells in the developing retina. We examined whether changes in the expression of purinergic molecules were instrumental in the proliferative phase after injury of adult zebrafish retinas with ouabain. P2RY1 messenger RNA (mRNA) increased early after injury and showed maximal levels at the time of peak progenitor cell proliferation. Extracellular nucleotides, mainly ADP, regulate P2RY1 transcriptional and protein expression. The injury-induced upregulation of P2RY1 is mediated by an autoregulated mechanism. After injury, the transcriptional expression of ecto-nucleotidases and ecto-ATPases also increased and ecto-ATPase activity inhibitors decreased Müller glia-derived progenitor cell amplification. Inhibition of P2RY1 endogenous activation prevented progenitor cell proliferation at two intervals after injury: one in which progenitor Müller glia mitotically activates and the second one in which Müller glia-derived progenitor cells amplify. ADPβS induced the expression of lin28a and ascl1a genes in mature regions of uninjured retinas. The expression of these genes, which regulate multipotent Müller glia reprogramming, was significantly inhibited by blocking the endogenous activation of P2RY1 early after injury. We consistently observed that the number of glial fibrillary acidic protein-BrdU-positive Müller cells after injury was larger in the absence than in the presence of the P2RY1 antagonist. Ecto-ATPase activity inhibitors or P2RY1-specific antagonists did not modify apoptotic cell death at the time of peak progenitor cell proliferation. The results suggested that ouabain injury upregulates specific purinergic signals which stimulates multipotent progenitor cell response.

Sox2 regulates Müller glia reprogramming and proliferation in the regenerating zebrafish retina via Lin28 and Ascl1a

Sox2 is a well-established neuronal stem cell-associated transcription factor that regulates neural development and adult neurogenesis in vertebrates, and is one of the critical genes used to reprogram differentiated cells into induced pluripotent stem cells. We examined if Sox2 was involved in the early reprogramming-like events that Müller glia undergo as they upregulate many pluripotency- and neural stem cell-associated genes required for proliferation in light-damaged adult zebrafish retinas. In the undamaged adult zebrafish retina, Sox2 is expressed in Müller glia and a subset of amacrine cells, similar to other vertebrates. Following 31 h of light damage, Sox2 expression significantly increased in proliferating Müller glia. Morpholino-mediated knockdown of Sox2 expression resulted in decreased numbers of proliferating Müller glia, while induced overexpression of Sox2 stimulated Müller glia proliferation in the absence of retinal damage. Thus, Sox2 is necessary and sufficient for Müller glia proliferation. We investigated the role of Wnt/β-catenin signaling, which is a known regulator of sox2 expression during vertebrate retinal development. While β-catenin 2, but not β-catenin 1, was necessary for Müller glia proliferation, neither β-catenin paralog was required for sox2 expression following retinal damage. Sox2 expression was also necessary for ascl1a (neurogenic) and lin28a (reprogramming) expression, but not stat3 expression following retinal damage. Furthermore, Sox2 was required for Müller glial-derived neuronal progenitor cell amplification and expression of the pro-neural marker Tg(atoh7:EGFP). Finally, loss of Sox2 expression prevented complete regeneration of cone photoreceptors. This study is the first to identify a functional role for Sox2 during Müller glial-based regeneration of the vertebrate retina.

Cone signals in monostratified and bistratified amacrine cells of adult zebrafish retina.

Strata within the inner plexiform layer (IPL) of vertebrate retinas are suspected to be distinct signaling regions. Functions performed within adult zebrafish IPL strata were examined through microelectrode recording and staining of stratified amacrine types. The stimulus protocol and analysis discriminated the pattern of input from red, green, blue, and UV cones as well as the light-response waveforms in this tetrachromatic species. A total of 36 cells were analyzed. Transient depolarizing waveforms at ON and OFF originated with bistratified amacrine types, whose dendritic planes branched either in IPL sublaminas a & b, or only within sublamina a. Monophasic-sustained depolarizing waveforms originated with types monostratified in IPL s4 (sublamina b). OFF responses hyperpolarized at onset, depolarized at offset, and in some cases depolarized during mid-stimulus. These signals originated with types monostratified in s1 or s2 (sublamina a). Bistratified amacrines received depolarizing signals only from red cones, at both ON and OFF, while s4 stratified ON cells combined red and green cone signals. The s1/s2 stratified OFF cells utilized hyperpolarizing signals from red, red and green, or red and blue cones at ON, but only depolarizing red cone signals at OFF. ON and OFF depolarizing transients from red cones appear widely distributed within IPL strata. C-type physiologies, depolarized by some wavelengths, hyperpolarized by others, in biphasic or triphasic spectral patterns, originated with amacrine cells monostratified in s5. Collectively, cells in this stratum processed signals from all cone types. J. Comp. Neurol. 525:1532-1557, 2017. © 2016 Wiley Periodicals, Inc.

Retinoblastoma binding protein 6 and crystallin lambda 1 are cadmium-responsive genes in zebrafish embryos and adults retinae

Nonessential metal cadmium is widely used and released in the environment, causing cell toxicity and posing a severe threat to wildlife. Zebrafish (Danio rerio) is one of the most commonly used animals in the investigation of environmental cadmium toxicity in vertebrates. In this study, we identified two cadmium-responsive genes, RBBP6 and CRYL1, in the early phases of zebrafish development, at the gastrula stage. The retinoblastoma binding protein 6 is associated with increased protein degradation and cell proliferation; crystallin-lambda 1 is a lens protein with redox activity. In situ hybridization analysis performed on adult zebrafish exposed to 1.5–40 μM cadmium for 30 days confirmed the ability of cadmium to up-regulate the expression of both genes in retinal cells in a dose-dependent manner. The over-expression was transient, being switched off when cadmium was removed. The involvement of RBBP6 and CRYL1 in the onset of cadmium-induced morphological alterations in adult zebrafish retina is discussed.

Comparison of proteomic profiles in the zebrafish retina during experimental degeneration and regeneration

Zebrafish spontaneously regenerate the retina after injury. Although the gene expression profile has been extensively studied in this species during regeneration, this does not reflect protein function. To further understand the regenerative process in the zebrafish, we compared the proteomic profile of the retina during injury and upon regeneration. Using two-dimensional difference gel electrophoresis (2D-DIGE) and label-free quantitative proteomics (quadrupole time of flight LC-MS/MS), we analysed the retina of adult longfin wildtype zebrafish at 0, 3 and 18 days after Ouabain injection. Gene ontology analysis indicates reduced metabolic processing, and increase in fibrin clot formation, with significant upregulation of fibrinogen gamma polypeptide, apolipoproteins A-Ib and A-II, galectin-1, and vitellogenin-6 during degeneration when compared to normal retina. In addition, cytoskeleton and membrane transport proteins were considerably altered during regeneration, with the highest fold upregulation observed for tubulin beta 2 A, histone H2B and brain type fatty acid binding protein. Key proteins identified in this study may play an important role in the regeneration of the zebrafish retina and investigations on the potential regulation of these proteins may lead to the design of protocols to promote endogenous regeneration of the mammalian retina following retinal degenerative disease.

Neurotransmitter-Regulated Regeneration in the Zebrafish Retina.

SummaryCurrent efforts to repair damaged or diseased mammalian retinas are inefficient and largely incapable of fully restoring vision. Conversely, the zebrafish retina is capable of spontaneous regeneration upon damage using Müller glia (MG)-derived progenitors. Understanding how zebrafish MG initiate regeneration may help develop new treatments that prompt mammalian retinas to regenerate. We show that inhibition of γ-aminobutyric acid (GABA) signaling facilitates initiation of MG proliferation. GABA levels decrease following damage, and MG are positioned to detect decreased ambient levels and undergo dedifferentiation. Using pharmacological and genetic approaches, we demonstrate that GABAA receptor inhibition stimulates regeneration in undamaged retinas while activation inhibits regeneration in damaged retinas.