Visual acuity in larval zebrafish: behavior and histology

BACKGROUND: Visual acuity, the ability of the visual system to distinguish two separate objects at a given angular distance, is influenced by the optical and neuronal properties of the visual system. Although many factors may contribute, the ultimate limit is photoreceptor spacing. In general, at least one unstimulated photoreceptor flanked by two stimulated ones is needed to perceive two objects as separate. This critical interval is also referred to as the Nyquist frequency and is according to the Shannon sampling theorem the highest spatial frequency where a pattern can be faithfully transmitted. We measured visual acuity in a behavioral experiment and compared the data to the physical limit given by photoreceptor spacing in zebrafish larvae. RESULTS: We determined visual acuity by using the optokinetic response (OKR), reflexive eye movements in response to whole field movements of the visual scene. By altering the spatial frequency we determined the visual acuity at approximately 0.16 cycles/degree (cpd) (minimum separable angle = 3.1 degrees ). On histological sections we measured the retinal magnification factor and the distance between double cones, that are thought to mediate motion perception. These measurements set the physical limit at 0.24 cpd (2.1 degrees ). CONCLUSION: The maximal spatial information as limited by photoreceptor spacing can not be fully utilized in a motion dependent visual behavior, arguing that the larval zebrafish visual system has not matured enough to optimally translate visual information into behavior. Nevertheless behavioral acuity is remarkable close to its maximal value, given the immature state of young zebrafish larvae

Управління трудовим потенціалом при створенні інноваційної продукції

Super-resolution microscopy (SRM) bypasses the diffraction limit, a physical barrier that restricts the optical resolution to roughly 250 nm and was previously thought to be impenetrable. SRM techniques allow the visualization of subcellular organization with unprecedented detail, but also confront biologists with the challenge of selecting the best-suited approach for their particular research question. Here, we provide guidance on how to use SRM techniques advantageously for investigating cellular structures and dynamics to promote new discoveries

Impaired retinal differentiation and maintenance in zebrafish laminin mutants

PURPOSE: To characterize morphologic and physiological alterations in the retina of three laminin mutant zebrafish, bashful (bal, lama1), grumpy (gup, lamb1), and sleepy (sly, lamc1), which were identified in forward genetic screens and were found to be impaired in visual functions. METHODS: Mutant larvae were observed for defects in visual behavior by testing their optokinetic response (OKR). In addition, electroretinograms (ERG) were measured and retinal morphology was examined by standard histology, immunocytochemistry, TUNEL assay, and electron microscopy. RESULTS: Both, gup and sly showed no OKR at any light intensity tested, whereas bal embryos showed some remaining OKR behavior at more than 40% of contrast. Consistent with the OKR result, gup and sly did not show an ERG response at any light intensity tested, whereas bal mutants exhibited small a- and b-waves at high light intensities. All three laminin mutants showed altered ganglion cell layers, optic nerve fasciculations, and lens defects. Again, bal showed the least severe morphologic phenotype with no additional defects. In contrast, both, gup and sly, showed severe photoreceptor outer segment shortening and synapse alteration (floating ribbons) as well as increased cell death. CONCLUSIONS: Lamb1 and lamc1 chains play an important role in the morphogenesis of photoreceptors and their synapses. In contrast, lama1 is not involved in outer retina development

Space-saving advantage of an inverted retina

Vertebrate eyes are of the simple or camera type with a single optical system that creates an image on the retina in the back of the eye. There, the visual information is encoded as nervous signals by photoreceptors, processed by retinal neurons, and then sent to the brain via the optic nerve. Surprisingly at first sight, the retinal neurons are located between the lens and the light-sensitive parts of the photoreceptors. The tissue scatters some light, which leads to loss of light and image blur. The inverted retina has, therefore, long been regarded as inferior. Here, we provide evidence that the inverted retina actually is a superior space-saving solution, especially in small eyes. The inverted retina has most likely facilitated the evolution of image-forming eyes in vertebrates, and it still benefits especially small and highly visual species

Acquisition speed comparison of microscope software programs

Reliable software is a prerequisite for successful operation of a modern wide field fluorescence microscope. When used for live cell imaging, acquisition speed is of particular interest. This is both because biological processes can be highly-dynamic, and to avoid unnecessary photobleaching and phototoxicity of living samples. This article shows that besides the hardware (microscope) components themselves, the acquisition control software is an important influencing factor of speed performance. We tested and compared the speed performance of five different generic applications (Image-Pro Plus, MetaMorph, Micro-Manager, SlideBook, and Volocity) using typical experimental setups involving a single specific state-of-the-art fluorescence microscope configuration. The test measurements included multichannel experiments, z-stacking, burst acquisition, as well as combinations of these measurements with time-lapse acquisitions. The measured data provided values for guiding the testing and analysis of other microscope systems with similar configurations. Despite the identical hardware settings, significant and surprisingly large speed differences were evident among the various software applications. Additionally, no application was identifiable as the fastest in all tests. Our work pinpoints the importance of the control software in determining a system`s ``real`` maximal imaging speed. The study could serve as basis for further tests, eventually influencing the system selection criteria for speed-sensitive applications. Microsc. Res. Tech., 2010. (c) 2010 Wiley-Liss, Inc

A rat model of Parkinsonism shows depletion of dopamine in the retina

The retinal dopamine (DA) deficiency is an important feature of the pathogenesis in Parkinson`s disease (PD) visual dysfunction. Systemic inhibition of complex I (rotenone) in rats has been proposed as a model of PD. In this study, we investigated whether systemic inhibition of complex I can induce impairment of DA-ergic cells in the retina, similar to the destruction of retinal cells found in PD patients. Rotenone (2.5mg/kg i.p., daily) was administered over 60 days. Neurochemically, rotenone treated rats showed a depletion of DA in the striatum and substantia nigra (SN). In addition, the number of retinal DA-ergic amacrine cells was significantly reduced in the rotenone treated animals. This study is the first one giving highlight towards a deeper understanding of systemic complex I inhibition (rotenone as an environmental toxin) and the connection between both, DA-ergic degeneration in the nigrostriatal pathway, and in the DA-ergic amacrine cells of the retina

Onset and time course of apoptosis in the developing zebrafish retina

In mammalian development, apoptosis spreads over the retina in consecutive waves and induces a remarkable amount of cell loss. No evidence for such consecutive waves has been revealed in the fish retina so far. As the zebrafish is of growing importance as a model for retinal development and for degenerative retinal diseases, we examined the onset and time course of apoptosis in the developing zebrafish retina and in adult fish. We found that apoptosis peaked in the ganglion cell layer (GCL) and inner nuclear layer (INL) in early developmental stages (3-4 days post-fertilization; dpf) followed by a second, but clearly smaller wave at 6-7dpf. Apoptosis in the outer nuclear layer (ONL) started at 5dpf and peaked at 7dpf. This late-onset high peak of apoptosis of photoreceptors is different from that of all other species examined to date. With 1.09% of cells in the GCL and 1.10% in the ONL being apoptotic, the rate of apoptosis in the developing zebrafish retina was conspicuously lower than that observed in other vertebrates (up to 50% in GCL). During development (2-21dpf), apoptotic waves were most obvious in the central retina, whereas in the periphery near the marginal zone (MZ), apoptosis was much lower; in adult animals, practically no apoptosis was present in the central retina but it still occurred near the MZ. Our data show that the onset and time course of apoptosis in the GCL and INL of the zebrafish is comparable with other vertebrates; however, the amount of apoptosis is clearly reduced. Thus, apoptosis in the zebrafish retina may serve more as a mechanism for the fine tuning of the retinal neuronal network after mitotic waves during development or in remaining mitotic areas than as a mechanism for eliminating large numbers of excess cells

Survival, excitability, and transfection of retinal neurons in an organotypic culture of mature zebrafish retina

Over the last 20 years, the zebrafish has become an important model organism for research on retinal function and development. Many retinal diseases do not become apparent until the later stages of life. This means that it is important to be able to analyze (gene) function in the mature retina. To meet this need, we have established an organotypic culture system of mature wild-type zebrafish retinas in order to observe changes in retinal morphology. Furthermore, cell survival during culture has been monitored by determining apoptosis in the tissue. The viability and excitability of ganglion cells have been tested at various time points in vitro by patch-clamp recordings, and retinal functionality has been assessed by measuring light-triggered potentials at the ganglion cell site. Since neurogenesis is persistent in adult zebrafish retinas, we have also monitored proliferating cells during culture by tracking their bromodeoxyuridine uptake. Reverse genetic approaches for probing the function of adult zebrafish retinas are not yet available. We have therefore established a rapid and convenient protocol for delivering plasmid DNA or oligonucleotides by electroporation to the retinal tissue in vitro. The organotypic culture of adult zebrafish retinas presented here provides a reproducible and convenient method for investigating the function of drugs and genes in the retina under well-defined conditions in vitro

Impaired retinal differentiation and maintenance in zebrafish laminin mutants

PURPOSE: To characterize morphologic and physiological alterations in the retina of three laminin mutant zebrafish, bashful (bal, lama1), grumpy (gup, lamb1), and sleepy (sly, lamc1), which were identified in forward genetic screens and were found to be impaired in visual functions. METHODS: Mutant larvae were observed for defects in visual behavior by testing their optokinetic response (OKR). In addition, electroretinograms (ERG) were measured and retinal morphology was examined by standard histology, immunocytochemistry, TUNEL assay, and electron microscopy. RESULTS: Both, gup and sly showed no OKR at any light intensity tested, whereas bal embryos showed some remaining OKR behavior at more than 40% of contrast. Consistent with the OKR result, gup and sly did not show an ERG response at any light intensity tested, whereas bal mutants exhibited small a- and b-waves at high light intensities. All three laminin mutants showed altered ganglion cell layers, optic nerve fasciculations, and lens defects. Again, bal showed the least severe morphologic phenotype with no additional defects. In contrast, both, gup and sly, showed severe photoreceptor outer segment shortening and synapse alteration (floating ribbons) as well as increased cell death. CONCLUSIONS: Lamb1 and lamc1 chains play an important role in the morphogenesis of photoreceptors and their synapses. In contrast, lama1 is not involved in outer retina development

Double cone dystrophy and RPE degeneration in the retina of the zebrafish gnn mutant

PURPOSE: To characterize morphologic alterations in the retina of the visual mutant zebrafish gantenbein (gnn) and to examine whether these alterations correlate with those present in human hereditary eye diseases. METHODS: The gnn mutant was isolated by behavioral and macroscopic screening. Retinas of gnn zebrafish larvae were examined at different developmental stages from 2 to 9 days postfertilization (dpf) by standard histologic staining techniques and by immunocytochemistry. Ultrastructural alterations were examined by electron microscopy. The genetic map position of the induced mutation was identified by mapping with two candidate primer pairs on single larvae. RESULTS: The gnn mutant exhibited shortened outer photoreceptor segments and altered RPE morphology. In the photoreceptor layer of the mutant, the total number of lectin-labeled cones was reduced in all developmental stages from 2 to 7 dpf, whereas the amount of rhodopsin-positive cells remained at the wild-type (WT) level. Labeling with zebrafish opsin antibodies revealed dystrophic red cones at 5 dpf, whereas the morphology of all other cone types was largely unaffected. Electron microscopy unveiled electron-dense deposits between the discs of the double cone outer segments. In addition, the onset of progressive RPE degeneration was observed at this stage of development. At later stages, all cone types and the RPE became degenerative. The morphology of distinct second-order neurons remained largely unaffected by the mutation. The gnn mutation was located approximately 4.3 cM from the simple sequence length polymorphism (SSLP) marker Z15453 on linkage group 16. CONCLUSIONS: In gnn mutant zebrafish, cones, and especially red cones, are dystrophic in early retinal development. Subsequent to this cone dystrophy, the RPE becomes dysfunctional and starts to degenerate in later stages of development. Thus, the early developmental morphology of gnn exhibits similarities to cone dystrophies most commonly seen in age-related macular degeneration (AMD) among humans, whereas the later stages of degeneration in gnn resemble RPE alterations in retinitis pigmentosa (RP) in humans. The gnn zebrafish mutant may therefore be a useful model for examining the possible interplay and connection between cone dystrophy and RPE degeneration