Visual acuity in larval zebrafish: behavior and histology

Background

Mating plugs that males place onto the female genital tract are generally assumed to prevent remating with other males. Mating plugs are usually explained as a consequence of male-male competition in multiply mating species. Here, we investigated whether mating plugs also have collateral effects on female fitness. These effects are negative when plugging reduces female mating rate below an optimum. However, plugging may also be positive when plugging prevents excessive forced mating and keeps mating rate closer to a females' optimum. Here, we studied these consequences in the gonochoristic nematode Caenorhabditis remanei. We employed a new CO₂-sedation technique to interrupt matings before or after the production of a plug. We then measured mating rate, attractiveness and offspring number.

Results

The presence of a mating plug did not affect mating rate or attractiveness to roving males. Instead, females with mating plugs produced more offspring than females without copulatory plugs.

Conclusions

Our experiment suggests that plugging might have evolved under male-male competition but represents a poor protection against competing males in our experiment. Even if plugging does not reduce mating rate, our results indicate that females may benefit from being plugged in a different sense than remating prevention.     

Funduscopy in adult zebrafish and its application to isolate mutant strains with ocular defects

Funduscopy is one of the most commonly used diagnostic tools in the ophthalmic practice, allowing for a ready assessment of pathological changes in the retinal vasculature and the outer retina. This non-invasive technique has so far been rarely used in animal model for ophthalmic diseases, albeit its potential as a screening assay in genetic screens. The zebrafish (Danio rerio) is well suited for such genetic screens for ocular alterations. Therefore we developed funduscopy in adult zebrafish and employed it as a screening tool to find alterations in the anterior segment and the fundus of the eye of genetically modified adult animals.A stereomicroscope with coaxial reflected light illumination was used to obtain fundus color images of the zebrafish. In order to find lens and retinal alterations, a pilot screen of 299 families of the F3 generation of ENU-treated adult zebrafish was carried out.Images of the fundus of the eye and the anterior segment can be rapidly obtained and be used to identify alterations in genetically modified animals. A number of putative mutants with cataracts, defects in the cornea, eye pigmentation, ocular vessels and retina were identified. This easily implemented method can also be used to obtain fundus images from rodent retinas.In summary, we present funduscopy as a valuable tool to analyse ocular abnormalities in adult zebrafish and other small animal models. A proof of principle screen identified a number of putative mutants, making funduscopy based screens in zebrafish feasible.     

Synaptic plasticity and functionality at the cone terminal of the developing zebrafish retina

Previous studies have analyzed photoreceptor development, some inner retina cell types, and specific neurotransmitters in the zebrafish retina. However, only minor attention has been paid to the morphology of the synaptic connection between photoreceptors and second order neurons even though it represents the transition from the light sensitive receptor to the neuronal network of the visual system. Here, we describe the appearance and differentiation of pre- and postsynaptic elements at cone synapses in the developing zebrafish retina together with the maturation of the directly connecting second order neurons and a dopaminergic third order feedback-neuron from the inner retina. Zebrafish larvae were examined at developmental stages from 2 to 7dpf (days postfertilization) and in the adult. Synaptic maturation at the photoreceptor terminals was examined with antibodies against synapse associated proteins. The appearance of synaptic plasticity at the so-called spinule-type synapses between cones and horizontal cells was assessed by electron microscopy, and the maturation of photoreceptor downstream connection was identified by immunocytochemistry for GluR4 (AMPA-type glutamate receptor subunit), protein kinase beta(1) (mixed rod-cone bipolar cells), and tyrosine hydroxylase (dopaminergic interplexiform cells). We found that developing zebrafish retinas possess first synaptic structures at the cone terminal as early as 3.5dpf. Morphological maturation of these synapses at 3.5-4dpf, together with the presence of synapse associated proteins at 2.5dpf and the maturation of second order neurons by 5dpf, indicate functional synaptic connectivity and plasticity between the cones and their second order neurons already at 5dpf. However, the mere number of spinules and ribbons at 7dpf still remains below the adult values, indicating that synaptic functionality of the zebrafish retina is not entirely completed at this stage of development.     

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.     

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.     

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.     

Knockdown of cone-specific kinase GRK7 in larval zebrafish leads to impaired cone response recovery and delayed dark adaptation

Phosphorylation of rhodopsin by rhodopsin kinase GRK1 is an important desensitization mechanism in scotopic vision. For cone vision GRK1 is not essential. However, cone opsin is phosphorylated following light stimulation. In cone-dominant animals as well as in humans, but not in rodents, GRK7, a cone-specific homolog of GRK1, has been identified in cone outer segments. To investigate the function of GRK7 in vivo, we cloned two orthologs of grk7 in zebrafish and knocked down gene expression of grk7a in zebrafish larvae by morpholino antisense nucleotides. Photoresponse recovery in Grk7a-deficient larvae was delayed in electroretinographic measurements, and temporal contrast sensitivity was reduced, particularly under bright-light conditions. These results show that function of a cone-specific kinase is essential for cone vision in the zebrafish retina and argue that pigment bleaching and spontaneous decay alone are not sufficient for light adaptation and rapid cone response inactivation.