Establishment of an adult zebrafish model of retinal neurodegeneration induced by NMDA.

AIM: To establish a model of retinal neurodegeneration induced by N-Methyl-D-aspartic acid (NMDA) in adult zebrafish. METHODS: We compared the effects of three different NMDA delivery methods on retinal neurodegeneration in adult zebrafish: immersion (I.M.), intravitreal injection (I.V.), and intraperitoneal injection (I.P.), and examined retinal pathology and degeneration by hematoxylin and eosin and TUNEL staining in the treated zebrafish. Effects of the NMDA receptor antagonist MK-801 and the natural product resveratrol on NMDA-induced retinal neurodegeneration were also assessed. RESULTS: The thickened inner retina was seen in histology with 100 µmol/L NMDA by I.M. administration. Significant apoptosis in the retinal ganglion cell layer and retinal thickness reduction occurred in 0.5 mol/L NMDA I.P. administration group.Seizure-like behavioral changes, but no retinal histological alteration occurred in 16 mg/kg NMDA I.P. administration group. Resveratrol and MK-801 prevented NMDA-induced retinal neurodegeneration in the zebrafish. CONCLUSION: Among the three drug administration methods, I.V. injection of NMDA is the most suitable for establishment of an acute retinal damage model in zebrafish. I.M. with NMDA is likely the best for use as a chronic retinal damage model. I.P. treatment with NMDA causes brain damage. Resveratrol and MK801 may be a clinically valuable treatment for retinal neurodegeneration.

Retrograde labeling of retinal ganglion cells in adult zebrafish with fluorescent dyes.

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

[Analysis of adult zebrafish OKR behavior].

As a means of visual function testing and visual related mutants screening, the optokinetic response (OKR) and the optomotor response (OMR) behaviour tests are simple and effective tools for visual functional testing, which have been widely used in studying zebrafish larvae. However, adult zebrafish OKR analysis method is rarely reported. In this study, the methods of inducing adult zebrafish OKR behaviour, as well as tracking the movement of eyes using Pattern Match approaches, are presented. The quantitative measurement of the adult zebrafish OKR behaviour was successfully established. Using these methods, the binocular vision area was found to make a certain contribution to OKR behaviour. Moreover, the monocular vision of adult zebrafish showed a certain degree of directional sensitivity to moving gratings. Such approaches can also be applied to the zebrafish larvae OKR. The abnormal OKR behaviour phenomenon of period1b mutant larvae fish was detected.

Using the optokinetic response to study visual function of zebrafish.

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zebrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.