Electroretinogram Recording in Larval Zebrafish using A Novel Cone-Shaped Sponge-tip Electrode

Summary

Here, we present a protocol that simplifies the measurement of light evoked electroretinogram responses from larval zebrafish. A novel cone-shaped sponge-tip electrode can help to make the study of visual development in larval zebrafish using the electroretinogram ERG easier to achieve with reliable outcomes and lower cost.

Abstract

The zebrafish (Danio rerio) is commonly used as a vertebrate model in developmental studies and is particularly suitable for visual neuroscience. For functional measurements of visual performance, electroretinography (ERG) is an ideal non-invasive method, which has been well established in higher vertebrate species. This approach is increasingly being used for examining the visual function in zebrafish, including during the early developmental larval stages. However, the most commonly used recording electrode for larval zebrafish ERG to date is the glass micropipette electrode, which requires specialized equipment for its manufacture, presenting a challenge for laboratories with limited resources. Here, we present a larval zebrafish ERG protocol using a cone-shaped sponge-tip electrode. The novel electrode is easier to manufacture and handle, more economical, and less likely to damage the larval eye than the glass micropipette. Like previously published ERG methods, the current protocol can assess outer retinal function through photoreceptor and bipolar cell responses, the a- and b-wave, respectively. The protocol can clearly illustrate the refinement of visual function throughout the early development of zebrafish larvae, supporting the utility, sensitivity, and reliability of the novel electrode. The simplified electrode is particularly useful when establishing a new ERG system or modifying existing small-animal ERG apparatus for zebrafish measurement, aiding researchers in the visual neurosciences to use the zebrafish model organism.

Introduction

The zebrafish (Danio rerio) has become a widely used genetic vertebrate model, including studies of the visual neurosciences. The increasing popularity of this species can be attributed to advantages including ease of genetic manipulation, the highly conserved vertebrate visual system (neuron types, anatomical morphology and organization, and underlying genetics), high fecundity and lower cost of husbandry compared to mammalian models¹. The non-invasive electroretinogram (ERG) has long been used clinically to assess human visual function, and in the laboratory setting to quantify vision in a range of large and small species including r....

Protocol

All electroretinogram (ERG) procedures were performed according to the provisions of the Australian National Health and Medical Research Council code of practice for the care and use of animals and were approved by the institutional animal ethics committee at the University of Melbourne.

1. Buffer Preparation

1. Prepare the 10x goldfish Ringer’s buffer (1.25 M NaCl, 26 mM KCl, 25 mm CaCl₂, 10 mM MgCl₂, 100 mM glucose, 100 mM HEPES) using reverse osmosis (RO) water. Adjust the buffer to pH 7.8 and sterilize the buffer using 0.22 μm filter. Store the 10x buffer at 4 °C as the solution stock

Results

This section provides representative results for ERG measurements taken daily from 4 to 7 dpf. From 4 dpf, ERG responses show robust a- and b-wave components, which arise from photoreceptors and bipolar cells, respectively. At each age tested, the amplitude of the b-wave increased with light intensity (Figure 2; Figure 3). Notably, the sensitivity of the larval zebrafish retina to dimmer flashes increased with age. The a- and b-wave were not .......

Discussion

Functional readouts such as the ERG have become increasingly important in the suite of tools used to study larval zebrafish^8,9,12,14. Due to the small size of the larval zebrafish eye, glass micropipettes have been adapted as recording electrodes in most published protocols^3,4,5,

Materials

Name	Company	Catalog Number	Comments
0.22 µm filter	Millex GP	SLGP033RS	Filters the 10× goldfish ringer's buffer for sterilizatio
1-mL syringe	Terumo	DVR-5175	With a 30G × ½" needle to add drops of saline to the electrode sponge tip to prevent drying and increased noisein the ERG signals.
30G × ½" needle	Terumo	NN*3013R	For adding saline toteh sopnge tip electrode.
Bioamplifier	ADInstruments	ML135	For amplifying ERG signals.
Bleach solution	King White	9333441000973	For an alternative method of sliver electrode chlorination. Active ingredient: 42 g/L sodium hypochlorite.
Circulation water bath	Lauda-Königshoffen	MGW Lauda	Used to make the water-heated platfrom.
Electrode lead	Grass Telefactor	F-E2-30	Platinum cables for connecting silver wire electrodes to the amplifier.
Faraday Cage	Photometric Solution International		For maintianing dark adaptation and enclosing the Ganzfeld setup to improve signal-to-noise ratio.
Ganzfeld Bowl	Photometric Solution International		Custom designed light stimulator: 36 mm diameter, 13 cm aperture size.
Luxeon LEDs	Phillips Light Co.		For light stimulation twenty 5W and one 1W LEDs.
Micromanipulator	Harvard Apparatus	BS4 50-2625	Holds the recording electrode during experiments.
Microsoft Office Excel	Microsoft	version 2010	Spreadsheet software for data analysis.
Moisturizing eye gel	GenTeal Gel	9319099315560	Used to cover zebrafish larvae during recordings to avoiding dehydration. Active ingredient: 0.3 % Hypromellose and 0.22 % carbomer 980.
Pasteur pipette	Copan	200C	Used to caredully transfer larval zebrafish.
Powerlab data acquisition system	ADInstruments	ML785	Controls the LEDs to generate stimuli.
PVA sponge	MeiCheLe	R-1675	For the placement of larval zebrafish and making the cone-shaped electrode ti
Saline solution	Aaxis Pacific	13317002	For electroplating silver wire electrode.
Scope Software	ADInstruments	version 3.7.6	Simultaneously triggers the stimulus through the Powerlab system and collects data
Silver (fine round wire)	A&E metal	0.3 mm	Used to make recording and reference ERG electrodes.
Stereo microscope	Leica	M80	Used to shape and measure the cone-shaped sponge apex (with scale bar on eyepiece). Positioned in the Faraday cage for electrode placement.
Tricaine	Sigma-aldrich	E10521-50G	For anaethetizing larval zebrafish.
Water-heated platform	custom-made		For maintianing the temperature of the sponge platform and the larval body during ERG recordings