The electroretinogram (ERG) is a noninvasive electrophysiological method that has been used extensively in the clinic for determining the function of the retina in humans. The electrical activity in response to a light stimulus is measured by placing recording electrodes on the outer surface of the cornea. The characteristics of the stimulus paradigm and the response waveform define the retinal neurons contributing to the response. This method has been adapted for use with a number of animal models including mice and zebrafish. The typical vertebrate ERG response has four principal components: the a-wave, which is a cornea-negative potential derived from photoreceptor cell activity; the b-wave, a cornea-positive potential derived from the ON bipolar cells; the d-wave, a cornea-positive potential interpreted as the activity of the OFF bipolar cells; and the c-wave, which occurs several seconds after the b-wave and reflects activity in Müller glia and the retinal pigment epithelium^1-4. Additional references for understanding the history and principles of ERG analysis in humans and model animals are the online textbook, Webvision, from the University of Utah and texts such as the Principles and Practice of Clinical Electrophysiology of Vision^4,5.

Daniorerio (zebrafish) has long been favored as a model for vertebrate development, due to its rapid maturation and transparency, which allows for noninvasive morphological analysis of organ systems, behavioral assays and both forward and reverse genetic screens (for review, see Fadool and Dowling⁶). Zebrafish larvae are highly amenable to genetic and pharmacological manipulation, which, when coupled with their high fecundity, make them an excellent animal model for high-throughput biological analyses. The higher ratio of cones to rods in larval zebrafish – roughly 1:1 compared to mice (~3% cones) – make them particularly useful for the study of cone function^7-9.

In the vertebrate retina, cones develop before rods¹⁰. Interestingly, zebrafish cones are operative as early as 4 dpf, allowing for selective electrophysiological analysis of cones at that stage^6,11,12. In contrast, ERG responses in rods appear between 11 and 21 dpf¹³. Therefore, zebrafish larvae at 4-7 dpf serve functionally as an all-cone retina. However, the native photopic ERG response of 4-7 dpf larvae is dominated by the b-wave. Application of pharmacological agents, such as L-(+)-2-amino-4-phosphono-butyric acid (L-AP4), an agonist for the metabotropic glutamate (mGluR6) receptor expressed by the ON bipolar cells, effectively blocks the generation of the b-wave and reveals the isolated cone mass receptor potential, (the “a-wave”)^14-17.

Here we describe a simple and reliable method for ERG analysis using commercially available ERG equipment designed for use with mice that have been adapted for use with zebrafish larvae. This system can be utilized on zebrafish larvae of varying genetic backgrounds, as well as those treated with pharmacological agents, to aid researchers in the identification of signaling pathways that contribute to visual sensitivity and light adaptation¹⁶. The experimental procedures outlined in this protocol will guide investigators in the use of ERG analysis to answer a variety of biological questions pertaining to vision, and demonstrate the construction of a flexible ERG setup.