Microelectrode Recordings in Zebrafish Retinal Neurons

This study aims to identify and characterize zebrafish retinal neuron morphology and physiology with the use of staining and membrane potential measurements during light stimulation. Zebrafish (Danio rerio) retina-eyecups were perfused with equilibrated Minimum Essential Medium (95% O2/5% CO₂) at a rate of 0.3 ml/min in a dark Faraday chamber. Sharp microelectrodes filled with 0.3 M KCl and Alexa Fluor 594 hydrazide were used to penetrate neurons in the retina. Two light protocols were run and collected via Clampex 8.2 and a computer-controlled Xenon arc lamp shutter and optical bench. Cells were photographed in wide-field z-axis stacks with a video camera and IrfanView software. Cell stacks were reconstructed in Adobe Photoshop 7.0.1 and ImageJ. Origin 7.0 was used to import, analyze, and display light responses from Clampex 8.2. Stains revealed a characteristic horizontal (H1/H_A) and a characteristic amacrine (A_off-s1n) cell. Horizontal cell membrane potentials displayed hyperpolarization to each wavelength with particular emphasis to the red light stimulus, typical of L-type cells, while amacrine cell membrane potentials exhibited depolarization at the onset and offset of the stimulus, as expected of transient cells. Cell dimensions revealed a horizontal cell with a long axon (393.103 µm), and an amacrine cell with an eccentric dendritic field with 2 extending axon-like dendrites (major axis of the dendritic field: 190.344 µm; minor axis of the dendritic field: 88.042 µm). The horizontal cell morphology is similar to that found by Connaughton et al. (2004) and Song et al. (2008) for H_A-2/H1 cells, while the amacrine cell morphology is similar to that found by Connaughton et al. (2004) for A_off-s1n transient cells.