In vivo physiological recording from the lateral line of juvenile zebrafish.

Jennifer Olt,Walter Marcotti,Claire E Allen

doi:10.1113/jp271794

Abstract

Key points Zebrafish provide a unique opportunity to investigate in vivo sensory transduction in mature hair cells.We have developed a method for studying the biophysical properties of mature hair cells from the lateral line of juvenile zebrafish.The method involves application of the anaesthetic benzocaine and intubation to maintain ventilation and oxygenation through the gills.The same approach could be used for in vivo functional studies in other sensory and non‐sensory systems from juvenile and adult zebrafish. Hair cells are sensory receptors responsible for transducing auditory and vestibular information into electrical signals, which are then transmitted with remarkable precision to afferent neurons. The zebrafish lateral line is emerging as an excellent in vivo model for genetic and physiological analysis of hair cells and neurons. However, research has been limited to larval stages because zebrafish become protected from the time of independent feeding under European law (from 5.2 days post‐fertilization (dpf) at 28.5°C). In larval zebrafish, the functional properties of most of hair cells, as well as those of other excitable cells, are still immature. We have developed an experimental protocol to record electrophysiological properties from hair cells of the lateral line in juvenile zebrafish. We found that the anaesthetic benzocaine at 50 mg l−1 was an effective and safe anaesthetic to use on juvenile zebrafish. Concentrations up to 300 mg l−1 did not affect the electrical properties or synaptic vesicle release of juvenile hair cells, unlike the commonly used anaesthetic MS‐222, which reduces the size of basolateral membrane K+ currents. Additionally, we implemented a method to maintain gill movement, and as such respiration and blood oxygenation, via the intubation of > 21 dpf zebrafish. The combination of benzocaine and intubation provides an experimental platform to investigate the physiology of mature hair cells from live zebrafish. More generally, this method would allow functional studies involving live imaging and electrophysiology from juvenile and adult zebrafish.

Highlights

The zebrafish is becoming an increasingly popular model to study the genetic basis of hearing and deafness (Grunwald & Eisen 2002; Nicolson, 2005) and to investigate the molecular mechanisms controlling the normal development and function of sensory hair cells
Zebrafish are known to become more sensitive to MS-222 with development (Rombough, 2007) and it has recently been shown to affect the biophysical properties of hair cells of the juvenile lateral line when used at 0.1% (Olt et al 2014)
We describe an experimental method to record from functionally mature hair cells of live juvenile zebrafish

Summary

Introduction

The zebrafish is becoming an increasingly popular model to study the genetic basis of hearing and deafness (Grunwald & Eisen 2002; Nicolson, 2005) and to investigate the molecular mechanisms controlling the normal development and function of sensory hair cells In vivo physiological experiments are normally performed at embryonic or larval stages when the majority of hair cells within a neuromast have an immature synaptic activity and basolateral current profile (Olt et al 2014). Oxygenation in > 21 dpf zebrafish is entirely dependent on gill function, which prevents in vivo experiments at older ages. This limits the use of zebrafish for studies on mature physiological characteristics, in the lateral line (Liao & Haehnel, 2012; Olt et al 2014) and in other systems such as the brain (Vargas et al 2011)

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