Techniques for Studying Neuromast Function in Zebrafish

Abstract

The mechano-sensitive hair cells of superficial neuromasts (SNs) of the zebrafish lateral line organ are mechanically coupled to the water motion via gelatinous cupulae. SNs transduce the water motion into electrical signals that can be measured with an extracellular electrode. In this chapter, we review the preparation and measurement techniques for quantifying cupular dynamics and extracellular receptor potentials (ERPs) of SNs. We compare the measuring techniques used in hair cell mechano-physiology and give instructions for building both an intensity-based and an interferometry-based microscope system. We compare the methods used for mechanical excitation of mechanoreceptors, including dipole sources, microfluid jets (FJ) and elastic as well as stiff microprobes. We present the caveats of the measurements of ERPs, especially the crosstalk from the stimulation device. We show that ERPs at twice the stimulation frequency of zebrafish SNs are a reliable measure of mechano-electrical coupling in a restricted range of both stimulus frequency and amplitude. We report the measurements of sub-micrometre motion of SN cupulae using a heterodyne laser interferometer microscope (HLIM) and continuous sinusoidal stimulation with a micro FJ device. Light interference signals were decoded with a phase- and frequency modulation scheme. We compare the robustness of both decoding strategies in terms of accuracy of the measured cupular displacement and velocity. Both approaches can faithfully monitor cupular movement down to a few nanometres, though the velocity decoding technique offered a slightly superior performance and is recommended for higher stimulation frequencies.