Chapter 11 - Physiological recordings from the zebrafish lateral line
Introduction
Our auditory system has developed the capacity to process and transmit information with remarkable precision and fidelity. Most of our current knowledge about sensory transduction in hair cells comes from in vitro experiments using altricial rodents and other vertebrates including the bullfrog and turtle (for reviews see Fettiplace and Kim, 2014, Howard et al., 1988, Pan and Holt, 2015). Currently, mice are still the preferred choice for investigating the development and function of the auditory and vestibular systems due to in part the availability of models linked to deafness and vestibular dysfunction in humans (Lenz & Avraham, 2011). However, while in vivo afferent neuron recordings have been performed in mammals, they have relied on tone delivery to stimulate hair cells in the ear (Buran et al., 2010, Taberner and Liberman, 2005) as direct access to innervated hair cells from an intact animal is technically very challenging. Recently, zebrafish (Danio rerio) have emerged as a powerful tool to study sensory hair-cell transduction in vivo (Nicolson et al., 1998, Olt et al., 2014, Ricci et al., 2013, Trapani and Nicolson, 2010) overcoming many of the technical challenges present in rodent models.
The lateral line is required in zebrafish to detect water movement and vibration up to about 300 Hz (Van Trump & McHenry, 2008), and the animal uses this information for schooling behavior, detection of prey, mating, and avoiding predators (Ghysen & Dambly-Chaudière, 2004). The hair cells in the lateral line are clustered in rosettelike structures termed neuromasts, which are deposited along the length of the fish during the first few days post fertilization (dpf) (Ghysen & Dambly-Chaudière, 2004). Each neuromast contains about 8 hair cells at around 4 dpf and increases to about 20–30 cells by 30 dpf (Kindt et al., 2012, Olt et al., 2014, Sheets et al., 2012).
On the apical surface of each hair cell is the mechanosensory hair bundle. Each hair bundle is composed of microvilli-like structures called stereocilia that are organized in a staircased fashion together with a single, taller kinocilium located adjacent to the tallest stereocilium in fish, amphibians, and mammalian vestibular hair cells. The stereocilia are arranged in rows and are connected by extracellular links of several types, with the topmost link, known as the tip link, being crucial for the opening of the mechanoelectrical transducer (MET) channels (Assad et al., 1991, Maeda et al., 2014, Pickles et al., 1984). While in mammalian systems, hair cells and associated hair bundles are encased in bone and are difficult to access, the hair cells within zebrafish neuromasts are located on the body surface. The accessibility of the hair cells and afferent neurons of the lateral-line system represents an ideal experimental model to investigate the molecular mechanisms underlying the development and function of this sophisticated sensory cell and how signals are encoded along the afferent neuronal pathway.
Here we describe the methods used to perform electrophysiological recordings from hair cells and afferent neurons in the intact larval zebrafish, which allow for functional studies of sensory transduction from the lateral line. Microphonic potentials are used as an assay for the functioning of the MET current at the top of the hair cell. We also show that this technique is sensitive to detect other transepithelial ionic currents, such as those through the light-gated ion channel, Channelrhodopsin-2. Single cell patch-clamp is used to investigate the biophysical properties of hair cells including basolateral membrane ion channels and vesicle fusion at ribbon synapses. Extracellular, loose-patch recordings of afferent neuron activity at rest and in response to stimulation allow for a better understanding of how hair cells accomplish the task of encoding sensory information into defined trains of action potentials.
Section snippets
Common Methods for Lateral Line Electrophysiology
In this section, we describe methods that are common to the three techniques used to examine hair-cell transduction and sensory encoding in the lateral line of larval zebrafish.
Stimulation of Neuromast Hair Cells
To investigate properties of hair cell mechanoelectrical transduction and hair cell–driven spiking in afferent neurons, the sensory receptor must be stimulated. Hair cells within lateral line neuromasts are traditionally stimulated with precisely controlled delivery of extracellular solution via a picospritzer, high-speed pressure clamp, or other fluid delivery system. Piezoelectric devices have also been used to deflect the neuromast cupula (Haehnel-Taguchi et al., 2014, Nicolson et al., 1998
Recording Microphonic Potentials
Microphonic potentials represent the collective, transepithelial flow of ions that occurs when hair cells are stimulated (Corey & Hudspeth, 1983). Following hair bundle deflection, the microphonic potential results from the current flowing through open MET channels and voltage-gated ion channels expressed in the basolateral membrane of hair cells, which in the zebrafish lateral line include both calcium and potassium channels (Olt et al., 2014). We have also observed microphonic potentials in
In Vivo Hair Cell Physiology
In larval zebrafish, the lateral line consists of an anterior lateral line situated on the head and a posterior lateral line extending down the trunk and tail of the fish. Because the hair cells are more visible and accessible along the trunk, it is most straightforward to perform electrophysiological recordings on hair cells within primary neuromasts of the posterior lateral line (L1–L4) originating from the first primordium (primI; (Gompel et al., 2001, Pujol-Martí and López-Schier, 2013)).
Afferent Neuron Action Currents
Hair bundle stimulation elicits a change in receptor potential that results in synaptic vesicle fusion at the ribbon synapse and the generation of action potentials (spikes) in the afferent neuron. Action potentials from an afferent neuron can be recorded using whole-cell patch-clamp (Liao, 2010, Liao and Haehnel, 2012) or with an extracellular “loose patch” in voltage clamp mode (v = 0) as action currents, which are equivalent to the first derivative of the neuron's action potential (dV/dt).
Summary
In this chapter, we have described three methods for recording sensory transduction and transmission from hair cells of the posterior lateral line of larval zebrafish. The recently developed technique of single hair cell patch-clamp has made an important contribution toward an understanding of the biophysical properties of hair cells in vivo. Together with microphonic and action current recordings, these methods—along with other established cell physiology techniques in other animal
Discussion
Zebrafish are an excellent vertebrate model to study hair-cell function and sensory transduction. A large number of auditory and vestibular mutants (Nicolson et al., 1998) and genome editing techniques including CRISPR-Cas9 (Hwang et al., 2013) are available for examining proteins of interest and continue to elucidate the molecular mechanisms underlying hearing and balance. Moreover, zebrafish are amenable for calcium imaging due to their optical transparency, which allows researchers to
Acknowledgments
This work was supported by grants from the Wellcome Trust (102892) to WM and a Faculty Research Award from the H. Axel Schupf '57 Fund for Intellectual Life to JGT. JO was supported by a PhD studentship from the University of Sheffield to WM.
References (60)
- et al.
Tip-link integrity and mechanical transduction in vertebrate hair cells
Neuron
(1991) - et al.
Development of the zebrafish lateral line
Current Opinion in Neurobiology
(2004) - et al.
Pattern formation in the lateral line of zebrafish
Mechanisms of Development
(2001) - et al.
Kinocilia mediate mechanosensitivity in developing zebrafish hair cells
Developmental Cell
(2012) - et al.
Blockage of the transduction channels of hair cells in the bullfrog's sacculus by aminoglycoside antibiotics
Hearing Research
(1989) - et al.
Hereditary hearing loss: from human mutation to mechanism
Hearing Research
(2011) - et al.
Directional cell migration establishes the axes of planar polarity in the posterior lateral-line organ of the zebrafish
Developmental Cell
(2004) - et al.
Genetic analysis of vertebrate sensory hair cell mechanosensation: the zebrafish circler mutants
Neuron
(1998) - et al.
The molecules that mediate sensory transduction in the mammalian inner ear
Current Opinion in Neurobiology
(2015) - et al.
Spike discharge patterns of spontaneous and continuously stimulated activity in the cochlear nucleus of anesthetized cats
Biophysical Journal
(1965)