Early Development of Hearing in Zebrafish

Abstract

The zebrafish (Danio rerio) has become a valuable vertebrate model for human hearing and balance disorders because it combines powerful genetics, excellent embryology, and exceptional in vivo visualization in one organism. In this study, we investigated auditory function of zebrafish at early developmental stages using the microphonic potential method. This is the first study to report ontogeny of response of hair cells in any fish during the first week post fertilization. The right ear of each zebrafish embedded in agarose was linearly stimulated with a glass probe that was driven by a calibrated piezoelectric actuator. Using beveled micropipettes filled with standard fish saline, extracellular microphonic potentials were recorded from hair cells in the inner ear of zebrafish embryos or larvae in response to 20, 50, 100, and 200-Hz stimulation. Saccular hair cells expressing green fluorescent protein of the transgenic zebrafish from 2 to 7 days post fertilization (dpf) were visualized and quantified using confocal microscopy. The otic vesicles' areas, otoliths' areas, and saccular hair cell count and density increased linearly with age and standard body length. Microphonic responses increased monotonically with stimulus intensity, stimulus frequency, and age of zebrafish. Microphonic threshold at 200 Hz gradually decreased with zebrafish age. The increases in microphonic response and sensitivity correlate with the increases in number and density of hair cells in the saccule. These results enhance our knowledge of early development of auditory function in zebrafish and provide the control data that can be used to evaluate hearing of young zebrafish morphants or mutants.

Manipulation of BK channel expression is sufficient to alter auditory hair cell thresholds in larval zebrafish

“…Consistent with prior studies using similar stimulation and recording methods (Starr et al, 2004;Tanimoto et al, 2009;Lu and DeSmidt, 2013), and of behavioral audiograms (Bhandiwad et al, 2013), wildtype larvae were responsive to vibratory stimuli over the range of frequencies tested (175-500 Hz, Fig. 2).…”

“…Microphonic potentials can be recorded from the saccule of developing zebrafish as young as 2 dpf (Tanimoto et al, 2009;Lu and DeSmidt, 2013) and are a valuable tool for determining the effect of mutations or MO treatments on auditory function (Lu and DeSmidt, 2013). Here, we used this larval zebrafish model to test the functional implications of manipulating the expression of BK channels encoded by two slo1 genes on hair cell auditory thresholds.…”

“…Saccular microphonic potentials were recorded from the otic vesicles of zebrafish larvae at 3 dpf, a time point by which the auditory circuit is functional (Tanimoto et al, 2009). Although saccular auditory thresholds decrease over 3-7 dpf as the hair cell population increases in number (Lu and DeSmidt, 2013), we did not want to delay our recordings until a later time point because MOs are most effective up to 3 dpf (Bill et al, 2009). This limitation of MOs does not change our prediction of the disruptive effect of slo1 MO treatment on hair cell function in zebrafish larvae in this initial investigation using this methodology.…”

“…KCl (Rohmann and Bass, 2011) was positioned medial to the posterior, saccular macula (Lu and DeSmidt, 2013) to record saccular microphonic potentials at 3 dpf from the otic vesicles of 13 non-manipulated wild-type, 24 slo1a MO-injected (total for three doses, see below), 12 slo1a mis-pair control MO-injected, 16 slo1b MO-injected and 16 slo1b mis-pair control-injected animals. Consistent with prior studies using similar stimulation and recording methods (Starr et al, 2004;Tanimoto et al, 2009;Lu and DeSmidt, 2013), and of behavioral audiograms (Bhandiwad et al, 2013), wildtype larvae were responsive to vibratory stimuli over the range of frequencies tested (175-500 Hz, Fig.…”

Vocal differentiation parallels development of auditory saccular sensitivity in a highly soniferous fish

“…Although relatively few studies have examined the structurefunction relationships in the developing fish ear, increases in auditory sensitivity during ontogeny in fish have been typically explained as a result of general inner ear development with continued addition of hair cells (Popper and Hoxter, 1984;Lombarte and Popper, 1994;Lu and DeSmidt, 2013), number of auditory nerve ganglion cells, innervation patterns of the eighth nerve (Corwin, 1983;Popper and Hoxter, 1984) and/or increase in swimbladder size and development of the morphological connections (Weberian ossicles) to the ear (Lechner et al, 2011). Another possibility could be the otolith enlargement with fish growth, providing increased inertial mass for the accelerometer-like auditory endorgan (Gauldie, 1988).…”