In vertebrates, sound and head movement are transformed into electrical signals in the inner ear hair cells, and the signals are transmitted to brain through the auditory (VIIIp) and vestibular (VIIIa) nerves, respectively. Although the electrophysiological properties of the VIIIth nerve have been extensively studied, the differentiation, development and molecular basis underlying auditory and vestibular systems remain to be fully elucidated. Here, we established a semi-in-vivo preparation of the VIIIth nerve organization in zebrafish larvae to elucidate them. Larval zebrafish at 2-5 days post-fertilization (dpf) were temporally anaesthetized and the brain was removed from the head to expose the VIIIth nerve cell bodies, with keeping inner ear intact, for loose patch recording.
In 5 dpf larvae, which sense sound and maintain body balance, VIIIp nerve cells, but not VIIIa, fired in response to sound (500 Hz). The spiking was locked to the sound phase at the same or twice the sound frequency.
In early larvae at 2 dpf, which do not escape from sound stimulus, VIIIp nerves already responded to sound but the sensitivity was low and the spike timing varied from trial to trial. Between the two stages, sound sensitivity and phase-locked firing are gradually improved. The time course of VIIIp nerve responsivity changes matches to that of developmental acquisition of acoustically evoked escape behavior (Kohashi et al., 2012).
Underlying mechanisms for development in sound-evoked VIIIp nerve cell response may involve improvement of hair cell mechanotransduction: increase in hair cell number (Haddon and Lewis, 1996) and improvement of their mechanosensitivity (Tanimoto et al., 2009) by the ear stone growth (Inoue et al., 2013). Maturations of temporally precise synaptic vesicle release from the hair cells, postsynaptic machinery and membrane properties in VIIIp nerves may also be important. The semi-in-vivo preparation we established here takes advantage to reveal the developmental processes of the VIIIth nerve system and their molecular basis.