The Role of the Mechanotransduction Ion Channel Candidate Nanchung-Inactive in Auditory Transduction in an Insect Ear

Abstract

Insect auditory receivers provide an excellent comparative resource to understand general principles of auditory transduction, but analysis of the electrophysiological properties of the auditory neurons has been hampered by their tiny size and inaccessibility. Here we pioneer patch-clamp recordings from the auditory neurons of Müller's organ of the desert locust Schistocerca gregaria to characterize dendritic spikes, axonal spikes, and the transduction current. We demonstrate that dendritic spikes, elicited by sound stimuli, trigger axonal spikes, and that both types are sodium and voltage dependent and blocked by TTX. Spontaneous discrete depolarizations summate upon acoustic stimulation to produce a graded transduction potential that in turn elicits the dendritic spikes. The transduction current of Group III neurons of Müller's organ, which are broadly tuned to 3 kHz, is blocked by three ion channel blockers (FM1-43, streptomycin, and 2-APB) that are known to block mechanotransduction channels. We investigated the contribution of the candidate mechanotransduction ion channel Nanchung-Inactive—which is expressed in Müller's organ—to the transduction current. A specific agonist of Nanchung-Inactive, pymetrozine, eliminates the sound-evoked transduction current while inducing a tonic depolarizing current of comparable amplitude. The Nanchung-Inactive ion channels, therefore, have the required conductance to carry the entire transduction current, and sound stimulation appears not to open any additional channels. The application of three mechanotransduction ion channel blockers prevented the pymetrozine-induced depolarizing current. This implies that either Nanchung-Inactive is, or forms part of, the mechanotransduction ion channel or it amplifies a relatively small current (<30 pA) produced by another mechanotransduction ion channel such as NompC.SIGNIFICANCE STATEMENT The mechanically activated ion channel underpinning hearing is not known. We have pioneered intracellular patch-clamp recordings from locust auditory neurons to unravel the role of the candidate mechanotransduction ion channel Nanchung-Inactive in auditory transduction in insects.

Physiological basis of noise-induced hearing loss in a tympanal ear

“…In order to quantitatively record tone-evoked currents and spikes from individual auditory neurons the ear was excised from the locust, placed in a recording chamber and the internal surface, with Müller's organ, perfused with saline. Immersion of Müller's organ in saline is necessary to gain access to, image and record from individual auditory neurons through whole-cell patch-clamp intracellular recordings (Warren & Matheson, 2018). Elizabeth quantified the reduction in tone-evoked tympanal vibrations when the tympanum is backed by water -as opposed to its tracheal air backing when in vivo (Figure 1Bi, Bii).…”

“…We recorded smaller discrete depolarisations in the auditory neurons of noise-exposed locust auditory neurons compared to control ( Figure 3D, 3E). Discrete depolarisations are assumed to be transient stochastic opening of mechanotransduction ion channels shown both in insects (Hill, 1983b;Warren & Matheson, 2018) and vertebrate auditory receptors (Beurg et al, 2015;Pan et al, 2013). There was a significant delay in the generation of the transduction current in noise-exposed locusts compared to their control counterparts ( Figure 3F) at the maximal sound amplitude of 110 dB SPL ( Figure 3G) and across sound amplitudes ( Figure 3H).…”

“…In Orthoptera, such as locusts and crickets, the transduction potential is converted into a small dendritic spike that travels along a single dendrite of the neuron (Hill, 1983b;Oldfield & Hill, 1986) to the soma. There, most probably in the axon hillock, it triggers a larger axonal spike that carries auditory information to the central nervous system (Warren & Matheson, 2018).…”

“…Here, we used the physiologically-accessible locust ear to measure electrophysiological changes in the auditory neurons directly after noise exposure, nerve potentials from the auditory nerve and mechanical changes in the vibrations of the tympanum. The locust ear permits a detailed characterisation of the soundelicited ionic currents because whole-cell patch-clamp recordings can be conducted during concurrent stimulation by airborne sound in an intact ear (Warren and Matheson, 2018) -an approach not possible in other hearing models. In addition, composite spikes can be recorded from the auditory nerve and the tympanum is easily assessable for mechanical analysis (Windmill et al, 2005).…”

“…The locust's auditory Müller's Organ is attached directly to the inside of the tympanum -like a splayed hand -and has three groups of neurons that attach onto discrete parts of the tympanum to exploit its heterogeneous resonant frequencies to detect sound from 200 Hz to 40 kHz (Jacobs et al, 1999). We focus our analyses on Group III auditory neurons that make up ~46 of the 80 auditory neurons and are broadly tuned to 3 kHz (Warren & Matheson, 2018). In response to acoustic overstimulation we measured physiological changes in the sound-evoked displacements of the tympanum, electrical responses from the auditory nerve, ionic currents in individual auditory neurons, and relative abundances of some putative sound-activated ion channel genes.…”