Connexin-Mediated Signaling in Nonsensory Cells Is Crucial for the Development of Sensory Inner Hair Cells in the Mouse Cochlea

Abstract

Mutations in the genes encoding for gap junction proteins connexin 26 (Cx26) and connexin 30 (Cx30) have been linked to syndromic and nonsyndromic hearing loss in mice and humans. The release of ATP from connexin hemichannels in cochlear nonsensory cells has been proposed to be the main trigger for action potential activity in immature sensory inner hair cells (IHCs), which is crucial for the refinement of the developing auditory circuitry. Using connexin knock-out mice, we show that IHCs fire spontaneous action potentials even in the absence of ATP-dependent intercellular Ca 2ϩ signaling in the nonsensory cells. However, this signaling from nonsensory cells was able to increase the intrinsic IHC firing frequency. We also found that connexin expression is key to IHC functional maturation. In Cx26 conditional knock-out mice (Cx26 Sox10-Cre ), the maturation of IHCs, which normally occurs at approximately postnatal day 12, was partially prevented. Although Cx30 has been shown not to be required for hearing in young adult mice, IHCs from Cx30 knock-out mice exhibited a comprehensive brake in their development, such that their basolateral membrane currents and synaptic machinery retain a prehearing phenotype. We propose that IHC functional differentiation into mature sensory receptors is initiated in the prehearing cochlea provided that the expression of either connexin reaches a threshold level. As such, connexins regulate one of the most crucial functional refinements in the mammalian cochlea, the disruption of which contributes to the deafness phenotype observed in mice and DFNB1 patients.

Coordinated calcium signalling in cochlear sensory and non‐sensory cells refines afferent innervation of outer hair cells

“…In mature OHCs (P10), the number of ribbons in Cx30 À/À OHCs was about half of that in wildtype OHCs (P < 0.0001, t-test: Fig 9C,D and E). As a comparison, we also looked at IHCs and found a similar number of ribbons between the two genotypes at both P4 (P = 0.30, t-test: Appendix Fig S8A,B and E) and P10 (P = 0.43, t-test: Appendix Fig S8C,D and E), further supporting the evidence that at this age immature IHCs are unaffected by the absence of Ca 2+ waves (Johnson et al, 2017). The requirement for OHC Ca 2+ signals for the maturation of the afferent synapses was further tested by using knockout mice for Ca v 1.3 Ca 2+ channels (Cav1.3 À/À ), which are required for hair cell exocytosis.…”

“…receptors have previously been implicated in early development in the peripheral and central nervous system (Kidd et al, 1998). Although ATP-induced Ca 2+ signalling from non-sensory cells is crucial for promoting the maturation of IHCs after the onset of hearing (Johnson et al, 2017), our data suggest that it does not contribute directly to OHC maturation. However, the time course of maturation of OHCs and IHCs is very different (Knirsch et al, 2007;Corns et al, 2014).…”

“…Similar Ca 2+ signalling mechanisms are used to drive the maturation of IHCs (Johnson et al, 2013), but they do not appear to be required for the refinement of the pre-synaptic ribbons and postsynaptic afferents. Moreover, in contrast to the ATP-dependent activity modulating IHC action potentials (Wang et al, 2015), that influencing OHC Ca 2+ signals is mediated by ATP-induced activation of P2X 3 receptors, which has distinct functional consequences for OHC maturation and is separated by developmental timing, with OHCs preceding IHCs (Johnson et al, 2011(Johnson et al, , 2017. The data suggest that several distinct patterns of spontaneous, experience-independent Ca 2+ activity across the auditory sensory epithelium orchestrate the differential maturation of OHCs (afferent innervation) and IHCs (sensory cells Johnson et al, 2013) to shape the final stages of auditory organ development.…”

“…OHC ribbon synapses and afferent fibres are reduced in Cx30 À/À and Cav1.3 À/À mice The above results demonstrate that pre-hearing Cx30 À/À mice, in which OHCs retain their intrinsic Ca 2+ activity but which have reduced and more spatially confined Ca 2+ waves in the GER, were able to develop functionally mature OHCs. During the same time window (~P0-P12), immature IHCs from Cx30 À/À mice have been shown to be normal (Johnson et al, 2017), indicating that Ca 2+ waves in the non-sensory cells do not interfere with the normal prehearing development of hair cells. Indeed, it has been suggested that the modulation of AP activity in IHCs by the Ca 2+ waves could be used to refine the afferent auditory pathway (Tritsch et al, 2007).…”

“…The refinement of sensory circuits during development is normally influenced by periods of experience-independent action potential (AP) activity before the onset of function (Katz & Shatz, 1996;Blankenship & Feller, 2010). Calcium-dependent APs have been shown to occur spontaneously in immature IHCs (Johnson et al, 2011(Johnson et al, , 2017 but not in OHCs (Oliver et al, 1997;Marcotti & Kros, 1999;Weisz et al, 2012). One study reported spontaneous APs in OHCs of wild-type and otoferlin mutant mice, but mostly using elevated extracellular Ca 2+ and high intracellular EGTA (Beurg et al, 2008).…”