Fast Ca2+ Transients of Inner Hair Cells Arise Coupled and Uncoupled to Ca2+ Waves of Inner Supporting Cells in the Developing Mouse Cochlea

Abstract

Before the onset of hearing, which occurs around postnatal day 12 (P12) in mice, inner hair cells (IHCs) of the immature cochlea generate sound-independent Ca²⁺ action potentials (APs), which stimulate the auditory pathway and guide maturation of neuronal circuits. During these early postnatal days, intercellular propagating Ca²⁺ waves elicited by ATP-induced ATP release are found in inner supporting cells (ISCs). It is debated whether IHCs are able to fire Ca²⁺ APs independently or require a trigger by an ISC Ca²⁺ wave. To identify the Ca²⁺ transients of IHCs underlying Ca²⁺ APs and to analyze their dependence on ISC Ca²⁺ waves, we performed fast Ca²⁺ imaging of Fluo-8 AM-loaded organs of Corti at P4/P5. Fast Ca²⁺ transients (fCaTs) generated by IHCs were simultaneously imaged with Ca²⁺ waves in ISCs. ISC Ca²⁺ waves frequently evoked bursts consisting of >5 fCaTs in multiple adjacent IHCs. Although Ca²⁺ elevations of small amplitude appeared to be triggered by ISC Ca²⁺ waves in IHCs of Ca_v1.3 knockout mice we never observed fCaTs, indicating their requirement for Ca²⁺ influx through Ca_v1.3 channels. The Ca²⁺ wave-triggered Ca²⁺ upstroke in wildtype IHCs occurred 0.52 ± 0.27 s later than the rise of the Ca²⁺ signal in the adjacent ISCs. In comparison, superfusion of 1 μM ATP elicited bursts of fCaTs in IHCs starting 0.99 ± 0.34 s prior to Ca²⁺ elevations in adjacent ISCs. PPADS irreversibly abolished Ca²⁺ waves in ISCs and reversibly reduced fCaTs in IHCs indicating differential involvement of P2 receptors. IHC and ISC Ca²⁺ signals were however unaltered in P2X2R/P2X3R double knockout or in P2X7R knockout mice. Together, our data revealed a fairly similar occurrence of fCaTs within a burst (56.5%) compared with 43.5% as isolated single fCaTs or in groups of 2-5 fCaTs (minibursts). We provide evidence that IHCs autonomously generate single fCaTs and minibursts whereas bursts synchronized between neighboring IHCs were mostly triggered by ISC Ca²⁺ waves. Neonatal IHCs thus spontaneously generate electrical and Ca²⁺ activity, which is enhanced and largely synchronized by activity of ISCs of Kölliker's organ indicating two sources of spontaneous activity in the developing auditory system.