Determining crosstalk of Ca2+ signals between hair cell synapses during the development of the mammalian inner ear.

MARCELO J. MOGLIE; PAUL A. FUCHS; A. BELEN ELGOYHEN; JUAN D. GOUTMAN

Congreso; 39th ARO MidWinter Meeting; 2016

Association for Research in Otolaryngology

BackgroundAltricial rodents do not respond to sound until their second postnatal week. Before the onset of hearing, cochlear inner hair cells (IHCs) fire sensory-independent action potentials that are crucial for the normal development of the auditory pathway. The influx of Ca2+ through voltage-dependent Ca2+ channels triggers the release of glutamate to afferent dendrites of the auditory nerve, determining an excitatory role for Ca2+ ions. At this stage, IHC are also innervated by efferent cholinergic neurons, projecting from the brainstem. This synapse combines the entry of Ca2+ through α9α10 nicotinic acetylcholine receptors with the activation of nearby SK2, Ca2+ dependent potassium channels, to hyperpolarize and inhibit IHCs. Thus, Ca2+ can have these two contrary roles within a diffusionally compact cell. We aim to investigate the possible crosstalk between these seemingly opposing signals and its effect in the precise temporal pattern of spontaneous activity. MethodsP9 IHC were 3D reconstructed from ultra-thin serial section electron micrographs. We performed electrophysiological recordings combined with simultaneous Ca2+ imaging acquisition in IHC from P9-P11 mice to investigate Ca2+ -dynamics during synaptic currents evoked by efferent axon electrical stimulation. IHC afferent dendrites currents were recorded in whole-cell patch clamp configuration following exogenous application of acetylcholine (ACh) or electrical stimulation of efferent fibers. ResultsElectron-micrographs of IHC exhibited thin near-membrane cisterns juxtaposed to efferent synaptic contacts, like those described in mature OHC. Moreover, we found close proximity between these cisterns and synaptic ribbons. Imaging experiments have shown multiple Ca2+ entry hotspots following activation of efferent fibers. During single synaptic events, these domains would be spatially segregated from those observed after IHC depolarization. On the other hand, high frequency stimulation evoked global Ca2+ signal increases. Therefore, we tested the effect of several stimulation frequencies to inhibit IHC action potential firing: 20 Hz stimulation protocols probed to be sufficient to completely abolish firing. Finally, we decided to evaluate if ribbon synase activity could be elicied by calcium diffusing from efferent synapses. Exogenous app of ach produced a robust increase in the e frequency ofepsc activation. Using more physiological stimuli, we electrically stimulated efferemt fibers and foun that only trains of shocks at freq produced crosstalk. Howhe effect of efferent fiber stimulation on afferent release. We found that upon high frequency stimulation of efferent fibers, there was a robust increase in the frequency of EPSPs. In that way, the exogenous application of ACh had the same effect, confirming the cholinergic nature of the release. ConclusionsBoth morphological and ca imaging data provide evidence for a close proximity between afferent and eff terminals in developinf IHC.