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

FUCHS P; MOGLIE M; GOUTMAN J; ELGOYHEN AB

San Diego, California

Congreso; 39th Midwinter Meeting, Association for Research in Otolaryngology; 2016

Association for Research in Otolaryngology

BackgroundBefore the onset of hearing (in altricial rodents, postnatal day 14) cochlear inner hair cells (IHCs) fire sensory-independent action potentials, crucial for the normal development of the auditory pathway. Ca2+influx 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. At this synapse Ca2+ has an inhibitory functional endpoint. The entry of Ca2+ through α9α10 cholinergic receptors activates SK2, Ca2+-dependent potassium channels, producing hyperpolarization, and modulating IHCs electrical activity. Segregation of Ca2+´s excitatory versus inhibitory effects within IHCs appears as a functional demand within a very small diffusional space. The aim of our work was to investigate this phenomenon at the cellular level.MethodsIHC were 3D reconstructed from ultra-thin serial section electron micrographs. Electrophysiological recordings were combined with Ca2+ imaging measurements in IHCs from P9-P11 mice to investigate Ca2+dynamics during synaptic currents evoked by efferent axon electrical stimulation. IHC afferent bouton currents were recorded in whole-cell patch clamp configuration while stimulating efferent synapses.ResultsElectron-micrographs of IHC exhibited thin near-membrane cisterns juxtaposed to efferent synaptic contacts, similar to those described in mature OHC. In addition, close proximity between these cisterns and synaptic ribbons was observed. Imaging experiments showed an average of 2.4 Ca2+ entry hotspots per IHC, using efferent fibers electrical stimulation. During single synaptic events, these hotspots were spatially segregated from those observed after IHC depolarizations (average minimal distance = 2.0 mm). In contrast, trains of stimuli at different frequencies, and ACh applications, evoked global Ca2+ signal increases.Surprisingly, recordings from afferent boutons showed that exogenous applications of ACh produced a strong increase in the frequency of EPSCs, presumably due to release triggered by Ca2+ influx throughα9α10. This was reproduced by electrical stimulation of efferent fibers with trains at high frequencies (80 Hz), which also inhibited IHC. In contrast, stimulation at lower frequencies produced a decrease in afferent pathway activity. Consequently, trains of stimuli at these frequencies produced either partial (2?10 Hz), or complete (20 Hz) inhibition of IHC action potential firing.ConclusionsBoth morphological and Ca2+ imaging data provide evidence for a close proximity between afferent and efferent synapses in developing IHC. Physical barriers, imposed by synaptic cisterns and/or strong Ca2+buffering in IHC prevent efferent to afferent synaptic crosstalk only at low frequency stimulation.