Compartmentalization of antagonistic Ca2+ signals in developing cohlear hair cells

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

Baltimore, Maryland

Congreso; 40th ARO MidWinter Meeting; 2017

Association for Research in Otolaryngology

BackgroundBefore the onset of hearing (postnatal day 14 in rodents) cochlear inner hair cells (IHCs) fire sensory-independent action potentials, crucial for the normal development of the auditory pathway. Ca2+ influx through voltage-dependent channels triggers the release of glutamate onto afferent dendrites of the auditory nerve. At this stage, efferent cholinergic neurons from the brainstem also innervate IHCs. This efferent synapse combines the entry of Ca2+ through cholinergic α9α10 receptors with the activation of nearby SK2, Ca2+-dependent potassium channels, to hyperpolarize the IHC. Thus, efferent Ca2+ signals are inhibitory, opposing IHC transmitter release. The aim of our work was to investigate the mechanisms that allow segregation of excitatory versus inhibitory Ca2+ effects within the limited diffusional space of the IHCs synaptic pole.  MethodsWe performed anatomical and functional IHC 3D reconstructions via electron microscopy (EM) of ultra-thin serial sections and swept-field confocal Ca2+ imaging of IHCs in cochlear explants. Electrophysiological recordings were combined with Ca2+ imaging measurements in IHCs from P9-P11 mice to investigate Ca2+ dynamics during synaptic transmission evoked by electrical stimulation of efferent axons. Electrophysiological recordings of type I afferent boutons were made while stimulating efferent synapses.ResultsCa2+ imaging experiments revealed an average of 6±2 Ca2+ entry hotspots per IHC upon efferent fiber electrical stimulation. This value represents a lower limit of the functional synaptic contacts that could be successfully distinguished and differed from EM counts (17±8), which included all morphologically discernible synapses. Afferent hotspots were visualized following IHC depolarization and were spatially segregated from those encountered during efferent activation. We estimated an average distance between efferent hotspots and closest afferent neighbors of 1.45 µm. This value agreed with the distance measured between nearest neighbor sub-synaptic cisterns and synaptic ribbons in electron micrographs. Finally, in order to establish a possible efferent to afferent crosstalk, recordings from afferent boutons were performed. Despite the close proximity, only high frequency (80 Hz) electrical stimulation of efferent fibers evoked an increase in the frequency of postsynaptic excitatory currents, attributable to glutamate release triggered by Ca2+ influx through α9α10 receptors. ConclusionsBoth morphological and Ca2+ imaging data provide evidence for close proximity between afferent and efferent synapses in developing IHCs. Although high frequency efferent stimulation could drive IHC transmitter release, physical barriers imposed by synaptic cisterns and/or strong Ca2+buffering in IHCs prevent efferent to afferent synaptic crosstalk during lower frequency stimulation that is sufficient to suppress spontaneous action potentials in the IHC.