Compartmentalization of antagonistic Ca2+ signals in developing cohlear hair cells

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

Congreso; Society for Neuroscience; 2018

During a critical developmental period, cochlear inner hair cells (IHCs) fire sensory-independent action potentials, crucial for the normal development of the auditory pathway. During each action potential, Ca2+ influx through voltage-dependent channels triggers the release of glutamate onto afferent dendrites of the auditory nerve. At this synapse, IHC opoerate as a presynaptic terminal. At the same developmental stage, IHCs are also innervated by efferent cholinergic neurons from the brainstem. 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, which operates as a postsynaptic terminal in this case. 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.  Electrophysiological recordings combined with swept?field confocal Ca2+ imaging experiments revealed the existence of multiple Ca2+ entry hotspots per IHC upon efferent fiber electrical stimulation. They were concentrated in the basal pole of the IHC in close apposition and intermingled with the afferent hotspots encountered following IHC depolarization. Using serial section electron micrographs we were able to perform IHC reconstructions at a nanometer scale. We estimated an average distance between efferent hotspots and closest afferent neighbors of 1.62 ± 1.11 µm. The intimate localization of antagonist Ca2+ entry sites encountered suggests that during normal operation of developing IHCs, efferent Ca2+ spread could potentially occur over other synapses. Finally, in order to evaluate a possible efferent to afferent crosstalk, recordings from afferent boutons were performed. Despite the close proximity, even high frequency (80 Hz) electrical stimulation of efferent fibers failed to evoke an increase in the frequency of postsynaptic excitatory currents. On the contrary, locally applied saturating concentrations of ACh elicited Ca2+ signals capable of cross-activating afferent release. Using Ca2+ imaging experiments in IHC, we showed that physical barriers imposed by efferent synaptic cisterns, Ca2+ extrusion mechanisms and strong Ca2+ buffering prevent efferent to afferent synaptic crosstalk during synaptic activation of cholinergic release. Thus, efferent fibers maintain its inhibitory signature and operate at low frequencies to modulate spontaneous action potential firing in the developing IHC.