Calcium signaling in interdental cells during the critical developmental period of the mouse cochlea

Abstract

The tectorial membrane (TM), a complex acellular structure that covers part of the organ of Corti and excites outer hair cells, is required for normal hearing. It consists of collagen fibrils and various glycoproteins, which are synthesized in embryonic and postnatal development by different cochlear cell types including the interdental cells (IDCs). At its modiolar side, the TM is fixed to the apical surfaces of IDCs, which form the covering epithelium of the spiral limbus. We performed confocal membrane imaging and Ca²⁺ imaging in IDCs of the developing mouse cochlea from birth to postnatal day 18 (P18). Using the fluorescent membrane markers FM 4-64 and CellMask™ Deep Red on explanted whole-mount cochlear epithelium, we identified the morphology of IDCs at different z-levels of the spiral limbus. Ca²⁺ imaging of Fluo-8 AM-loaded cochlear epithelia revealed spontaneous intracellular Ca²⁺ transients in IDCs at P0/1, P4/5, and P18. Their relative frequency was lowest on P0/1, increased by a factor of 12.5 on P4/5 and decreased to twice the initial value on P18. At all three ages, stimulation of IDCs with the trinucleotides ATP and UTP at 1 and 10 μM elicited Ca²⁺ transients of varying amplitude and shape. Before the onset of hearing, IDCs responded with robust Ca²⁺ oscillations. At P18, after the onset of hearing, ATP stimulation either caused Ca²⁺ oscillations or an initial Ca²⁺ peak followed by a plateau while the UTP response was unchanged from that at pre-hearing stage. Parameters of spontaneous and nucleotide-evoked Ca²⁺ transients such as amplitude, decay time and duration were markedly reduced during cochlear development, whereas the kinetics of the Ca²⁺ rise did not show relevant changes. Whether low-frequency spontaneous Ca²⁺ transients are necessary for the formation and maintenance of the tectorial membrane e.g. by regulating gene transcription needs to be elucidated in further studies.

Keywords: Ca(2+) transient; Cochlea; Interdental cell; Membrane imaging; P2Y receptor; Tectorial membrane.