Research report
Developmental expression of Cav1.3 (α1d) calcium channels in the mouse inner ear

https://doi.org/10.1016/j.devbrainres.2004.03.007Get rights and content

Abstract

Voltage-gated calcium channels are important for neurotransmission at the level of inner hair cells (IHCs) and outer hair cells (OHCs). These channels open when mechanical stimulation depolarises the hair cell membrane and the resulting calcium influx triggers neurotransmitter release. Voltage-gated calcium channels expressed in hair cells are known to be of the L-type with a predominance of the Cav1.3 subunit. The present study describes the developmental expression of the Cav1.3 protein in the cochlea and the vestibular system using immunohistochemical technique. In the adult organ of Corti (OC), Cav1.3 was localized in both sensory and non-sensory cells with a more intense expression in IHCs and Deiters cells when compared to OHCs. In both hair cell types, immunoreactivity was observed in the apical pole, basolateral membrane and at the basal pole (synaptic zone). Similar results were obtained in the vestibular organs. During development, Cav1.3 immunoreactivity was observed in the cochlea as early as embryonic day 15, with expression increasing at birth. At these early stages of cochlear development, Cav1.3 was expressed in all cell types surrounding the scala media. In the OC, the labeling was observed in IHCs, OHCs and supporting cells. The Cav1.3 expression reached an adult-like pattern by the end of the second postnatal week. The present findings suggested that, in addition to their implication in hair cells synaptic transmission, Cav1.3 calcium channels also play an important role in vesicle recycling and transport, as suggested by their extrasynaptic location at the apical pole of the hair cells. The Cav1.3 channels in Deiters cells could participate in active calcium-induced changes in micromechanics of these supporting cells. An early expression during development suggested that these calcium channels are in addition important in the development of the cochlear and vestibular sensory epithelium.

Introduction

Hearing sensory cells are responsible for the transduction of sound into spikes at the afferent auditory fiber. Two different types of sensory cells are found in the cochlea (hearing organ): the outer hair cells (OHCs) which serve to amplify the incoming sound waves, and the inner hair cells (IHCs) which transduce sound signals into neuron discharges by secreting glutamate onto the afferent nerve fibers. Neurotransmitter release by IHCs has long been recognized to involve L-type voltage-gated calcium channel [4], [7], [9], [11], [23], [33], [34], [41], [42], [51], [52]. These calcium channels have also been also described in OHCs where their role remains more obscure [19], [30], [35].

Based on physiological and pharmacological actions, seven different types of calcium voltage-gated channels have been identified [1]. They are classified as P/Q, N, R, L and T types. The first five are activated by strong depolarization, while the T types are activated by a lower depolarization. The L type channel pore is formed by different α1 subunit isoforms {Cav1.1 (α1S), Cav1.2 (α1C), Cav1.3 (α1D), Cav1.4 (α1F)} and with auxiliary subunits (α2-δ, β). All these isoforms give rise to L-type calcium currents. The physiological properties of these channels are determined by the α1 subunits [18]. In sensory hair cells, L-type calcium currents are believed to be carried mainly by Cav1.3 subunit channels [6]. In mutant mice, the absence of Cav1.3 channels causes deafness and degeneration of developing OHCs and IHCs, suggesting their importance in hair cell survival [13], [37]. Several molecular studies have described mRNA expression of Cav1.3 L type subunit in chicken [21], [22] and mice cochlea [14], but the distribution of this channel subunit at the protein level has not yet been investigated in detail during development. The goal of the present work was to characterize the cellular distribution of the Cav1.3 channel protein in the developing and adult mouse cochlea and vestibular organ.

Section snippets

Materials and methods

Mice were deeply anaesthetized with sodium Chloral hydrate (10%) and were fixed by perfusion through the heart at postnatal day 0 (P0), P6, P12 and adult, or by immersion [Embryonic day 15 (E15) and E17] with a 4% paraformaldehyde solution. Dated pregnant mice were purchased from the Charles-River (France). Cochleae were then dissected and post-fixed overnight in the same fixative. labyrinth from P6 and older animals were decalcified in a 10% EDTA solution before immunochemical staining.

Cav1.3 in the adult cochlea

Immunoreactivity to Cav1.3 within the cochlea was observed in stria vascularis, spiral ligament, spiral limbus, OC and spiral ganglion neurons (Fig. 1a). Within the OC, the staining was observed in both IHCs, OHCs, and supporting cells, with IHCs exhibiting the most intense immunostaining (Fig. 1b). Intense labeling was also localized to the apical pole (just below the cuticular plate) of both sensory hair cells. Within the IHC, the Cav1.3 immunoreactivity was observed in the synaptic area, and

Discussion

The present study describes for the first time the immunolocalization of Cav1.3 subunit channel in the adult and developing cochlea. This subtype channel was expressed in a variety of cells within the adult cochlea, including the stria vascularis, the ligament limbus, the organ of Corti (supporting and sensory cells) and the spiral ganglion neurons.

A differential intensity of staining was observed between IHCs and OHCs, with stronger expression of Cav1.3 in IHCs. These results are in agreement

Acknowledgments

We would like to thank Dr. J. Saunders for his comments and English correction of the manuscript.

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