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