Maturation of NaV and KV channel topographies in the auditory nerve spike initiator before and after developmental onset of hearing function

Auditory nerve excitation and thus hearing depend on spike-generating ion channels and their placement along the axons of auditory nerve fibers (ANFs). The developmental expression patterns and native axonal locations of voltage-gated ion channels in ANFs are unknown. Therefore, we examined the development of heminodes and nodes of Ranvier in the peripheral axons of type I ANFs in the rat cochlea with immunohistochemistry and confocal microscopy. Nodal structures presumably supporting presensory spiking formed between postnatal days 5 (P5) and P7, including Ankyrin-G, NaV1.6, and Caspr. These immature nodal structures lacked low-voltage-activated KV1.1 which was not enriched at juxtaparanodes until approximately P13, concurrent with the developmental onset of acoustic hearing function. Anatomical alignment of ANF spike-initiating heminodes relative to excitatory input from inner hair cell (IHC) ribbon synapses continued until approximately P30. High-voltage-activated KV3.1b and KV2.2 were expressed in mutually exclusive domains: KV3.1b was strictly localized to nodes and heminodes, whereas KV2.2 expression began at the juxtaparanodes and continued centrally along the first internode. At spike-initiating heminodes in the distal osseous spiral lamina, NaV1.1 partly overlapped NaV1.6 and ankyrin-G. ANFs displayed KV7.2 and KV7.3 at heminodes, nodes, internodes, and the unmyelinated synaptic terminal segments beneath IHCs in the organ of Corti. In response to sound, spikes are initiated at the heminode, which is tightly coupled to the IHC ribbon synapse ∼20–40 μm away. These results show that maturation of nodal alignment and ion channel content may underlie postnatal improvements of ANF excitability and discharge synchrony.SIGNIFICANCE STATEMENTAcoustic and electrical hearing depends on rapid, reliable, and precise spike generation in auditory nerve fibers. A limitation of current models and therapies is a lack of information on the identities and topographies of underlying ion channels. We report the developmental profile of the auditory nerve spike generator with a focus on NaV1.1, NaV1.6, KV1.1, KV2.2, KV3.1b, KV7.2, and KV7.3 in relation to the scaffold ankyrin-G. Molecular anatomy of the spike generator matures in the weeks after developmental onset of hearing function. Subcellular positioning of voltage-gated ion channels will enable multicompartmental modeling of auditory nerve responses elicited by afferent chemical neurotransmission from hair cells and modulated by efferent neurotransmitters or evoked by extracellular field stimulation from a cochlear implant.</jats:p

Sodium selectivity of Reissner's membrane epithelial cells

<p>Abstract</p> <p>Background</p> <p>Sodium absorption by Reissner's membrane is thought to contribute to the homeostasis of the volume of cochlear endolymph. It was previously shown that the absorptive transepithelial current was blocked by amiloride and benzamil. The most commonly-observed target of these drugs is the epithelial sodium channel (ENaC), which is composed of the three subunits α-,β- and γ-ENaC. However, other less-selective cation channels have also been observed to be sensitive to benzamil and amiloride. The aim of this study was to determine whether Reissner's membrane epithelial cells could support parasensory K⁺absorption via amiloride- and benzamil-sensitive electrogenic pathways.</p> <p>Results</p> <p>We determined the molecular and functional expression of candidate cation channels with gene array (GEO GSE6196), RT-PCR, and whole-cell patch clamp. Transcript expression analysis of Reissner's membrane detected no amiloride-sensitive acid-sensing ion channels (ASIC1a, ASIC2a, ASIC2b) nor amiloride-sensitive cyclic-nucleotide gated channels (CNGA1, CNGA2, CNGA4, CNGB3). By contrast, α-,β- and γ-ENaC were all previously reported as present in Reissner's membrane. The selectivity of the benzamil-sensitive cation currents was observed in whole-cell patch clamp recordings under Cl^--free conditions where cations were the only permeant species. The currents were carried by Na⁺but not K⁺, and the permeability of Li⁺was greater than that of Na⁺in Reissner's membrane. Complete replacement of bath Na⁺with the inpermeable cation NMDG⁺led to the same inward current as with benzamil in a Na⁺bath.</p> <p>Conclusions</p> <p>These results are consistent with the amiloride/benzamil-sensitive absorptive flux of Reissner's membrane mediated by a highly Na⁺-selective channel that has several key characteristics in common with αβγ-ENaC. The amiloride-sensitive pathway therefore absorbs only Na⁺in this epithelium and does not provide a parasensory K⁺efflux route from scala media.</p

Slc26a7 chloride channel activity and localization in mouse Reissner’s membrane epithelium

Several members of the SLC26 gene family have highly-restricted expression patterns in the auditory and vestibular periphery and mutations in mice of at least two of these (SLC26A4 and SLC26A5) lead to deficits in hearing and/or balance. A previous report pointed to SLC26A7 as a candidate gene important for cochlear function. In the present study, inner ears were assayed by immunostaining for Slc26a7 in neonatal and adult mice. Slc26a7 was detected in the basolateral membrane of Reissner’s membrane epithelial cells but not neighboring cells, with an onset of expression at P5; gene knockout resulted in the absence of protein expression in Reissner’s membrane. Whole-cell patch clamp recordings revealed anion currents and conductances that were elevated for NO[subscript 3]ˉ over Clˉ and inhibited by Iˉ and NPPB. Elevated NO[subscript 3]ˉ currents were absent in Slc26a7 knockout mice. There were, however, no major changes to hearing (auditory brainstem response) of knockout mice during early adult life under constitutive and noise exposure conditions. The lack of Slc26a7 protein expression found in the wild-type vestibular labyrinth was consistent with the observation of normal balance. We conclude that SLC26A7 participates in Clˉ transport in Reissner’s membrane epithelial cells, but that either other anion pathways, such as ClC-2, possibly substitute satisfactorily under the conditions tested or that Clˉ conductance in these cells is not critical to cochlear function. The involvement of SLC26A7 in cellular pH regulation in other epithelial cells leaves open the possibility that SLC26A7 is needed in Reissner’s membrane cells during local perturbations of pH

Vesicular glutamatergic transmission in noise-induced loss and repair of cochlear ribbon synapses

Noise-induced excitotoxicity is thought to depend on glutamate. However, the excitotoxic mechanisms are unknown, and the necessity of glutamate for synapse loss or regeneration is unclear. Despite absence of glutamatergic transmission from cochlear inner hair cells in mice lacking the vesicular glutamate transporter-3

Onset of expression of Slc26a7.

<p>A–D) immunolocalization of <i>Slc26a7</i> (red) in sections of Reissner’s membrane at the ages P1, P4, P5 and P16. The first expression was detected at P5. E–G) surface preparation of Reissner’s membrane of a P8 mouse stained for Slc26a7 and F-actin. The actin ring is known to associate with the lateral junctions.</p

Nitrate currents in Reissner’s membrane epithelial cells from wild-type mice.

<p>A) Whole-cell patch clamp currents from Reissner’s membrane epithelial cells from <i>Slc26a7^+/+</i> mice were recorded with Cl^–-rich pipette solution and either Cl^–-rich or NO₃^–-rich bath solution. B) The command voltage was held at 0 mV and stepped for 300 ms to +80 mV through −80 mV in 20 mV increments. Representative recordings of step responses before <i>(left)</i> and at the end <i>(right)</i> of the NO₃⁻ bath perfusion are shown. C) Summary of the mean currents at the end of each step are plotted with SEM error bars. Light green panel shows values used for statistical tests of differences in currents and conductances. Green triangles point to the reversal voltages. D) A representative continuous recording during perfusion of Cl^–-rich and NO₃^–-rich bath solutions at the times marked.</p

Nitrate currents exceed chloride currents in wild-type but not knockout mice.

<p>Currents in NO₃^–-rich bath minus currents in Cl^–-rich bath are plotted for wild-type and for knockout mice. Excess NO₃⁻ currents (at +80 mV) are significantly greater in wild-type animals, consistent with the contribution of anion currents mediated by <i>Slc26a7</i> channels.</p