Electrical properties and functional expression of ionic channels in cochlear inner hair cells of mice lacking the alpha10 nicotinic cholinergic receptor subunit.
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ABSTRACT:
SUBMITTER: Gomez-Casati ME
PROVIDER: S-EPMC2674205 | biostudies-other | 2009 Jun
REPOSITORIES: biostudies-other
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Project description:BackgroundOuter hair cells are the specialized sensory cells that empower the mammalian hearing organ, the cochlea, with its remarkable sensitivity and frequency selectivity. Sound-evoked receptor potentials in outer hair cells are shaped by both voltage-gated K(+) channels that control the membrane potential and also ligand-gated K(+) channels involved in the cholinergic efferent modulation of the membrane potential. The objectives of this study were to investigate the tonotopic contribution of BK channels to voltage- and ligand-gated currents in mature outer hair cells from the rat cochlea.Methodology/principalFindings In this work we used patch clamp electrophysiology and immunofluorescence in tonotopically defined segments of the rat cochlea to determine the contribution of BK channels to voltage- and ligand-gated currents in outer hair cells. Although voltage and ligand-gated currents have been investigated previously in hair cells from the rat cochlea, little is known about their tonotopic distribution or potential contribution to efferent inhibition. We found that apical (low frequency) outer hair cells had no BK channel immunoreactivity and little or no BK current. In marked contrast, basal (high frequency) outer hair cells had abundant BK channel immunoreactivity and BK currents contributed significantly to both voltage-gated and ACh-evoked K(+) currents.Conclusions/significanceOur findings suggest that basal (high frequency) outer hair cells may employ an alternative mechanism of efferent inhibition mediated by BK channels instead of SK2 channels. Thus, efferent synapses may use different mechanisms of action both developmentally and tonotopically to support high frequency audition. High frequency audition has required various functional specializations of the mammalian cochlea, and as shown in our work, may include the utilization of BK channels at efferent synapses. This mechanism of efferent inhibition may be related to the unique acetylcholine receptors that have evolved in mammalian hair cells compared to those of other vertebrates.
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Project description:We report the cloning and characterization of rat alpha10, a previously unidentified member of the nicotinic acetylcholine receptor (nAChR) subunit gene family. The protein encoded by the alpha10 nAChR subunit gene is most similar to the rat alpha9 nAChR, and both alpha9 and alpha10 subunit genes are transcribed in adult rat mechanosensory hair cells. Injection of Xenopus laevis oocytes with alpha10 cRNA alone or in pairwise combinations with either alpha2-alpha6 or beta2-beta4 subunit cRNAs yielded no detectable ACh-gated currents. However, coinjection of alpha9 and alpha10 cRNAs resulted in the appearance of an unusual nAChR subtype. Compared with homomeric alpha9 channels, the alpha9alpha10 nAChR subtype displays faster and more extensive agonist-mediated desensitization, a distinct current-voltage relationship, and a biphasic response to changes in extracellular Ca(2+) ions. The pharmacological profiles of homomeric alpha9 and heteromeric alpha9alpha10 nAChRs are essentially indistinguishable and closely resemble those reported for endogenous cholinergic eceptors found in vertebrate hair cells. Our data suggest that efferent modulation of hair cell function occurs, at least in part, through heteromeric nAChRs assembled from both alpha9 and alpha10 subunits.
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Project description:Mammalian cochlear inner hair cells (IHCs) are specialized to process developmental signals during immature stages and sound stimuli in adult animals. These signals are conveyed onto auditory afferent nerve fibres. Neurotransmitter release at IHC ribbon synapses is controlled by L-type Ca(V)1.3 Ca(2+) channels, the biophysics of which are still unknown in native mammalian cells. We have investigated the localization and elementary properties of Ca(2+) channels in immature mouse IHCs under near-physiological recording conditions. Ca(V)1.3 Ca(2+) channels at the cell pre-synaptic site co-localize with about half of the total number of ribbons present in immature IHCs. These channels activated at about 70 mV, showed a relatively short first latency and weak inactivation, which would allow IHCs to generate and accurately encode spontaneous Ca(2+) action potential activity characteristic of these immature cells. The Ca(V)1.3 Ca(2+) channels showed a very low open probability (about 0.15 at 20 mV: near the peak of an action potential). Comparison of elementary and macroscopic Ca(2+) currents indicated that very few Ca(2+) channels are associated with each docked vesicle at IHC ribbon synapses. Finally, we found that the open probability of Ca(2+) channels, but not their opening time, was voltage dependent. This finding provides a possible correlation between presynaptic Ca(2+) channel properties and the characteristic frequency/amplitude of EPSCs in auditory afferent fibres.
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Project description:The transduction of sound in the auditory periphery, the cochlea, is inhibited by efferent cholinergic neurons projecting from the brainstem and synapsing directly on mechanosensory hair cells. One fundamental question in auditory neuroscience is what role(s) this feedback plays in our ability to hear. In the present study, we have engineered a genetically modified mouse model in which the magnitude and duration of efferent cholinergic effects are increased, and we assess the consequences of this manipulation on cochlear function. We generated the Chrna9L9'T line of knockin mice with a threonine for leucine change (L9'T) at position 9' of the second transmembrane domain of the alpha9 nicotinic cholinergic subunit, rendering alpha9-containing receptors that were hypersensitive to acetylcholine and had slower desensitization kinetics. The Chrna9L9'T allele produced a 3-fold prolongation of efferent synaptic currents in vitro. In vivo, Chrna9L9'T mice had baseline elevation of cochlear thresholds and efferent-mediated inhibition of cochlear responses was dramatically enhanced and lengthened: both effects were reversed by strychnine blockade of the alpha9alpha10 hair cell nicotinic receptor. Importantly, relative to their wild-type littermates, Chrna9(L9'T/L9'T) mice showed less permanent hearing loss following exposure to intense noise. Thus, a point mutation designed to alter alpha9alpha10 receptor gating has provided an animal model in which not only is efferent inhibition more powerful, but also one in which sound-induced hearing loss can be restrained, indicating the ability of efferent feedback to ameliorate sound trauma.
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Project description:Noise-induced hearing loss has gained relevance as one of the most common forms of hearing impairment. The anatomical correlates of hearing loss, principally cell damage and/or death, are relatively well-understood histologically. However, much less is known about the physiological aspects of damaged, surviving cells. Here we addressed the functional consequences of noise exposure on the capacity of inner hair cells (IHCs) to release synaptic vesicles at synapses with spiral ganglion neurons (SGNs). Mice of either sex at postnatal day (P) 15-16 were exposed to 1-12 kHz noise at 120 dB sound pressure level (SPL), for 1 h. Exocytosis was measured by tracking changes in membrane capacitance (ΔCm) from IHCs of the apical cochlea. Upon IHC depolarization to different membrane potentials, ΔC m showed the typical bell-shaped curve that mirrors the voltage dependence of Ca2+ influx, in both exposed and unexposed cells. Surprisingly, from IHCs at 1-day after exposure (d.a.e.), we found potentiation of exocytosis at the peak of the bell-shaped curve. The increase in exocytosis was not accompanied by changes in whole-cell Ca2+ influx, suggesting a modification in coupling between Ca2+ channels and synaptic vesicles. Consistent with this notion, noise exposure also changed the Ca2+-dependence of exocytosis from linear to supralinear. Noise exposure did not cause loss of IHCs, but did result in a small reduction in the number of IHC-SGN synapses at 1-d.a.e. which recovered by 14-d.a.e. In contrast, a strong reduction in auditory brainstem response wave-I amplitude (representing synchronous firing of SGNs) and distortion product otoacoustic emissions (reflecting outer hair cell function) indicated a profound hearing loss at 1- and 14-d.a.e. To determine the role of glutamate release in the noise-induced potentiation of exocytosis, we evaluated vesicular glutamate transporter-3 (Vglut3) knock-out (KO) mice. Unlike WT, IHCs from Vglut3 KO mice showed a noise-induced reduction in ΔC m and Ca2+ influx with no change in the Ca2+-dependence of exocytosis. Together, these results indicate that traumatic noise exposure triggers changes of IHC synaptic function including a Vglut3-dependent potentiation of exocytosis.
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Project description:The ?9?10 nicotinic acetylcholine receptor (nAChR) plays a fundamental role in inner ear physiology. It mediates synaptic transmission between efferent olivocochlear fibers that descend from the brainstem and hair cells of the auditory sensory epithelium. The ?9 and ?10 subunits have undergone a distinct evolutionary history within the family of nAChRs. Predominantly in mammalian vertebrates, the ?9?10 receptor has accumulated changes at the protein level that may ultimately relate to the evolutionary history of the mammalian hearing organ. In the present work, we investigated the responses of ?9?10 nAChRs to choline, the metabolite of acetylcholine degradation at the synaptic cleft. Whereas choline is a full agonist of chicken ?9?10 receptors it is a partial agonist of the rat receptor. Making use of the expression of ?9?10 heterologous receptors, encompassing wild-type, heteromeric, homomeric, mutant, chimeric, and hybrid receptors, and in silico molecular docking, we establish that the mammalian (rat) ?10 nAChR subunit underscores the reduced efficacy of choline. Moreover, we show that whereas the complementary face of the ?10 subunit does not play an important role in the activation of the receptor by ACh, it is strictly required for choline responses. Thus, we propose that the evolutionary changes acquired in the mammalian ?9?10 nAChR resulted in the loss of choline acting as a full agonist at the efferent synapse, without affecting the triggering of ACh responses. This may have accompanied the fine-tuning of hair cell post-synaptic responses to the high-frequency activity of efferent medial olivocochlear fibers that modulate the cochlear amplifier.
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Project description:The transcriptome is the complete set of all RNA transcripts produced by the genome in a cell and reflects the genes that are being actively expressed. Transcriptome analysis is essential for understanding the genetic mechanism controlling the phenotype of a cell. Using DNA microarray technique we examined transcriptomes of 2,000 individually collected inner (IHCs) and outer hair cells (OHCs), two types of auditory sensory cells critical for hearing. Among approximately 16,645 and 17,711 transcripts considered to be expressed, 1,296 and 256 genes showed significant differential expression in IHCs and OHCs, respectively. The top ten differentially expressed genes include Slc17a8, Dnajc5b, Slc1a3, Atp2a3, Osbpl6, Slc7a14, Bcl2, Bin1, Prkd1, Map4k4 in IHCs, and Slc26a5, C1ql1, Strc, Dnm3, Plbd1, Lbh, Olfm1, Plce1, Tectb, Ankrd22 in OHCs. Many unknown sequences and non-coding RNAs were also expressed in hair cells. The differentially expressed genes underlie the genetic mechanism for unique functions of IHCs and OHCs. The total RNA was extracted from the collected pools of single outer hair cell (OHC) and inner hair cell (IHC). Their whole-genome transcriptome expression was detected using GeneChip microarray analysis. The analysis and comparison between OHC and IHC allow us to determine what genes are expressed and what genes are uniquely or differential expressed in each population.
2014-08-12 | E-GEOD-56866 | biostudies-arrayexpress
Project description:Spiral ganglion neurons (SGNs) are primary sensory afferent neurons that relay acoustic information from the cochlear inner hair cells (IHCs) to the brainstem. The response properties of different SGNs diverge to represent a wide range of sound intensities in an action-potential code. This biophysical heterogeneity is established during pre-hearing stages of development, a time when IHCs fire spontaneous Ca2+ action potentials that drive glutamate release from their ribbon synapses onto the SGN terminals. The role of spontaneous IHC activity in the refinement of SGN characteristics is still largely unknown. Using pre-hearing otoferlin knockout mice (Otof-/-), in which Ca2+-dependent exocytosis in IHCs is abolished, we found that developing SGNs fail to upregulate low-voltage-activated K+-channels and hyperpolarisation-activated cyclic-nucleotide-gated channels. This delayed maturation resulted in hyperexcitable SGNs with immature firing characteristics. We have also shown that SGNs that synapse with the pillar side of the IHCs selectively express a resurgent K+ current, highlighting a novel biophysical marker for these neurons. RNA-sequencing showed that several K+ channels are downregulated in Otof-/- mice, further supporting the electrophysiological recordings. Our data demonstrate that spontaneous Ca2+-dependent activity in pre-hearing IHCs regulates some of the key biophysical and molecular features of the developing SGNs. KEY POINTS: Ca2+-dependent exocytosis in inner hair cells (IHCs) is otoferlin-dependent as early as postnatal day 1. A lack of otoferlin in IHCs affects potassium channel expression in SGNs. The absence of otoferlin is associated with SGN hyperexcitability. We propose that type I spiral ganglion neuron functional maturation depends on IHC exocytosis.
2024-09-27 | GSE242669 | GEO
Project description:Inner hair cells (IHCs) and outer hair cells (OHCs) are the two types of sensory receptor cells that are critical for hearing in the mammalian cochlea. IHCs and OHCs have different morphology and function. The genetic mechanisms that define their morphological and functional specializations are essentially unknown. The transcriptome reflects the genes that are being actively expressed in a cell and holds the key to understanding the molecular mechanisms of the biological properties of the cell. Using DNA microarray, we examined the transcriptome of 2000 individually collected IHCs and OHCs from adult mouse cochleae. We show that 16,647 and 17,711 transcripts are expressed in IHCs and OHCs, respectively. Of those genes, ?73% are known genes, 22% are uncharacterized sequences, and 5.0% are noncoding RNAs in both populations. A total of 16,117 transcripts are expressed in both populations. Uniquely and differentially expressed genes account for <15% of all genes in either cell type. The top 10 differentially expressed genes include Slc17a8, Dnajc5b, Slc1a3, Atp2a3, Osbpl6, Slc7a14, Bcl2, Bin1, Prkd1, and Map4k4 in IHCs and Slc26a5, C1ql1, Strc, Dnm3, Plbd1, Lbh, Olfm1, Plce1, Tectb, and Ankrd22 in OHCs. We analyzed commonly and differentially expressed genes with the focus on genes related to hair cell specializations in the apical, basolateral, and synaptic membranes. Eighty-three percent of the known deafness-related genes are expressed in hair cells. We also analyzed genes involved in cell-cycle regulation. Our dataset holds an extraordinary trove of information about the molecular mechanisms underlying hair cell morphology, function, pathology, and cell-cycle control.
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