María Eugenia Gómez-Casati

Developmental Regulation of Nicotinic Synapses on Cochlear Inner Hair Cells

In the mature cochlea, inner hair cells (IHCs) transduce acoustic signals into receptor potentials, communicating to the brain by synaptic contacts with afferent fibers. Before the onset of hearing, a transient efferent innervation is found on IHCs, mediated by a nicotinic cholinergic receptor that may contain both α9 and α10 subunits. Calcium influx through that receptor activates calcium-dependent (SK2-containing) potassium channels. This inhibitory synapse is thought to disappear after the onset of hearing [after postnatal day 12 (P12)]. We documented this developmental transition using whole-cell recordings from IHCs in apical turns of the rat organ of Corti. Acetylcholine elicited ionic currents in 88-100% of IHCs between P3 and P14, but in only 1 of 11 IHCs at P16-P22. Potassium depolarization of efferent terminals caused IPSCs in 67% of IHCs at P3, in 100% at P7-P9, in 93% at P10-P12, but in only 40% at P13-P14 and in none of the IHCs tested between P16 and P22. Earlier work had shown by in situ hybridization that α9 mRNA is expressed in adult IHCs but thatα10 mRNA disappears after the onset of hearing. In the present study, antibodies toα10 and to the associated calcium-dependent (SK2) potassium channel showed a similar developmental loss. The correlated expression of these gene products with functional innervation suggests that Alpha10 and SK2, but not Alpha9, are regulated by synaptic activity. Furthermore, this developmental knock-out of α10, but not α9, supports the hypothesis that functional nicotinic acetylcholine receptors in hair cells are heteromers containing both these subunits.

Biophysical and pharmacological characterization of nicotinic cholinergic receptors in rat cochlear inner hair cells.

Before the onset of hearing, a transient efferent innervation is found on inner hair cells (IHCs). This synapse is inhibitory and mediated by a nicotinic cholinergic receptor (nAChR) probably formed by the α9 and α10 subunits. We analysed the pharmacological and biophysical characteristics of the native nAChR using whole‐cell recordings from IHCs in acutely excised apical turns of the rat organ of Corti. Nicotine did not activate but rather blocked the acetylcholine (ACh)‐evoked currents with an IC50 of 1 ± 0.1 μm. Antagonists of non‐cholinergic receptors such as strychnine, bicuculline and ICS‐205930 blocked ACh‐evoked responses with an IC50 of 8.6 ± 0.8 nm, 59 ± 4 nm and 0.30 ± 0.02 μm, respectively. The IHC nAChR was both permeable to (PCa/PNa= 8 ± 0.9) and modulated by external Ca2+. ACh‐evoked currents were potentiated by Ca2+ up to 500 μm but were reduced by higher concentrations of this cation. Ba2+ mimicked the effects of Ca2+ whereas Mg2+ only blocked these currents. In addition, elevation of extracellular Ca2+ reduced the amplitude of spontaneous synaptic currents without affecting their time course. The receptor had an EC50 for ACh of 60.7 ± 2.8 μm in 0.5 mm Ca2+. In the absence of Ca2+, the EC50 for ACh increased, suggesting that potentiation by Ca2+ involves changes in the apparent affinity for the agonist. These pharmacological and biophysical characteristics of the IHC nAChR closely resemble those of the recombinant α9α10 nAChR, reinforcing the hypothesis that the functional nAChR at the olivocochlear efferent–IHC synapse is composed of both the α9 and α10 subunits.

Stoichiometry of the α9α10 nicotinic cholinergic receptor

The α9 and α10 nicotinic cholinergic subunits assemble to form the receptor that mediates synaptic transmission between efferent olivocochlear fibers and hair cells of the cochlea. They are the latest vertebrate nicotinic cholinergic receptor (nAChR) subunits that have been cloned, and their identification has established a distant early divergent branch within the nAChR gene family. The α10 subunit serves as a “structural” component leading to heteromeric α9α10 nAChRs with distinct properties. We now have probed the stoichiometry of recombinant α9α10 nAChRs expressed in Xenopus oocytes. We have made use of the analysis of the population of receptors assembled from a wild-type subunit and its partner α9 or α10 subunit bearing a reporter mutation of a valine to threonine at position 13′ of the second transmembrane domain (TM2). Because the mutation increased the sensitivity of the receptor for acetylcholine (ACh) but mutations at different subunits were not equivalent, the number of α9 and α10 subunits could be inferred from the number of components in compound concentration-response curves to ACh. The results were confirmed via the analysis of the effects of a mutation to threonine at position 17′ of TM2. Because at this position the mutations at different subunits were equivalent, the stoichiometry was inferred directly from the shifts in the ACh EC50 values. We conclude that the recombinant α9α10 receptor is a pentamer with a (α9)2(α10)3 stoichiometry.

A Novel α-Conotoxin, PeIA, Cloned from Conus pergrandis, Discriminates between Rat α9α10 and α7 Nicotinic Cholinergic Receptors

The α9 and α10 nicotinic cholinergic subunits assemble to form the receptor believed to mediate synaptic transmission between efferent olivocochlear fibers and hair cells of the cochlea, one of the few examples of postsynaptic function for a non-muscle nicotinic acetylcholine receptor (nAChR). However, it has been suggested that the expression profile of α9 and α10 overlaps with that of α7 in the cochlea and in sites such as dorsal root ganglion neurons, peripheral blood lymphocytes, developing thymocytes, and skin. We now report the cloning, total synthesis, and characterization of a novel toxin α-conotoxin PeIA that discriminates between α9α10 and α7 nAChRs. This is the first toxin to be identified from Conus pergrandis, a species found in deep waters of the Western Pacific. α-Conotoxin PeIA displayed a 260-fold higher selectivity for α-bungarotoxin-sensitive α9α10 nAChRs compared with α-bungarotoxin-sensitive α7 receptors. The IC50 of the toxin was 6.9 ± 0.5 nm and 4.4 ± 0.5 nm for recombinant α9α10 and wild-type hair cell nAChRs, respectively. α-Conotoxin PeIA bears high resemblance to α-conotoxins MII and GIC isolated from Conus magus and Conus geographus, respectively. However, neither α-conotoxin MII nor α-conotoxin GIC at concentrations of 10 μm blocked acetylcholine responses elicited in Xenopus oocytes injected with the α9 and α10 subunits. Among neuronal non-α-bungarotoxin-sensitive receptors, α-conotoxin PeIA was also active at α3β2 receptors and chimeric α6/α3β2β3 receptors. α-Conotoxin PeIA represents a novel probe to differentiate responses mediated either through α9α10 or α7 nAChRs in those tissues where both receptors are expressed.

A novel alpha-conotoxin, PeIA, cloned from Conus pergrandis, discriminates between rat alpha9alpha10 and alpha7 nicotinic cholinergic receptors.

The α9 and α10 nicotinic cholinergic subunits assemble to form the receptor believed to mediate synaptic transmission between efferent olivocochlear fibers and hair cells of the cochlea, one of the few examples of postsynaptic function for a non-muscle nicotinic acetylcholine receptor (nAChR). However, it has been suggested that the expression profile of α9 and α10 overlaps with that of α7 in the cochlea and in sites such as dorsal root ganglion neurons, peripheral blood lymphocytes, developing thymocytes, and skin. We now report the cloning, total synthesis, and characterization of a novel toxin α-conotoxin PeIA that discriminates between α9α10 and α7 nAChRs. This is the first toxin to be identified from Conus pergrandis, a species found in deep waters of the Western Pacific. α-Conotoxin PeIA displayed a 260-fold higher selectivity for α-bungarotoxin-sensitive α9α10 nAChRs compared with α-bungarotoxin-sensitive α7 receptors. The IC50 of the toxin was 6.9 ± 0.5 nm and 4.4 ± 0.5 nm for recombinant α9α10 and wild-type hair cell nAChRs, respectively. α-Conotoxin PeIA bears high resemblance to α-conotoxins MII and GIC isolated from Conus magus and Conus geographus, respectively. However, neither α-conotoxin MII nor α-conotoxin GIC at concentrations of 10 μm blocked acetylcholine responses elicited in Xenopus oocytes injected with the α9 and α10 subunits. Among neuronal non-α-bungarotoxin-sensitive receptors, α-conotoxin PeIA was also active at α3β2 receptors and chimeric α6/α3β2β3 receptors. α-Conotoxin PeIA represents a novel probe to differentiate responses mediated either through α9α10 or α7 nAChRs in those tissues where both receptors are expressed.

Stoichiometry of the alpha9alpha10 nicotinic cholinergic receptor.

The α9 and α10 nicotinic cholinergic subunits assemble to form the receptor that mediates synaptic transmission between efferent olivocochlear fibers and hair cells of the cochlea. They are the latest vertebrate nicotinic cholinergic receptor (nAChR) subunits that have been cloned, and their identification has established a distant early divergent branch within the nAChR gene family. The α10 subunit serves as a “structural” component leading to heteromeric α9α10 nAChRs with distinct properties. We now have probed the stoichiometry of recombinant α9α10 nAChRs expressed in Xenopus oocytes. We have made use of the analysis of the population of receptors assembled from a wild-type subunit and its partner α9 or α10 subunit bearing a reporter mutation of a valine to threonine at position 13′ of the second transmembrane domain (TM2). Because the mutation increased the sensitivity of the receptor for acetylcholine (ACh) but mutations at different subunits were not equivalent, the number of α9 and α10 subunits could be inferred from the number of components in compound concentration-response curves to ACh. The results were confirmed via the analysis of the effects of a mutation to threonine at position 17′ of TM2. Because at this position the mutations at different subunits were equivalent, the stoichiometry was inferred directly from the shifts in the ACh EC50 values. We conclude that the recombinant α9α10 receptor is a pentamer with a (α9)2(α10)3 stoichiometry.

Cochlear hair cells: the sound-sensing machines

The sensory epithelium of the mammalian inner ear contains two types of mechanosensory cells: inner (IHC) and outer hair cells (OHC). They both transduce mechanical force generated by sound waves into electrical signals. In their apical end, these cells possess a set of stereocilia representing the mechanosensing organelles. IHC are responsible for detecting sounds and transmitting the acoustic information to the brain by converting graded depolarization into trains of action potentials in auditory nerve fibers. OHC are responsible for the active mechanical amplification process that leads to the fine tuning and high sensitivity of the mammalian inner ear. This active amplification is the consequence of the ability of OHC to alter their cell length in response to changes in membrane potential, and is controlled by an efferent inhibitory innervation. Medial olivocochlear efferent fibers, originating in the brainstem, synapse directly at the base of OHC and release acetylcholine. A very special type of nicotinic receptor, assembled by α9α10 subunits, participates in this synapse. Here we review recent knowledge and the role of both afferent and efferent synapse in the inner ear.

Activation of presynaptic GABA(B(1a,2)) receptors inhibits synaptic transmission at mammalian inhibitory cholinergic olivocochlear-hair cell synapses.

The synapse between olivocochlear (OC) neurons and cochlear mechanosensory hair cells is cholinergic, fast, and inhibitory. The inhibitory sign of this cholinergic synapse is accounted for by the activation of Ca2+-permeable postsynaptic α9α10 nicotinic receptors coupled to the opening of hyperpolarizing Ca2+-activated small-conductance type 2 (SK2)K+ channels. Acetylcholine (ACh) release at this synapse is supported by both P/Q- and N-type voltage-gated calcium channels (VGCCs). Although the OC synapse is cholinergic, an abundant OC GABA innervation is present along the mammalian cochlea. The role of this neurotransmitter at the OC efferent innervation, however, is for the most part unknown. We show that GABA fails to evoke fast postsynaptic inhibitory currents in apical developing inner and outer hair cells. However, electrical stimulation of OC efferent fibers activates presynaptic GABAB(1a,2) receptors [GABAB(1a,2)Rs] that downregulate the amount of ACh released at the OC–hair cell synapse, by inhibiting P/Q-type VGCCs. We confirmed the expression of GABABRs at OC terminals contacting the hair cells by coimmunostaining for GFP and synaptophysin in transgenic mice expressing GABAB1–GFP fusion proteins. Moreover, coimmunostaining with antibodies against the GABA synthetic enzyme glutamic acid decarboxylase and synaptophysin support the idea that GABA is directly synthesized at OC terminals contacting the hair cells during development. Thus, we demonstrate for the first time a physiological role for GABA in cochlear synaptic function. In addition, our data suggest that the GABAB1a isoform selectively inhibits release at efferent cholinergic synapses.

Electrical Properties and Functional Expression of Ionic Channels in Cochlear Inner Hair Cells of Mice Lacking the α10 Nicotinic Cholinergic Receptor Subunit

Cochlear inner hair cells (IHCs) release neurotransmitter onto afferent auditory nerve fibers in response to sound stimulation. During early development, synaptic transmission is triggered by spontaneous Ca2+ spikes which are modulated by an efferent cholinergic innervation to IHCs. This synapse is inhibitory and mediated by the α9α10 nicotinic cholinergic receptor (nAChR). After the onset of hearing, large-conductance Ca2+-activated K+ channels are acquired and both the spiking activity and the efferent innervation disappear from IHCs. In this work, we studied the developmental changes in the membrane properties of cochlear IHCs from α10 nAChR gene (Chrna10) “knockout” mice. Electrophysiological properties of IHCs were studied by whole-cell recordings in acutely excised apical turns of the organ of Corti from developing mice. Neither the spiking activity nor the developmental functional expression of voltage-gated and/or calcium-sensitive K+ channels is altered in the absence of the α10 nAChR subunit. The present results show that the α10 nAChR subunit is not essential for the correct establishment of the intrinsic electrical properties of IHCs during development.

Positive Selection and Adaptation of Novel Inner Ear Genes in the Mammalian Lineage

The mammalian inner ear possesses functional and morphological innovations that contribute to its unique hearing capacities. The genetic bases underlying the evolution of this mammalian landmark are poorly understood. We propose that the emergence of morphological and functional innovations in the mammalian inner ear could have been driven by adaptive molecular evolution. In this work, we analyzed the complete inner ear transcriptome in order to identify genes that show signatures of adaptive evolution in this lineage. We analyzed approximately 1,300 inner ear expressed genes and found that 13 % show signatures of positive selection in the mammalian lineage. Several of these genes are known to play an important function in the inner ear. In addition, we identified that a significant proportion of genes showing signatures of adaptive evolution in mammals have not been previously reported to participate in inner ear development and/or physiology. We focused our analysis in two of these novel genes: STRIP2 and ABLIM2 by generating null mutant mice and analyzed their auditory function. We found that mice lacking Strip2 displayed a decrease in neural response amplitudes. In addition, we observed a reduction in the number of afferent synapses, suggesting a potential cochlear neuropathy. Thus, this study shows the usefulness of pursuing a high-throughput evolutionary approach followed by functional studies to track down novel genes that are important for inner ear function. Moreover, this approach sheds light on the genetic basis underlying the evolution of the mammalian inner ear.