Constitutive Expression of the α10 Nicotinic Acetylcholine Receptor Subunit Fails to Maintain Cholinergic Responses in Inner Hair Cells After the Onset of Hearing
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Efferent inhibition of cochlear hair cells is mediated by α9α10 nicotinic cholinergic receptors (nAChRs) functionally coupled to calcium-activated, small conductance (SK2) potassium channels. Before the onset of hearing, efferent fibers transiently make functional cholinergic synapses with inner hair cells (IHCs). The retraction of these fibers after the onset of hearing correlates with the cessation of transcription of the Chrna10 (but not the Chrna9) gene in IHCs. To further analyze this developmental change, we generated a transgenic mice whose IHCs constitutively express α10 into adulthood by expressing the α10 cDNA under the control of the Pou4f3 gene promoter. In situ hybridization showed that the α10 mRNA is expressed in IHCs of 8-week-old transgenic mice, but not in wild-type mice. Moreover, this mRNA is translated into a functional protein, since IHCs from P8-P10 α10 transgenic mice backcrossed to a Chrna10 −/− background (whose IHCs have no cholinergic function) displayed normal synaptic and acetylcholine (ACh)-evoked currents in patch-clamp recordings. Thus, the α10 transgene restored nAChR function. However, in the α10 transgenic mice, no synaptic or ACh-evoked currents were observed in P16-18 IHCs, indicating developmental down-regulation of functional nAChRs after the onset of hearing, as normally observed in wild-type mice. The lack of functional ACh currents correlated with the lack of SK2 currents. These results indicate that multiple features of the efferent postsynaptic complex to IHCs, in addition to the nAChR subunits, are down-regulated in synchrony after the onset of hearing, leading to lack of responses to ACh.
Keywordsnicotinic cholinergic receptors efferent medial olivocochlear SK2 channel acetylcholine transgenic mice
Efferent inhibition of cochlear hair cells is mediated by the release of acetylcholine (ACh) from neurons originating in the superior olivary complex of the brainstem. In the mature cochlea, the medial olivocochlear (OC) efferent pathway projects to outer hair cells (OHCs) where large synaptic contacts are formed (Guinan 1996). Activation of this pathway reduces cochlear sensitivity through the action of ACh on nicotinic receptors (nAChRs) at the base of OHCs. Significant progress has been made in defining the cellular mechanisms of hair cell inhibition: α9 and α10 nAChR subunits arrange into a pentameric assembly with a likely (α9)2(α10)3 stoichiometry (Elgoyhen et al. 1994, 2001; Lustig et al. 2001; Plazas et al. 2005; Sgard et al. 2002) and activation of the α9α10 nAChR leads to an increase in intracellular Ca2+ and the subsequent opening of small conductance Ca2+-activated K+ (SK2) channels, thus leading to hyperpolarization of hair cells (Dulon et al. 1998; Fuchs and Murrow 1992; Housley and Ashmore 1991; Oliver et al. 2000).
Although adult inner hair cells (IHCs) receive very few (if any) direct axosomatic contacts from efferent fibers, before the onset of hearing [until about postnatal (P) day 12 in rats and mice], a transient efferent innervation is found on IHCs (Simmons 2002; Katz et al. 2004). These transient efferent axosomatic synapses with IHCs most likely play a role in the modulation of the Ca2+ spiking activity, a characteristic of immature IHCs, which may drive rhythmic or bursting activity of neurons at higher levels of the auditory pathway (Glowatzki and Fuchs 2000). Previous studies have suggested that this transient efferent innervation may play a role in the ultimate functional maturation of cochlear hair cells (Simmons 2002). Most impressively, surgical lesion of the efferent nerve supply causes kittens to fail to develop normal hearing (Walsh et al. 1998).
With maturation of the cochlea, a number of changes in the expression of voltage-gated channels tend to reduce IHC spiking (Kros et al. 1998; Marcotti et al. 2003a, b). These changes signal the transformation from a developing epithelium with active formation of synaptic contacts to a sensing epithelium where receptor potentials represent the mechanical input in a graded fashion. These changes are accompanied by the loss of direct efferent innervation to IHCs, and this is directly correlated to the cessation of transcription in IHCs of the gene coding for the α10 (Chrna10) but not the α9 (Chrna9) nAChR subunit (Elgoyhen et al. 1994, 2001; Morley and Simmons 2002; Simmons 2002). In fact, Chrna9 continues to be transcribed into adult stages (Elgoyhen et al. 1994). To further analyze this critical developmental change near the onset of hearing, we generated a transgenic mice whose IHCs constitutively express α10 into adulthood by expressing the α10 cDNA under the control of the mouse Pou4f3 gene promoter, a hair cell transcription factor (Erkman et al. 1996). We reasoned that if the lack of responses to ACh after the onset of hearing was due to the cessation in transcription of Chrna10, constitutive expression of the α10 subunit would result in functional receptors. However, ACh sensitivity was lost on schedule, as in wild-type animals. Thus, we found that expression of the α10 nAChR subunit into adult ages is not sufficient to sustain cholinergic function, and efferent innervation, of IHCs after the onset of hearing.
Materials and methods
Generation of Pou4f3-α10 transgenic mice
For transgenic genotyping, primers tgα10 β-globin (5′-CATGAGGGTCCATGGTGATAC-3′) and tgα10 Pou4f3 (5′-GCATCAGGCTCTCAGATGGCG-3′) were used, producing a 494-bp fragment (Fig. 1B). Polymerase chain reaction (PCR) was performed using 94°C for 2 min followed by 94°C for 30 s, 58°C for 1 min, and 72°C for 1.5 min for 30 cycles with 20 ng of genomic DNA obtained from tail biopsies, buffer Mix D (Epicentre, Madison, WI, USA), and Qiagen Taq polymerase (Qiagen, Valencia, CA, USA). The genotyping of the Chrna10 −/− mice was performed as previously described (Vetter et al. 2007).
Reverse transcriptase PCR
Eight-week-old mice were killed, the cochleae removed, and immediately frozen in liquid nitrogen. For each genotype, total RNA was extracted using Trizol reagent (Invitrogen, Buenos Aires, Argentina) following the manufacturer’s instructions after grinding the tissue in a TH-1 homogenizer (OMNI, Marietta, Gainsville) and centrifuging at 12,000×g at 4°C to remove bone fragments. A total of 1 μg of RNA was used to reverse transcribe using Superscript II reverse transcriptase (Invitrogen) and oligo dT (Invitrogen). One microliter of this reaction was used to amplify the transgenic α10 cDNA using one amplimer that anneals to the FLAG sequence (5′-CCATGGTCACATTCTCCACA-3′) and another to the α10 cDNA sequence (5′-CTTGTCATCGTCGTCCTTGTAGTC-3′). In transgenic mice, a 446-bp fragment was obtained.
In situ hybridization
In situ hybridization experiments used previously published protocols (Hiel et al. 1996). Briefly, the temporal bones of 8-week-old mice were fixed, decalcified, and embedded for cryosectioning. Postfixed, acetylated, and dehydrated 14-μm cryosections were hybridized with 35S-labeled riboprobe (989 bp; 1.2 × 106 cpm/slide) for 16 h at 56°C. After high-stringency washes and dehydration, tissue sections were coated with photographic emulsion NBT-2 (Eastman Kodak, Rochester, NY, USA) and developed for 2–5 weeks at 4°C. Relative expression levels for α10 mRNA in OHCs and IHCs were compared by counting grains in a box placed over selected regions of cochlear cross-sections. Background counts were repeatedly collected, averaged, and subtracted for each cross-section. Labeled sections from base, middle, and apical cochlear turns were obtained from one wild-type (five sections) and two α10 Pou4f3-α10 transgenic mice (six sections).
Electrophysiological recordings from cochlear hair cells
Mice were killed by decapitation. All experimental protocols were carried out in accordance with the AVMA Guidelines on Euthanasia (June 2007). Apical turns of the organ of Corti were excised from mice and used within 3 h. Day of birth was considered postnatal day 0, P0. Cochlear preparations were mounted under a Leica DMLFS microscope (Leica Microsystems, Wetzlar, Germany) and viewed with differential interference contrast using a 40× water immersion objective and a Hamamatsu C7500-50 camera with contrast enhancement (Hamamatsu, Hamamatsu City, Japan). Methods to record from IHCs were essentially as described previously (Glowatzki and Fuchs 2000; Oliver et al. 2000).
Briefly, IHCs were identified visually with the 40× objective and during recordings by the size of their capacitance (7 to 12 pF) and their characteristic voltage-dependent Na+ and K+ currents (Kros et al. 1998). Some cells were removed to access IHCs, but mostly, the pipette moved through the tissue under positive pressure. The extracellular solution was as follows (in mM): 155 NaCl, 5.8 KCl, 1.3 CaCl2, 0.9 MgCl2, 0.7 NaH2PO4, 5.6 d-glucose, and 10 HEPES buffer, pH 7.4. The pipette solution contained (in mM): 150 KCl, 3.5 MgCl2, 0.1 CaCl2, 5 ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-teraacetic acid (EGTA), 5 HEPES buffer, 2.5 Na2ATP, pH 7.2 (KCl-EGTA saline). Solutions containing ACh or high K+ were applied by a gravity-fed multichannel glass pipette (∼150-μM tip diameter) positioned about 300 μM from the recorded cell. All working solutions containing either ACh or elevated K+ or both were made up in a saline containing low Ca2+ (0.5 mM) and no Mg2+ so as to optimize the experimental conditions for measuring currents flowing through the α9α10 receptors (Weisstaub et al. 2002).
Recording pipettes, 1.2-mm I.D, had resistances of 5–8 MΩ. Currents in IHCs were recorded in the whole-cell patch-clamp mode with an Axopatch 200B amplifier, low-pass-filtered at 2–10 kHz, and digitized at 5–20 kHz with a Digidata 1322A board (Molecular Devices, California, USA). Recordings were made at room temperature (22–25°C). Holding potentials were not corrected for liquid junction potentials or for the voltage drop across the uncompensated series resistance.
IHCs of Pou4f3-α10 mice express α10 mRNA after the onset of hearing
It has been reported that the Pou4f3 transcription factor is expressed in hair cells from early embryonic until adult stages (Erkman et al. 1996) and that Cre expression driven by the Pou4f3 promoter starts as early as embryonic day 13.5 in a transgenic mouse (Sage et al. 2006). Thus, the use of the Pou4f3 promoter has become a useful tool for transgenic expression of genes in cochlear hair cells. The generation of a transgenic mouse in which the expression of the α10 cDNA is driven by Pou4f3 is explained in “Materials and methods”, the construct shown in Figure 1A and genotyping in Figure 1B. In order to assess Pou4f3-α10-driven expression of α10 RNA, reverse transcriptase PCR (RT-PCR) was performed from total RNA extracted from 8-week-old mice. As shown in Figure 1C, a 446-bp fragment was obtained in Pou4f3-α10 transgenic mice. Since one of the amplimers anneals to the FLAG sequence, this fragment can only derive from the RNA that has been correctly transcribed from the α10 transgenic cDNA. Amplification from genomic DNA is precluded since no band was observed in the control reaction without reverse transcriptase (lane b). Lanes c and d are control reactions, minus oligo dT and minus RNA, respectively.
Backcross of the Pou4f3-α10 to a α10 nAChR knockout background restores ACh responses and synaptic currents before the onset of hearing
In order to learn whether the transgenic α10 RNA is effectively translated into a functional protein, Pou4f3-α10 transgenic mice were backcrossed into a Chrna10 null background. It has been reported that Chrna10 −/− mice lack responses to ACh and the synaptic cholinergic currents observed in normal neonatal mice before the onset of hearing (Vetter et al. 2007). Therefore, we reasoned that responses to ACh in Pou4f3-α10 × Chrna10 −/− would only be observed if the α10 transgene was translated into a α10 protein subunit that restored normal function when co-assembled with the endogenous α9 subunit.
IHCs from Chrna10 +/+ mice also exhibit outward currents at −40 mV (281 ± 47 pA, n = 3; Fig. 3B), indicating functional coupling to SK2 channels (Katz et al. 2004; Vetter et al. 2007), whereas no response is found at −40 mV in IHCs from Chrna10 −/− mice (n = 0/8 cells tested; Fig. 3B, middle panel). As shown in Figure 3B (right panel), the backcrossing of the Pou4f3-α10 transgenic mice into the α10 null background restored the normal outward responses to 1 mM ACh (325 ± 48 pA, n = 3 cells, 3 mice) at P8-9 in all IHCs tested.
Finally, when the preparation is superfused with a buffer containing 40 mM K+ to depolarize the efferent terminals, thus increasing the frequency of ACh release (Glowatzki and Fuchs 2000; Katz et al. 2004), synaptic currents are observed in IHCs of Chrna10 +/+ mice (n = 3/3 cells tested; Fig. 3C, left panel), but not in IHCs from Chrna10 −/− mice (n = 0/8 cells tested; Fig. 3C, middle panel; Vetter et al. 2007). Even when adding 1 mM ACh in the presence of 40 mM K+, a procedure that enhances small responses to ACh (due to the change in the K+ equilibrium potential and the concomitant increase in the driving force for K+ ions at the holding voltage of −90 mV; Katz et al. 2004), no responsive IHCs are observed in Chrna10 −/− mice (Fig. 3C, middle panel). When the preparation was superfused with a buffer containing 40 mM K+ to depolarize the efferent terminals, synaptic currents were observed in all Pou4f3-α10 × Chrna10 −/− IHCs (Fig. 3C, right panel), and the addition of 1 mM ACh in the presence of 40 mM K+ produced an inward current similar to that observed in IHCs of Chrna10 +/+ mice. Figure 3D shows boxes in C at an expanded timescale.
Thus, the experiments described so far demonstrate that the Pou4f3-α10 transgene is indeed transcribed into RNA and then translated into a functional α10 protein that can assemble with the endogenous α9 subunit, leading to normal ACh responses and synaptic currents.
IHCs of Pou4f3-α10 mice fail to respond to ACh and to elicit synaptic currents after the onset of hearing
Since IHCs continue to express the α9 mRNA after the onset of hearing (Elgoyhen et al. 1994) and the Pou4f3-α10 transgenic mice shown here constitutively express α10 mRNA even after the onset of hearing, one would expect to find functional α9α10 receptors after this critical developmental period if the lack of responses of IHCs to ACh is solely due to the cessation in the expression of Chrna10.
However, as observed in Figure 4 (lower panels), the constitutive expression of the α10 subunit was not sufficient to maintain functional α9α10 receptors after the onset of hearing. Thus, at P17-20, IHCs from Pou4f3-α10 mice failed to respond to 1 mM ACh both at −40 and −90 mV (Fig. 4A, B, n = 9 cells, 4 mice), failed to exhibit synaptic currents at 40 mM K+ (Fig. 4C, D), and failed to respond to 1 mM ACh under conditions in which the driving force for K+ was increased (Fig. 4C).
IHCs of Pou4f3-α10 mice lack functional SK currents after the onset of hearing
Responses of IHCs to ACh prior to the onset of hearing is strictly dependent upon the expression of both the α9 and the α10 nAChR subunits, as demonstrated by the generation of subunit specific null mutant mice (Vetter et al. 2007). Since the expression of the α10, but not that of the α9 subunit, is developmentally regulated (Elgoyhen et al. 2001; Katz et al. 2004), the present work tested the hypothesis that the lack of cholinergic currents in IHCs after the onset of hearing might result from the cessation of the transcription of the Chrna10 gene. To that end, we generated the Pou4f3-α10 line of transgenic mice, which drove expression of the α10 nAChR subunit after the onset of hearing. Nevertheless, this was not sufficient for the formation of functional α9α10 channels, leading to either ACh responses or efferent synaptic currents after P12.
The absence of functional responses after the onset of hearing in the transgenic mice could have alternative explanations. Lack of transcription of the transgene or misexpression of the transgenic RNA can result from the generation of transgenic mice (Haruyama et al. 2009; Su et al. 2004). This is precluded in the case of the Pou4f3-α10 line, since transgenic RNA was present in the cochlea as assessed by RT-PCR and it was localized to the IHC region as demonstrated by in situ hybridization. Alternatively, the α10 cDNA plasmid used to engineer the transgene construct could have led to a non-functional α10 protein subunit, particularly since a FLAG tag was added before the stop codon of the cDNA. However, this was not the case since the Pou4f3-α10 transgene rescued the α10 null phenotype, demonstrated by the presence of normal responses to ACh and synaptic currents in the Pou4f3-α10 × Chrma10−/− backcross, thus indicating that in vivo, the transgenic α10 subunit efficiently assembles with the endogenous α9 to form functional channels. Importantly, taken together, these results indicate that lack of cholinergic responses of IHCs after the onset of hearing goes beyond the transcription of the Chrna10 gene.
The possibility exists that after the onset of hearing, genes other than Chrna10 also cease transcription and/or translation and that these genes lead to proteins which form part of a macromolecular synaptic complex that includes, but extends beyond the nAChR and which is necessary for assembly, trafficking and/or anchorage of the nAChR to the plasma membrane at the base of the IHC. For example, RIC-3, a transmembrane protein which acts as a molecular chaperone, is required for efficient receptor folding, assembly, and functional expression of the α7 nAChR (Millar 2008). Similar chaperon proteins have not been described in the case of α9α10 receptors. However, it is known that activation of the α9α10 nAChR leads to an increase in intracellular Ca2+ and the subsequent opening of small conductance Ca2+-activated K+ SK channels, thus leading to hyperpolarization of hair cells (Dulon et al. 1998; Fuchs and Murrow 1992; Housley and Ashmore 1991; Oliver et al. 2000). Moreover, SK channels and α9α10 are known to co-localize in the same functional microdomain, and through such close coupling, the gating kinetics of the SK channels determine the time course of synaptic action, outlasting the driving calcium signals (Oliver et al. 2000). In addition, through the generation of a KCNN2 (gene coding for the SK2 protein) knockout mice, it has been demonstrated recently that the KCNN2 gene is solely responsible for encoding this class of small conductance, calcium-activated potassium channel in cochlear hair cells (Johnson et al. 2007; Kong et al. 2008) and that it cannot be replaced by the later developmental arrival of rapidly activating, iberiotoxin-sensitive “BK”-type potassium channels shown in mammals (Hafidi et al. 2005; Kros et al. 1998; Langer et al. 2003) and birds (Fuchs and Sokolowski 1990). The present results demonstrate that as observed in wild-type mice, the Pou4f3-α10 transgenic also lack functional SK2 currents after the onset of hearing. This observation, together with the fact that KCNN2 knockout mice totally lack ACh responses in hair cells (Johnson et al. 2007; Kong et al. 2008), might indicate the SK2 protein as fundamentally required for the assembly, trafficking, and/or anchorage of the nAChR macromolecular synaptic complex. Alternatively, proteins known to form a macromolecular complex with SK2 channels, such as calmodulin, protein kinase CK2, and protein phosphatase 2A (Bildl et al. 2004), might also be developmentally regulated and be the linking molecules of the SK2 channel with the nAChR macromolecular complex.
Although the lack of ACh responses in the KCNN2 knockout mice points towards the SK2 protein as a key player, the fact that in these mice a concomitant OC fiber degeneration is also observed (Kong et al. 2008; Murthy et al. 2009) does not allow an unequivocal conclusion. Thus, lack of pre- and postsynaptic cross talk in the absence of innervation, rather than lack of the SK2 protein as a stabilizing component of the macromolecular synaptic complex, could also lead to the same result. The need of presynaptic neuronal input for the correct assembly of the postsynaptic apparatus has been described at the neuromuscular junction where motor nerve terminals seem to organize postsynaptic differentiation by releasing a proteoglycan called agrin. Agrin activates a receptor tyrosine kinase called muscle-specific kinase on the myotube surface, which leads to clustering of nAChRs and other postsynaptic components through association with the cytoplasmic linker protein rapsyn (Sanes and Lichtman 2001).
Finally, we cannot preclude the possibility of a developmental regulation of the translation of the α10 mRNA, which might prevent α10 protein synthesis after the onset of hearing. Emerging studies show that translational control in eukaryotic cells is critical for gene regulation during nutrient deprivation and stress, development and differentiation, nervous system function, aging, and disease (Sonenberg and Hinnebusch 2009). For example, microRNAs are major regulators of gene expression and function at the posttranscriptional level (Carthew and Sontheimer 2009). Further work is required in order to dissect the alternative possibilities proposed as the underlying mechanisms for the developmental regulation of the cholinergic responses of IHCs.
The authors want to thank the laboratory of Marcelo Rubinstein at INGEBI for generating the transgenic mouse line. This work was supported by the National Institutes of Deafness and other Communication Disorders (NIDCD) grant R01DC001508 to P.A.F. and A.B.E, an International Research Scholar Grant from the Howard Hughes Medical Institute, The National Organization for Hearing Research, a Research Grant from ANPCyT (Argentina), and a Grant from the University of Buenos Aires (Argentina) to A.B.E., NIDCD R01DC006258 to D.E.V, and a CONICET grant to EK.
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