Assessment of the expression and role of the α1-nAChR subunit in efferent cholinergic function during the development of the mammalian cochlea

Abstract

Hair cell (HC) activity in the mammalian cochlea is modulated by cholinergic efferent inputs from the brainstem. These inhibitory inputs are mediated by calcium-permeable nicotinic acetylcholine receptors (nAChRs) containing α9- and α10-subunits and by subsequent activation of calcium-dependent potassium channels. Intriguingly, mRNAs of α1- and γ-nAChRs, subunits of the "muscle-type" nAChR have also been found in developing HCs (Cai T, Jen HI, Kang H, Klisch TJ, Zoghbi HY, Groves AK. J Neurosci 35: 5870-5883, 2015; Scheffer D, Sage C, Plazas PV, Huang M, Wedemeyer C, Zhang DS, Chen ZY, Elgoyhen AB, Corey DP, Pingault V. J Neurochem 103: 2651-2664, 2007; Sinkkonen ST, Chai R, Jan TA, Hartman BH, Laske RD, Gahlen F, Sinkkonen W, Cheng AG, Oshima K, Heller S. Sci Rep 1: 26, 2011) prompting proposals that another type of nAChR is present and may be critical during early synaptic development. Mouse genetics, histochemistry, pharmacology, and whole cell recording approaches were combined to test the role of α1-nAChR subunit in HC efferent synapse formation and cholinergic function. The onset of α1-mRNA expression in mouse HCs was found to coincide with the onset of the ACh response and efferent synaptic function. However, in mouse inner hair cells (IHCs) no response to the muscle-type nAChR agonists (±)-anatoxin A, (±)-epibatidine, (-)-nicotine, or 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) was detected, arguing against the presence of an independent functional α1-containing muscle-type nAChR in IHCs. In α1-deficient mice, no obvious change of IHC efferent innervation was detected at embryonic day 18, contrary to the hyperinnervation observed at the neuromuscular junction. Additionally, ACh response and efferent synaptic activity were detectable in α1-deficient IHCs, suggesting that α1 is not necessary for assembly and membrane targeting of nAChRs or for efferent synapse formation in IHCs.

Keywords: cochlea; efferent cholinergic synapse; hair cell; synapse formation; α1-nicotinic acetylcholine receptor; α9α10-nicotinic acetylcholine receptor.

Fig. 1.

Generation of the reporter Chrna1^lacZ and knockout Chrna1^Δ4 alleles. A, top: genomic region of Chrna1. Middle: Chrna1^tm1a allele was obtained by insertion of the “Knock Out first allele” cassette flanked by frt sites, into intron 3 of Chrna1, the gene coding for the α₁-nicotinic acetylcholine receptor (α₁-nAChR) subunit. This cassette encodes β-galactosidase (β-gal; lacZ) preceded by an internal ribosome entry site (IRES) and a promoter driven neomycin resistance cassette (neo). Three loxP sites were also introduced: 5′ of the neo cassette, and 5′ and 3′ of exon 4 (EMMA Consortium). Bottom: Chrna1^lacZ allele was generated after excision of the neo cassette and exon 4 by germline Cre recombination between the 2 loxP sites further apart. SA, splice acceptor and pA, polyadenylation site. Chrna1^fl allele was generated after excision of the “Knock Out first allele” cassette by germline FLPe recombination between the 2 frt sites flanking this cassette. Chrna1^Δ4 allele was subsequently generated after excision of exon 4 by germline Cre recombination between the two loxP sites 5′ and 3′ of this exon. B: genotyping of Chrna1^lacZ/+ mice by PCR. The primers lacZF and lacZR were used to detect lacZ-containing alleles (1,170-bp amplicon). Lack of amplification with primers neoF/neoR and appearance of a 724 bp amplicon with primers T3F/T3R attest of the excision of the neo cassette and exon 4 in Chrna1^lacZ/+ mice. Note a 536-bp amplicon with primers neoF/neoR was present in Chrna1^tm1a/+, but the expected 3,417-bp amplicon with primers T3F/T3R was not detected with the PCR conditions used here. C: genotyping of embryonic day (E)18.5 wild-type (Chrna1^+/+), heterozygote (Chrna1^Δ4/+), and homozygote knockout (Chrna1^Δ4/Δ4) mouse littermates by PCR. Deletion of exon 4 leads to the appearance of a 310-bp amplicon instead of the 909-bp amplicon detected in wild-type allele with primers Efbis and L3r. D: an E18.5 Chrna1^Δ4/Δ4 embryo compared with a Chrna1^Δ4/+ control littermate. Note the hunched posture of Chrna1^Δ4/Δ4 mouse. E: confirmation of the absence of exon 4 from Chrna1 transcripts in Chrna1^Δ4/Δ4 mice. RT-PCR analysis of Chrna1 expression in inner ear tissue of E18.5 wild-type (Chrna1^+/+), heterozygote (Chrna1^Δ4/+), and homozygote knockout (Chrna1^Δ4/Δ4) mouse littermates, using primers located in exons 2 and 5. As expected, a smaller amplification product was found in Chrna1^Δ4/Δ4 mice. The 223-bp amplicon reflects the 110-bp deletion of exon 4 with respect to the 333-bp RT-PCR product detected in Chrna1^+/+ mice. Note the persistence of Chrna1 mRNA in Chrna1^Δ4/Δ4 mice, also shown with primers located in exons 5 and 7. RT-PCR amplifications of Hprt, a constitutively expressed housekeeping gene, and of Chrng, the gene that codes for γ-nAChR subunit, were used as positive controls. The presence of 2 amplicons for Chrng reflects the presence of alternatively spliced transcripts with or without exon 5, also found in muscle (Mileo et al. 1995). +/−RT indicates the presence (+) or the absence (−) of reverse transcriptase in the cDNA synthesis reaction. All studies were performed on a C57BL/6 genetic background.

Fig. 2.

Developmental expression pattern of Chrna1 in cochlear hair cells. A-B: developmental expression pattern of Chrna1 in the cochlea as revealed by X-gal staining of Chrna1^lacZ/+ reporter mice. A: X-gal staining of Chrna1^lacZ/+ whole-mount cochlear preparation shows Chrna1 expression in the organ of Corti all along the cochlea at postnatal day (P)8. Scale bar: 100 μm. B: close-up views of the apical region of the cochlea at different ages. X-gal staining (blue dots) was detected in the organ of Corti in the inner hair cell (IHC) region at P4 and in the IHC and outer hair cell (OHC) regions at P8. From P16 onward, X-gal staining was restrained again to the IHC region and was still detectable at 6 wk, 6 mo, and 18 mo. No staining was detected in wild-type (WT) littermates (here shown at 18 mo). Scale bars = 10 μm. C–E: confocal analysis of Chrna1^lacZ/+ and WT littermates whole-mount cochlear preparations labeled with antibodies directed against β-gal (green) and myosin VI (hair cell marker, red). C: examples of maximal intensity projection (xy plane) at P8. Scale bar = 10 μm. D: single plane images in 3 perpendicular planes showing that β-gal immunoreactivity is localized in IHCs and OHCs. E: quantification of the percentage of IHCs and OHCs with β-gal labeling in the apical, medial, and basal regions of the cochlea at different ages. The number of HCs analyzed is indicated above the histogram bars.

Fig. 3.

Functional development of nAChRs and cholinergic efferent synapses in mouse apical IHCs. A: whole cell patch-clamp recordings from IHCs in excised apical cochlear coils; holding potential −94 mV. Application of 1 mM ACh evoked an inward current, and application of 80 mM K⁺ activated synaptic currents (*) in IHCs at P4. However, no ACh response or synaptic current was evoked at P0 or at 6 wk. 80 mM K⁺ induced a steady inward current in IHCs at all ages due to the change in potassium equilibrium potential. B: percentage of apical IHCs with ACh responses and synaptic currents at different ages. ACh responses were detected earliest at P0, and synaptic currents at P2. By P4, 100% of IHCs exhibited ACh responses and synaptic currents. At P16 and P21, the percentage of IHCs showing ACh responses and synaptic currents declined and reached 0% by 6 wk. The number of IHCs tested per age is indicated at the top of the histogram bars.

Fig. 4.

Test for a functional correlate associated with the expression of the α₁-nAChR subunit in developing IHCs. Whole cell voltage-clamp recordings from IHCs in excised apical cochlear coils at P4. A: sample traces of recordings from 2 IHCs. ACh (1 mM) evoked inward currents in all IHCs tested at P4. However, the “muscle-type” α₁-nAChR agonists 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), (±)-anatoxin A, (±)-epibatidine, and (−)-nicotine, which do not or only poorly activate α₉-containing nAChRs, did not activate any response in IHCs. Holding potential was −94 mV. B: response to 1 mM ACh was not significantly blocked by αA-conotoxin OIVA (αA-OIVA), a peptide toxin that blocks the fetal muscle form of α₁-nAChRs by specifically binding to its α₁γ interface. However, α-conotoxin RgIA (α-RgIA), a subtype specific blocker of α₉-containing nAChRs, did mostly block the ACh response. Holding potential of −80 mV.

Fig. 5.

Test for the influence of α₁-nAChR subunit on efferent cholinergic fiber patterning. Comparison of the cholinergic efferent innervation of ChAT-IRES-Cre^+/−;mTmG^+/−;Chrna1^Δ4/Δ4 and ChAT-IRES-Cre^+/−;mTmG^+/−;Chrna1^+/+ mouse littermates at E18. A-C and E: maximum intensity projections of whole-mount cochlear preparations labeled with antibodies directed against EGFP (green) and myosin VI (hair cell marker, white) analyzed by confocal microscopy. A and B: basal cochlear turn. Scale bar = 30 μm. C and E: higher magnification of cholinergic fibers in the HC area. D and F: single confocal image of the same area showing bouton like endings (arrowheads) (green) in close vicinity of the IHCs (white) for both genotypes. C–F: scale bar = 10 μm. Mice lacking α₁-expression did not show a marked change in the cochlear efferent innervation at the level of the radial fibers (RF) or in the organ of Corti compared with their control littermates.

Fig. 6.

Test for a role of the α₁-nAChR subunit in setting up functional nAChRs and efferent synapses in hair cells. A–D: whole cell recordings from basal IHCs in Chrna1^Δ4/Δ4 and control heterozygote littermates (Chrna1^Δ4/+) at E18.5. A: both Chrna1^Δ4/Δ4 and Chrna1^Δ4/+ IHCs showed similar current-voltage relationships dominated by delayed rectifier potassium currents. Voltage step protocol (inset): from a holding potential of −84 mV, 200-ms voltage steps to values between −104 and −36 mV were applied in 10-mV increments. B: IHCs of both genotypes fired calcium action potentials in current clamp. C: in both genotypes, in a subset of IHCs, 1 mM ACh induced an inward current response at a holding potential of −94 mV and an outward current at −34 mV, suggesting that α₉α₁₀-nAChRs were coupled to SK2 channels. D: in both genotypes, in a subset of IHCs, application of 80 mM K⁺ evoked synaptic currents. Together these data indicate that α₁-nAChR expression in IHCs is not necessary for the formation of functional efferent synapses.