Developmental regulation of nicotinic synapses on cochlear inner hair cells

Abstract

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 alpha9 and alpha10 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 alpha9 mRNA is expressed in adult IHCs but that alpha10 mRNA disappears after the onset of hearing. In the present study, antibodies to alpha10 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 alpha10, but not alpha9, supports the hypothesis that functional nicotinic acetylcholine receptors in hair cells are heteromers containing both these subunits.

Figure 1.

ACh-evoked currents and K⁺-evoked IPSCs in IHCs throughout development. A, Representative records of currents (nAChR + SK) evoked by 100 μm ACh in IHCs voltage-clamped at -90 mV at the different postnatal ages. B, Representative records of currents evoked by 40 mm external K⁺ (at -90 mV). Note that at P16-P22, 40 mm K⁺ produced the expected change in the holding current but no IPSCs. Responses shown in A and B at each age were taken from the same IHC. C, Synaptic currents enclosed by box shown in B (top record), plotted at a slower time scale. D, Bar diagram illustrating the mean amplitude ± SEM of the currents evoked by 100 μm ACh, normalized to the mean capacitance (C_m) at each age group. Numbers between brackets are the number of cells in which 100 μm ACh elicited a current per number of cells tested. The mean amplitude includes only the ACh-positive cells. Amplitudes at P3 and at P13-P14 were significantly different from those at P7-P9 (p < 0.0019). Mean capacitance values used to normalize amplitudes were (in picofarads, mean ± SEM): P3, 10.4 ± 0.7; P7-P9, 9.5 ± 0.4; P10-P12, 10.0 ± 0.4; P13-P14, 12.6 ± 0.6 and P16-P22, 13.0 ± 1.0. E, Bar diagram representing the number of IHCs that had IPSCs after superfusing the preparation with saline containing 40 mm K⁺. Numbers between brackets are the number of cells studied at each age group. F, Bar diagram illustrating the fraction of Ach-sensitive IHCs that also had K⁺-evoked IPSCs at the different postnatal ages studied. Numbers between brackets are the number of cells positive to ACh that were superfused with high potassium to evaluate synaptic activity. In all cases the intracellular solution contained KCl and EGTA.

Figure 2.

Expression of the α10 nAChR subunit before and after the onset of hearing. α10 nAChR (red, closed arrow) in IHCs at P10 (A) and P15 (B) of the middle cochlear turn. The section is counterstained with synaptophysin to demarcate the efferent fibers (syn, green, open arrow). The IHC is delineated with a dotted line. Note the disappearance of the α10 nAChR on P15. Scale bars, 5 μm.

Figure 3.

Coupling of the nAChR current to the activation of SK channels. A, Representative recordings of the currents evoked by ACh in IHCs at P3 and P13, voltage-clamped at the holding potentials indicated under each record. In all cases the intracellular solution contained KCl and EGTA. Notice that currents were inward at -90 mV and outward at -50 mV, indicating that SK potassium currents were present. Illustrated records are representative of those found in three to six different IHCs at each age. B, Representative traces of K⁺-evoked combined nAChR plus SK IPSCs at P9-P11 by 40 mm K⁺ (E_K, -35 mV). C, Representative traces of K⁺-evoked isolated nAChR synaptic currents at P9-P11 by 40 mm K⁺ (E_K, -35 mV). D, Representative records of one combined (nAChR + SK) IPSC (gray trace) and one isolated (nAChR) postsynaptic current (black trace) superimposed to illustrate the much slower kinetics of the combined nAChR plus SK IPSC. E, bar diagrams showing that combined nAChR plus SK IPSCs have a larger amplitude and slower kinetics than those of the isolated nAChR postsynaptic currents (p < 0.0001). In B, recordings were made with a KCl-EGTA intracellular solution; in C, recordings were made with an intracellular solution in which EGTA was replaced by BAPTA to prevent the activation of SK channels. Recordings in B-D were made at a V_hold of -90 mV.

Figure 4.

Amplitude and kinetics of IPSCs during development. A, Representative traces of K⁺-evoked (40 mm K⁺) combined nAChR plus SK IPSCs throughout development. Insets at the right of each trace are the average of 30-40 individual IPSCs from each record. B, Bar diagrams illustrating that both the amplitude and kinetics of nAChR + SK IPSCs remained constant from P3 to P11 and that at P13-P14, both parameters decreased by ∼25% with respect to previous ages (p < 0.001). P3, 225 events, 5 cells; P9-P11, 213 events, 10 cells; P13-P14, 365 events, 6 cells. Amplitudes are represented in absolute values.

Figure 5.

Expression of the SK2 channel during postnatal development. SK2 channels (red, closed arrow) in IHCs at P3 (A), P9 (B), P12 (C), and P15 (D) of the middle cochlear turn. The section is counterstained with neurofilament-200 (green, open arrow) and DAPI (nuclear staining, blue). The IHC is delineated with a dotted line. Note the disappearance of SK2 from the basolateral pole of IHCs between P9 and P15. Scale bars, 5 μm.