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. 2013 Nov;3(11):130163.
doi: 10.1098/rsob.130163.

Cholinergic efferent synaptic transmission regulates the maturation of auditory hair cell ribbon synapses

Stuart L Johnson, Carolina Wedemeyer, Douglas E Vetter, Roberto Adachi, Matthew C Holley, Ana Belén Elgoyhen, Walter Marcotti

Cholinergic efferent synaptic transmission regulates the maturation of auditory hair cell ribbon synapses

Stuart L Johnson et al. Open Biol. 2013 Nov.

Abstract

Spontaneous electrical activity generated by developing sensory cells and neurons is crucial for the maturation of neural circuits. The full maturation of mammalian auditory inner hair cells (IHCs) depends on patterns of spontaneous action potentials during a 'critical period' of development. The intrinsic spiking activity of IHCs can be modulated by inhibitory input from cholinergic efferent fibres descending from the brainstem, which transiently innervate immature IHCs. However, it remains unknown whether this transient efferent input to developing IHCs is required for their functional maturation. We used a mouse model that lacks the α9-nicotinic acetylcholine receptor subunit (α9nAChR) in IHCs and another lacking synaptotagmin-2 in the efferent terminals to remove or reduce efferent input to IHCs, respectively. We found that the efferent system is required for the developmental linearization of the Ca(2+)-sensitivity of vesicle fusion at IHC ribbon synapses, without affecting their general cell development. This provides the first direct evidence that the efferent system, by modulating IHC electrical activity, is required for the maturation of the IHC synaptic machinery. The central control of sensory cell development is unique among sensory systems.

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Figures

Figure 1.
Figure 1.
Efferent activity in IHCs from control and α9nAChR KO mice. (a,b) Whole-cell voltage-clamp recordings from immature IHCs (P7–P9) in control (a) and α9nAChR KO (b) mice during the superfusion of ACh. Note that 1 mM ACh did not elicit an inward current from a holding potential of −90 mV in α9nAChR KO mice. Experiments were performed on three IHCs for each genotype. (c,d) IPSCs evoked with 30 mM extracellular KCl during long-lasting recordings from a P9 control (c) and a P10 α9nAChR KO (d) IHC (holding potential: −84 mV). Note that IPSCs were absent in the α9nAChR KO IHC. Similar effects were seen in all six P9–P10 controls and four P10 KO IHCs. (e,f) Action potential activity recorded from late postnatal (P8) IHCs in control and α9nAChR KO mice, respectively. Whole-cell current-clamp recordings were obtained by injecting a depolarizing current from the IHC resting membrane potential. Note that the extracellular superfusion of 30 μM ACh caused hyperpolarization and cessation of the firing activity only in the control IHC. Similar effects were seen in six control and seven KO IHCs (P8–P10).
Figure 2.
Figure 2.
Efferent input is required for the development of the IHC synaptic machinery. Membrane capacitance recordings from apical coil IHCs of control and α9nAChR KO adult mice (P15–P32). (a) ICa and corresponding ΔCm recordings in response to 50 ms voltage steps (10 mV increments) from −81 mV. For clarity, only the peak responses at −11 mV are shown. (b) Average ICa–voltage and ΔCm–voltage curves in control and α9nAChR KO IHCs. (c) Synaptic transfer curves obtained by plotting the average ΔCm against the corresponding ICa for membrane potentials between −71 and −11 mV (see shaded area in (b)). Fits in (c) are according to a power function formula image, where c is a scaling coefficient and the power is N. The ΔCm traces shown on the left are averaged from 11 IHCs for both control and α9nAChR KO mice.
Figure 3.
Figure 3.
The amplitude of evoked efferent IPSCs is reduced in Syt-2 KO IHCs. (a) Examples of spontaneous action potential activity recorded using whole-cell current clamp from early postnatal IHCs in both control and Syt-2 KO mice. Spontaneous IPSPs are indicated by arrows. (b) Whole-cell voltage-clamp recordings of IPSCs from a control (black) and a Syt-2 KO IHC (red) made at the holding potential of −84 mV. The release of ACh from efferent fibres was evoked by depolarizing efferent terminals with 15 mM extracellular KCl. (c) IPSCs shown in (b) but on an expanded time scale. (d) IPSCs from a control and a Syt-2 KO IHC made as in (b) but using 30 mM extracellular KCl.
Figure 4.
Figure 4.
Exocytotic Ca2+ dependence is normal in immature Syt-2 KO IHCs. Data are from apical coil control (black) and Syt-2 KO (red) immature IHCs (P5–P8). (a) ICa and corresponding ΔCm recordings as described in figure 2. (b) Average ICa–voltage (bottom) and ΔCm–voltage (top) curves in Syt-2 control (n = 14) and KO (n = 8) IHCs. (c) Synaptic transfer curves obtained as described in figure 2. Fits in (c) are according to formula image.
Figure 5.
Figure 5.
Exocytotic Ca2+ dependence is abnormal in mature Syt-2 KO IHCs. Data are from apical coil control (black) and Syt-2 KO (red) mature mouse IHCs (P16–P18). (a) ICa and corresponding ΔCm recordings as described in figure 2. (b) Average ICa–voltage (bottom) and ΔCm–voltage (top) curves in Syt-2 control (n = 8) and KO (n = 14) IHCs. (c) Synaptic transfer curves obtained as described in figure 2. Fits in (c) are according to formula image.
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