Fig 1: Cholinergic efferent synapses are differentially affected in apical and basal OHCs of CaV1.3-/- mice A, inward (top) and outward (bottom) currents in wild-type apical-coil OHCs elicited during the extracellular application of 100 µM extracellular ACh at –90 mV and –40 mV, respectively. Note that at –90 mV the current was reversibly blocked by 1 µM strychnine, indicating the direct involvement of a9a10-nAChRs; at –40 mV, the outward current was prevented by the absence of Ca2+ in the extracellular solution, indicating the presence of SK2 channels. B, same experiments as in panel A but performed in apical-coil OHCs from CaV1.3-/- mice. Note that ACh produced very little or no responses at both potentials. C and D, maximum intensity projections of confocal z-stack images that were taken from mature P11 apical (C) and basal coil (D) of wild-type and CaV1.3-/- mice. Immunostaining for SK2 channels (green) and ChAT, which is used to visualize the efferent olivocochlear innervation of OHCs (red); Myo7a (blue) was used as the hair-cell marker. Scale bars: 10 µm. E–H, whole-cell voltage-clamp recordings obtained from mature OHCs in wild-type (E) and CaV1.3-/- (F–H) mice during the superfusion of 40 mm extracellular K+. Lower panels in E and H show an expanded time scale of the area shown in the panels above. I–K, maximum intensity projections of confocal z-stack images taken at two different frequencies along the cochlea (apical: 8 kHz; basal: 32 kHz) from P11 wild-type (J) and CaV1.3-/- mice (K). Immunostaining for the OHC marker prestin (I: green) and ChAT (I: white), which is used to visualize the efferent terminals and fibres below the IHCs, tunnel crossing fibres (arrows), and terminals below the OHCs. In the cochlear apical coil of CaV1.3-/- mice (K) there were fewer ChAT-labelled tunnel crossing fibres and OHC terminals than wild-type mice (J). The ChAT-labelled OHC terminals at 8 kHz were also disordered and larger than in the wild type. In the basal coil, the efferent innervation was visually comparable between the two genotypes. Scale bars: 10 µm.
Fig 2: IHCs from gata3fl/flotof-cre+/- mice retain an immature basolateral membrane current profile A, outward K+ currents recorded from P9 gata3fl/fl (control) and gata3fl/flotof-cre+/- (gata3 null) IHCs; voltage protocol as described in Fig. 2 C. B, current-voltage curves measured at 160 ms from the voltage-step onset from IHCs of gata3fl/fl (black: P9–P10: n = 19), gata3+/flotof-cre+/- (blue: P10: n = 3) and gata3fl/flotof-cre+/- (red: P9: n = 3) mice. C, inward K+ currents (I K1) elicited from P9 IHCs using hyperpolarizing voltage steps in 10 mV nominal increments from –64 mV. D, steady-state I K1-voltage curves measured at 160 ms from the onset of the voltage step applied to IHCs from control gata3fl/fl (black), gata3+/flotof-cre+/- (blue) and gata3fl/flotof-cre+/- (red) mice; number of cells as in panel B. E, K+ currents from adult P39 IHCs elicited by 10 mV hyperpolarizing and depolarizing voltage steps from –84 mV. F, example of K+ currents recorded from a P52 gata3fl/flotof-cre+/- IHC showing evidence for the expression of SK2 channels, which are normally expressed only in immature cells; voltage protocol as in panel E. G and H, size of the total K+ current (G) and isolated I K,f (H) recorded from IHCs of all three genotypes just after the onset of hearing (P16–P19) and at adult (P38–P52) stages. I, voltage responses recorded from IHCs elicited by applying depolarizing current injections from their respective membrane potentials.
Fig 3: Efferent innervation is retained in IHCs from adult gata3 deficient mice A and B, maximum intensity projections of confocal z-stacks taken from the apical cochlear region of control (left columns: A, gata3fl/fl, P29; B, gata3+/+, P49; n = 4 mice in each) and gata3 deficient mice (right columns: A, gata3fl/flotof-cre+/-, P29; B, gata3+/-, P49; n = 4 mice in each) using antibodies against SK2 (green) and ChAT (red). Each panel represents a different mouse. Lower panels show the IHC synaptic region at a higher magnification. Note that the immature-type SK2 channels are still expressed in the adult gata3+/- and gata3fl/flotof-cre+/- IHCs. Myosin 7a (Myo7a, blue) was used as a hair cell marker. Scale bars 10 µm.
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