Fig 2: Disruption of type II spiral ganglion afferents, the outer spiral fibers (OSF), in the mid-basal region of the Prph knockout (KO) delineated using ß-III tubulin immunofluorescence. (A) In the WT cochlea, the type II spiral ganglion afferents exit from the basal aspect of the inner spiral plexus (ISP) beneath the inner hair cells (IHC), cross the floor of the tunnel of Corti (tC) (arrowheads), pass between the outer pillar cells (OPC) and turn basally to form parallel sheets of OSF along the sides of the Deiters' cells (DC), as the outer spiral bundles (OSB). The OSF branch periodically and turn apically to terminate along the three rows of outer hair cells (OHC). The medial olivocochlear (MOC) efferent fibers cross the mid-region of the tC to innervate the OHC. (B) In the PrphKO image, the OSF density is substantially reduced, while equivalent MOC efferent fiber projections extend to the OHC. OSL, osseous spiral lamina. Transparent-mode confocal reconstructed images of batch-processed WT and PrphKO cryosections; 21–25 µm depth z stacks. See also Supplementary material 2.

Fig 3: Loss of afferent innervation of outer hair cells (OHC) in Prph(-/-) cochlea.(a) Type II spiral ganglion neuron (SGN) outer spiral bundle (OSB) innervation of the OHC runs alongside the Deiters' cells (DC) and is resolved in adult Prph(+/+) organ of Corti via neurofilament 200 kDa immunofluorescence (red). This immunolabelling (confocal optical slice with transmitted light overlay) of a cryosection (50 µm) through the organ of Corti also identifies the medial olivocochlear (MOC) efferent fibres crossing the tunnel of Corti to innervate the OHC. CtBP2 immunofluorescence (green puncta) labels the presynaptic ribbons at the base of the OHC nuclei and at the inner spiral plexus (ISP) region of the inner hair cell (IHC) (the IHC nucleus is also labelled by this antibody). Blue fluorescence is DAPI nuclear staining. OSL, osseous spiral lamina. (b) Neurofilament 200 kDa immunofluorescence image of adult Prph(-/-) organ of Corti in a cryosection, as for a, showing the absence of the type II SGN innervation of the OHC via the OSB. (c) The type II SGN OSB fibres were more fully resolved in the adult Prph(+/+) organ of Corti mid-cochlear level whole mount via the neurofilament immunofluoresence (three-dimensional reconstruction of optical slices imaged from the basilar membrane; colour coded for depth (z)). Multiple fibres are shown crossing the floor of the tunnel of Corti, to enter the OSB and turn basally in parallel tracks (arrow). Deeper in the tissue, the MOC efferent fibres project to the OHC, terminating with bulbous synaptic boutons (VAChT immunolabelling; green–blue). (d) OSB fibres were absent in the adult Prph(-/-) organ of Corti (crossed arrow); whole mount as for c. Note the retention of the MOC innervation to the OHC.

Fig 4: Prph(-/-) mice lack ipsilateral suppression.(a) 5 s of 82 dB SPL 10–25 kHz ipsilateral suppressor sound produced a sustained reduction in the amplitude of the ipsilateral f2–f1 DPOAE (60 dB SPL ~20 kHz) in Prph(+/+) and Prph(+/-) mice (**P=0.004 (SA3), 21–45 s post noise (lower bar, comparing mean values); n=9). In contrast, Prph(-/-) mice had only a small transient modulation (upper bar; P=0.461 (SA4); n=6). Inset in a is a control experiment in Prph(+/+) mice that confirmed the involvement of the MOC reflex by sectioning the olivocochlear bundle at the floor of the fourth ventricle (*P=0.049, 0.022, 0.028 for sequential pre- versus post-cut comparisons as indicated (SA7), n=3). (b) Comparison of 30 s of 82 dB 15–25 kHz ipsilateral suppressor sound on f2–f1 DPOAE (60 dB SPL ~20 kHz) in Prph(+/+) and Prph(+/-) versus Prph(-/-) mice (*P=0.032 (SA7), n=17 for (+/+)/(+/-), n=6 for (-/-)). Breaks in the data indicate a 6 min interval. Inset in b extracts the OHC—type II SGN—MOC efferent reflex from the initial co-incident off-sound transient (WT/HET—KO). Data are shown as mean±s.e.m.

Fig 5: Prph(-/-) mice have baseline hearing indistinguishable from Prph(+/+) littermate mice.(a,b) Cubic (2f1–f2) distortion product otoacoustic emission (DPOAE) thresholds and 8 kHz input–output functions. (c,d) Auditory brainstem response (ABR) threshold and 8 kHz input–output functions for the N1-P2 wave, which reflects cochlear nerve recruitment (n=5). Data are shown as mean±s.e.m.; P>0.05 (SA8).