Supplementary Figure 1. Identification of the type II spiral ganglion neurons (SGN) via immunofluorescence of peripherin protein (PRPH). (a), (b), PRPH immunolabelling of cryosections from post-natal day four (P4) and adult Prph (+/+) cochleae, with transmitted light overlay. Insets in (a) and (left - b) show detail of the organ of Corti PRPH (+ve) immunofluorescence. Single confocal sections. The type II SGN somata are evident as a subpopulation of SGN within Rosenthal s canal (Rc). The type II SGN neurites project to the organ of Corti via the osseous spiral lamina (OSL). These afferent fibres pass through the lower region of the inner spiral plexus (ISP) beneath the inner hair cells (IHC), cross the tunnel of Corti and form the outer spiral bundle (OSB), before innervating the outer hair cells (OHC). Absence of PRPH labelling of SGN is confirmed in the adult Prph (-/-) cochlea ((b), right - inset). (c), PRPH immunofluorescence labelling type II SGN in post-natal day 8 (P8) Prph (+/+) cochlea. (d), Detail of P8 PRPH-immunopositive OSB fibres along the sides of the Deiters cells (DC), with ascending branches to the OHC. 3D reconstruction, with separated autofluorescence shown in gray for structure. (e), P8 β-tubulin immunofluorescence labels all neural elements in the organ of Corti, including the type II SGN OSB, the type I SGN 1
innervation of the IHC via the ISP, and the medial olivocochlear (MOC) efferent fibres innervating the OHC. 3D reconstruction, with separated autofluorescence shown in gray for structure. (f), (g), PRPH immunofluorescence of the OSB in P8 is more pronounced than in the adult (surface mount, 3D reconstructions), although the prominence of the OSB in the adult organ of Corti is evident from neurofilament 200 immunolabelling (see Fig. 1a). (h), Adult type II SGN PRPH immunofluorescence (single optical slice of cryosection). Note the loss of PRPH labelling in the OSB compared with P8. Inset confirms antibody specificity /loss of PRPH in adult Prph (-/-) organ of Corti and spiral ganglion (3D reconstruction). 2
Supplementary Figure 2. Loss of the type II spiral ganglion neuron afferent innervation of the outer hair cells (OHC) in Prph (-/-) detected with serial blockface scanning electron microscopy. (a), Base of an OHC from a Prph (+/+) mouse, showing the small type II afferent (aff) bouton (green) adjacent to the larger medial olivocochlear efferent (eff) bouton (blue). DC, Deiters cell; nu, nucleus; * indicate postsynaptic cistern electron density at efferent synapse. (b), Base of an OHC from a Prph (-/-) mouse, showing the retention of the efferent bouton, but absence of the afferent bouton. (c), Reconstruction of the afferent and efferent boutons at the base of an OHC. (d), Reconstruction of the synaptic boutons at the base of all three rows of OHCs in a stack of ~ 500 x 50 nm sections. The OHC in (c) is indicated in (d). (e) Prph (-/-) mouse OHC lacks an afferent bouton, but retains the medial olivocochlear efferent boutons. (f), Reconstruction of ~ 500 x 50 nm sections, including the OHC shown in (e). Note the absence of type II SGN afferent boutons. (g) Boxplots of the reconstructed volumes of afferent and efferent boutons at the base of OHCs obtained from serial 50 nm image stacks from Prph (+/+) and Prph (-/-) mice (three of each); boundaries indicate 25 th and 75 th percentile, solid line is median, dashed line is mean, with individual data overlaid; error bars indicate 95% confidence limits. (see also Supplementary Table 1). 3
Supplementary Figure 3. Effect of contralateral suppressor frequency band on ipsilateral distortion product otoacoustic emission (DPOAE). (a), (b), Contralateral suppression was tested across a range of contralateral sound stimulus frequencies in CBA/129 mice. The contralateral suppressor intensity was constant at 82 db SPL, and f 2 f 1 DPOAE primary tones were 65 db SPL about 28 khz. A contralateral stimulus of 10 17 khz, an octave below the primary DPOAE stimulus, was found to elicit the strongest contralateral suppression response (***P < 0.001; one-way repeated measures ANOVA). Insets in (a) show individual responses; data are mean ± s.e.m.. Box plots in (b) have boundaries indicating 25 th and 75 th percentile, solid line is median, dashed line is mean, with individual data overlaid (n = 8). 4
Supplementary Figure 4. Prph (-/-) mice lack contralateral suppression. (a), Examples of absence of contralateral suppression in Prph (-/-) mice compared with Prph (+/+) controls (129/BL6 mice). The contralateral suppressor noise (varying between 5s and 30 s) was 96 db SPL, 15 25 khz, with 60 db SPL f 1 and f 2 levels for the quadratic (f 2 f 1 ) distortion product otoacoustic emission (DPOAE) about 20 khz. (b), Box plots with boundaries indicating 25 th and 75 th percentile (95 % with bars), solid line is median, dashed line is mean, with data overlaid. Average starting f 2 f 1 DPOAE amplitudes were 13.7 ± 1.4 db (+/+) and 13.4 ± 1.9 db (-/-). * P = 0.012, unpaired t - test, n as indicated in (a). 5
Supplementary Figure 5. Contralateral suppression was blocked by sectioning the medial olivocochlear (MOC) efferent pathway. (a), (b), Repeated presentation of noise to the left ear (82 db SPL, 10 17 khz) produced comparable (contralateral) suppression of the f 2 f 1 distortion product otoacoustic emission (DPOAE; 65 db SPL about 28 khz) in the right ear (baseline and repeat). This was lost following sectioning of the olivocochlear bundle (OCB) at the floor of the fourth ventricle (4V) (*P = 0.036 baseline vs. OCB-cut and P = 0.043 repeat vs. OCB-cut; one-way repeated measures ANOVA with Holm-Sidak multiple pairwise comparisons, n = 3). (c), Darkfield microscopy of a coronal brain cryosection (50 µm) demonstrating the dorsomedial incision that disrupted the OCB (arrow). Overlay indicates contralateral suppression reflex pathway (derived from 1-3 ); dashed line indicates central drive to MOC efferent neurons, dotted line indicates OCB pathway. CN, cochlear nucleus. (a) Data are mean ± s.e.m.; insets in (a) show example traces; box plots in (b) have boundaries indicating 25 th and 75 th percentiles, solid line is median, dashed line is mean, with data overlaid. 6
Supplementary Table 1. SBF-SEM Analysis of Prph (+/+) vs Prph (-/-) Outer Hair Cell Synaptic Boutons Number of 50 nm thick sections analyzed Number of full OHCs present 1 Volume ( m 3 ) of afferent boutons Volume ( m 3 ) of efferent boutons Prph (+/+) 278 1020 19 1.38 ± 0.27 (17) 13.65 ± 1.35 (47) 375 920 17 1.19 ± 0.19 (15) 13.15 ± 1.16 (39) 110811 1001 19 1.02 ± 0.14 (19) 15.85 ± 1.49 (41) collectively 2941 56 1.03 ± 0.18 (51) 14.21 ± 0.78 (127) Prph (-/-) 556 855 20 0 19.58 ± 1.55 (43) 698 1072 22 0 17.20 ± 0.97 (54) 699 572 12 0 20.36 ± 1.45 (31) collectively 2499 54 (0) 18.76 ± 0.75 (128) Mean ± s.e.m. (n) 1 As OHC nuclei in the section series with individual bouton contacts. NB i. Afferent synaptic boutons were, in general, more electron dense cf/. efferents; whilst efferent boutons contained high densities of synaptic vesicles cf/. afferents and a denser synaptic region, consistent with a postsynaptic cistern.. ii. Membrane densities were also present between afferent-efferent; and efferent-efferent boutons. 7
Supplementary References 1 Liberman, M. C. & Brown, M. C. Physiology and anatomy of single olivocochlear neurons in the cat. Hear. Res. 24, 17-36 (1986). 2 Warren, E. H., 3rd & Liberman, M. C. Effects of contralateral sound on auditorynerve responses. II. Dependence on stimulus variables. Hear. Res. 37, 105-121. (1989). 3 Brown, M. C., de Venecia, R. K. & Guinan, J. J., Jr. Responses of medial olivocochlear neurons. Specifying the central pathways of the medial olivocochlear reflex. Exp. Brain Res. Experimentelle Hirnforschung 153, 491-498 (2003). 8