Supplemental table

Primer sequences

First PCR forward primer (5’3’) First PCR reverse primer (5’3’) Syt1 ACCGTGGGCCTTAATTGCCAT GGAAGACTTTGACGTATGGATCA Syt2 AAGAACAAGAAGGAGAAGGGCAA CCTCCCCGATGATGTCATGCT Syt3 GCCTCTGTGGTAGCTGCTG TCCTCTCCACCCGGCAGA Syt5 GCCTGGGTCCTGGCTACC CAACGCAGCCAGTCCCTGA Syt6 CCCTGCTTCAGAGACTCTC TGGGCTGCTCGGCGGCA Syt7 GAGGAACCTGAACCCCATCTT GGTGCCACTGGGCCACAG Syt9 GACCCACCTACACCCTGC GACCTCTGTTTGTATAACTCTGGT Syt10 AAAGCCAGCACCCAAAGCCAT CGCTAAAGTCGACGCTGGAGA Nested PCR forward primer (5’3’) Nested PCR reverse primer (5’3’) Syt1 CTAGTCGTGACCTGCTGCTT TTCAGCAGCCTGGATGATTCC Syt2 AGGCATGAAGAACGCCATGAAC CACCAGGGTCTTGCCTCCT Syt3 AGCCAGACATCCCCTGAGC GTGCAGGCGGTCGCTCCT Syt5 TCGGGCCTCCTGGTCTTCA AATACTGCAGTCGGCCTAGCT Syt6 GCTCCAGAGGCAACATGGC TCATTGCCATAGTCCACGCTG Syt7 AGTCTTTCGCCTTCGACATACC GACGAGCGATCATGTCCTTCC Syt9 GACATTCCCCTCTCCACCCA ATTCTACCAATCCCAGTCAGC Syt10 CCTGCAATAAAAATCAGCCACACAT CATTCATTTGCCTTGGTAGGTGT

Supplementary Figures

2

Supplementary Figure 1: Spontaneous firing activity in IHCs, afferent terminals and cochlear ganglion neurons during the early postnatal developmental period

A-B, Examples of a train of spontaneous APs in IHCs recorded (in absence of current injection) in P2 control (A) and Otof-/- (B) mice. Spontaneous AP frequency distribution for the same IHCs are shown in histograms (bin width 0.4 Hz). The average IHC firing frequency of spontaneous APs was 4.0 ± 0.6 Hz ; 3.5 ± 0.5 Hz for Otof-/- (n = 4) and Otof+/- (n = 3) mice, respectively. C, D, Examples of spontaneous EPSCs in a P2 control (C) and Otof-/- (D) afferent boutons with enlarged views (bottom traces) of two events. EPSC amplitude distribution for the same cells (bin width 2 pA). Spontaneous EPSCs could be observed in six and four fibers from Otof-/- and control mice respectively. Their frequency of occurrence was randomly distributed, with bursts of EPSCs separated by long silent intervals. Mean amplitude of small events was 17.5 ± 0.4 pA (Otof+/-, n = 759 events) and 14 ± 0.3 pA (Otof-/-

, n = 1630 events) E-F, Spontaneous APs recorded in cochlear ganglion neurons from P2 control (E) and Otof-/- (F) mice. Occurrence frequency distribution of spontaneous APs in the same neurons (bin width 0.5 Hz). The frequency of AP occurrence was 4.2 ± 0.3 Hz (n = 2) and 3.2 ± 1.8 Hz (n = 2 neurons) in Otof-/- and control mice, respectively.

Supplementary Figure 2: Specificity of the Syt1 and Syt2 immunostainings in the organ of Corti

In the organ of Corti from a wild-type mouse (at P0 and P1, respectively), Syt1 (A) and Syt2

(B) are mainly detected in IHCs, whereas the IHCs from a Syt1-/- or a Syt2-/- mouse are not immunoreactive for the anti-Syt1 or anti-Syt2 antibody, respectively. The CtBP2 immunostaining indicates the presence of synaptic ribbons both in the wild-type and Syt1-null or Syt2-null mice.

Supplementary Figure 3: Confocal microscopy images of whole mounts of the organ of

Corti at P10, labeled for Syt1 and viewed from the surface (xy plan, A), in cross-section views

(xz plan, B and yz plan, C). At P10 the staining is still observed along the membrane of the

IHCs and its synaptic region. Note that a Syt1 staining is no longer observed in the OHC region (A,B). Scale bars: 5 µm.