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.