DOI: 10.5061/dryad.fxpnvx0sb
title: Sensory transduction is required for development of hair cell synapses

description: Acoustic overexposure and aging can damage auditory synapses in the inner
 ear by a process known as synaptopathy.  These insults may also
 damage hair bundles and the sensory transduction apparatus in auditory
 hair cells.  However, a connection between sensory transduction
 and synaptopathy has not been established.  To evaluate potential
 contributions of sensory transduction to synapse formation and
 development, we assessed inner hair cell synapses in several genetic
 models of dysfunctional sensory transduction, including mice lacking
 Transmembrane Channel-like (Tmc) 1, Tmc2 or both, in Beethoven mice which
 carry a dominant Tmc1 mutation and in Spinner mice which carry a recessive
 mutation in Transmembrane inner ear (Tmie).  Our analyses reveal
 loss of synapses in the absence of sensory transduction and preservation
 of synapses in Tmc1-null mice following restoration of sensory
 transduction via Tmc1 gene therapy.  These results provide
 insight into the requirement of sensory transduction for hair cell synapse
 development and maturation.
description: <i>ABR acquisition.  </i>ABR recordings were
 performed, as previously described (Nist-Lund et al. 2019).  Mice were
 anesthetized with 0.5 mg of ketamine and 0.15 mg of xylazine per 10 g body
 weight via intraperitoneal injection.  Subcutaneous needle electrodes were
 inserted at the vertex (reference electrode), pinna (active electrode),
 and rump (ground electrode).  Acoustic stimuli were delivered directly
 into the ear through a custom probe tube speaker/microphone system (EPL
 PXI Systems) consisting of two electrostatic earphones (CUI Miniature
 Dynamics) to generate primary tones and a Knowles microphone (Electret
 Condenser) to record sound pressures from the ear canal. In a sound-proof
 chamber, mice were presented 5 ms pure tone stimuli of 8, 11.3, 16, 22.6,
 and 32 kHz at sound pressure levels (SPL) of 10 to 115 dB in 5 dB
 increment steps.  512 responses of alternating stimulus polarity were
 collected and averaged for each SPL.  ABR potentials were amplified
 (10,000x), band-pass filtered (0.3-10 kHz), and digitized using custom
 data acquisition software from the Eaton-Peabody Laboratories Cochlear
 Function Test Suite.  Waveforms with peak to trough amplitudes greater
 than 15 µV were discarded by an artifact-reject function.  Sound stimuli
 and electrode voltages were sampled at 40 µs increments using a National
 Instruments digital input-output board and stored for offline
 analyses.  <i>Tissue Dissection,
 Immunohistochemistry, and Imaging.  </i>Temporal bones were
 dissected and fixed in 4% paraformaldehyde for 1 h at room temperature. 
 Temporal bones were then decalcified in 120 mM EDTA for 2 h for 7-day-old
 mice and up to 20 h for 4-week-old mice.  Following decalcification, the
 entire length of the organ of Corti was microdissected in PBS for
 whole-mount processing.  Tissues were then permeabilized by freezing on
 dry ice in 30% sucrose and blocked for 1 h at room temperature in PBS with
 0.3% Triton X + 5% normal horse serum.  Tissues were then stained with the
 following primary antibodies and incubated at 37°C overnight: 1) mouse
 isotype IgG1 anti-C-terminal binding protein 2 (CtBP2, 1:200, BD
 Transduction Laboratories #612044), 2) mouse isotype IgG2a anti-glutamate
 receptor 2 (GluA2, 1:2000, Millipore #MABN1189), and 3) rabbit anti-myosin
 VIIa (Myo7a, 1:200: Proteus Biosciences #25-6790).  Tissues were washed in
 PBS and incubated for 2 h at 37°C with the following secondary antibodies
 diluted in 1% normal horse serum + 0.3% Triton X: 1) goat anti-mouse IgG1
 Alexa Fluor 546 (1:1000, Thermo Fisher #A-21123), 2) goat anti-mouse IgG2a
 Alexa Fluor 488 (1:1000, Thermo Fisher #A-21131), and 3) donkey
 anti-rabbit Alexa Fluor 647 (1:200, Thermo Fisher #A-31573).  Finally,
 samples were mounted on glass coverslips with Vectashield mounting medium
 (Vector Laboratories).  Using the 10x air objective on a
 LSM 800 (Carl Zeiss), low-power images of the myosin channel were obtained
 from each microdissected piece.  Using a custom ImageJ plugin, a cochlear
 frequency map was generated by measuring the full length of the cochlea
 from apex to base using all microdissected pieces (Muller et al.
 2005). 
 Full z-stacks were then acquired at cochlear regions
 corresponding to five frequencies (8, 11.3, 16, 22.6, 32 kHz) using a 63 x
 1.4 NA oil objective lens (Carl Zeiss, z step = 0.36 μm, scaling per
 pixel: 0.068 μm x 0.068 μm).  9-12 IHCs per field were imaged with
 z-stacks spanning the entire length of the hair cells.  For volume
 estimations, confocal z-stacks were acquired using the 63x oil objective
 with AiryScan processing (Carl Zeiss, z step = 0.18 μm, scaling per pixel:
 0.034 μm x 0.034 μm).  <i>Ribbon Synapse Counts
 and Volume Distribution Measurements.  </i>Confocal z-stacks were
 ported to Imaris, an image analysis software, for creation of 3D
 projections and quantitative analyses of synapse counts and volumes.  The
 “Spots” module in Imaris was used for automated identification and
 counting of all ribbons in a given z-stack.  All counts were manually
 reviewed and verified.  Synapses were defined as juxtaposition of
 presynaptic ribbons labeled with anti-CtBP2 with postsynaptic AMPA
 receptor puncta labeled with anti-GluA2.  Juxtaposition was verified
 manually for every ribbon identified using the “Spots” module.  The total
 number of synapses was divided by the number of IHCs in the image to
 calculate the average number of synapses/IHC.  For estimation of ribbon
 volume distributions, confocal z-stacks of ribbon synapses were obtained
 from P28 control, <i>Tmc1<sup>Δ/Δ</sup></i>, and
 <i>Tmc1<sup>Δ/Δ</sup></i> mice injected with
 AAV2/9-PHP.B-CMV-TMC1ex1-WPRE using Airyscan processing and ported to
 Imaris. 3D projections of Airyscan z-stacks were generated and ribbon
 volumes were segmented from each projection using the “Surfaces” module in
 Imaris. Identical settings were applied across image stacks, including the
 threshold for “background subtraction (local contrast)”. This threshold
 was fixed to ensure digital analyses in Imaris were consistent across
 images (despite potential variability in immunostaining quality and image
 acquisition confocal settings) and to prevent subjective biases from
 affecting ribbon volume calculations. As with the synapse counts, ribbons
 identified using the “Surfaces” module were manually verified to be
 juxtaposed with anti-GluA2 staining. Calculated volumes from each z-stack
 for each mouse were normalized to the median volume of all ribbons in the
 z-stack to account for differences in immunostaining quality and image
 acquisition settings across mice, genotypes, and cochlear regions.
 Standard deviations of normalized ribbon volumes from each z-stack for
 each mouse were compared to determine if significant differences in ribbon
 volume distributions were evident between groups.
 <i>Experimental Design and Statistical Analyses.</i>
 The Wilcoxon rank sum test was used for comparison of P2 WT and
 <i>Tmc1<sup>Δ/Δ</sup>;Tmc2<sup>Δ/Δ</sup></i> CtBP2+ puncta counts. Mixed-model ANOVA was used to compare synapse counts across different genotypes and timepoints. Six paired comparisons were made between the four groups in Figures 3A-C and three paired comparisons were made between the three groups in Figures 4A-C and 6A. The Bonferroni correction was applied to correct for the multiple comparisons and the reported p values are the original p values divided by the number of paired comparisons made. The Wilcoxon rank sum test was used to compare average ABR thresholds in WT and <i>Tmc1<sup>Δ/Δ</sup></i> mice injected with <i>Tmc1 </i>gene therapy. To evaluate whether the size of ribbon volume distribution differed between control,<i> Tmc1<sup>Δ/Δ  </sup></i>and <i>Tmc1<sup>Δ/Δ</sup></i> mice injected with <i>Tmc1 </i>gene therapy, standard deviations of normalized ribbon volumes from each z-stack were compared using Kruskal Wallis tests followed by Dunn’s multiple comparisons tests.  Exact <i>p </i>values are reported.  Statistical analyses were performed in GraphPad Prism and figures were created using OriginLab, OriginPro. 
description: Original raw data files from Figures 1,2,3,4 and 6 are confocal
 images acquired using Zeiss, Zen software.  Zen software or any image
 analysis software that can open *.CZI files can be used to view these
 images.  Original raw data files from Figure 5 include ABR data acquired
 using custom data acquisition software from the Eaton-Peabody Laboratories
 Cochlear Function Test Suite.  The data can be viewed using Excel or other
 data analysis software.