Synaptic activity is not required for establishing heterogeneity of inner hair cell ribbon synapses

Neural sound encoding in the mammalian cochlea faces the challenge of representing audible sound pressures that vary over six orders of magnitude. The cochlea meets this demand through the use of active micromechanics as well as the diversity and adaptation of afferent neurons and their synapses. Mechanisms underlying neural diversity likely include heterogeneous presynaptic input from inner hair cells (IHCs) to spiral ganglion neurons (SGNs) as well as differences in the molecular profile of SGNs and in their efferent control. Here, we tested whether glutamate release from IHCs, previously found to be critical for maintaining different molecular SGN profiles, is required for establishing heterogeneity of active zones (AZs) in IHCs. We analyzed structural and functional heterogeneity of IHC AZs in mouse mutants with disrupted glutamate release from IHCs due to lack of a vesicular glutamate transporter (Vglut3) or impaired exocytosis due to defective otoferlin. We found the variance of the voltage-dependence of presynaptic Ca2+ influx to be reduced in exocytosis-deficient IHCs of otoferlin mutants. Yet, the spatial gradients of maximal amplitude and voltage-dependence of Ca2+ influx along the pillar-modiolar IHC axis were maintained in both mutants. Further immunohistochemical analysis showed an intact spatial gradient of ribbon size in Vglut3–/– mice. These results indicate that IHC exocytosis and glutamate release are not strictly required for establishing the heterogeneity of IHC AZs.


Introduction
The mammalian auditory system responds to sound pressures ranging over six orders of magnitude.Downstream of dynamic range compression by active cochlear micromechanics, inner hair cells (IHCs)-the sensory receptors-represent the entire audible range.Yet, the rate of sound-evoked firing in each of their postsynaptic spiral ganglion neurons (SGNs) encodes only a fraction of it.SGNs tuned to a given sound frequency differ in their molecular profile and morphology as well as spontaneous and sound-evoked firing.They tile the audible intensity range and segregate their central projections.SGNs are often classified according to their spontaneous rate (SR) into high, medium, and low SR fibers (Kiang et al., 1965;Sachs and Abbas, 1974;Liberman, 1978;Winter et al., 1990).High SR fibers display low sound thresholds and therefore encode soft sounds, while low SR fibers with higher thresholds present stronger sound intensities.Recent transcriptomic studies identified three molecularly distinct classes of type I SGNs (I a , I b , I c ), which have been suggested to correspond to the above described physiological subtypes of SGNs (Petitpré et al., 2018;Shrestha et al., 2018;Sun et al., 2018;Li et al., 2020).Differences in type I SGNs are also reflected in the spatial innervation pattern of their presynaptic partner IHCs.Each IHC is innervated by 5-30 type I SGN (review in Meyer and Moser, 2010), whereby low SR and high threshold SGNs are said to predominantly contact the modiolar (neural) side of the IHC as opposed to the high-spontaneous rate low threshold SGNs, which primarily synapse on the pillar (abneural) side of the cell (Liberman, 1982;Merchan-Perez and Liberman, 1996).Similarly, SGNs with type I a molecular profile preferentially synapse on the pillar side of an IHC (Shrestha et al., 2023;Siebald et al., 2023), while type I b/c SGNs synapse on the modiolar side (Sherrill et al., 2019) and have lower SR (Siebald et al., 2023).
The molecular mechanisms that shape the heterogeneity of AZs in IHCs remain largely unknown.Previous studies provided preliminary evidence for an impact of efferent innervation (Yin et al., 2014;Hickman et al., 2015), transsynaptic signaling (Sherrill et al., 2019;Shrestha et al., 2023), and intrinsic planar polarity signaling (Jean et al., 2019).Moreover, disruption of the key AZ protein bassoon reduced the extent of AZ heterogeneity (Frank et al., 2010).Other manipulations, such as disruption of the AZ proteins RIM, RIM-BP, and RIBEYE or impaired mechanotransduction, affected synapses globally yet without obviously altering AZ heterogeneity (Jung et al., 2015;Krinner et al., 2017Krinner et al., , 2021;;Becker et al., 2018;Jean et al., 2018).Yet, to our knowledge, the impact of abolished IHC exocytosis has not been tested.
Here, we explored the hypothesis that synaptic transmission is relevant for establishing AZ heterogeneity and setting the spatial gradients of AZ properties along the modiolar-pillar axis of the IHCs.For that purpose, we used mice lacking the vesicular glutamate transporter 3 (Vglut3) as a model for abolished glutamatergic signaling from IHCs despite maintained exocytosis (Ruel et al., 2008;Seal et al., 2008).Previous studies on Vglut3 KO mice have shown that glutamatergic transmission at the afferent IHC synapse is required for maintaining molecular identities of type I b and I c SGNs (Shrestha et al., 2018;Sun et al., 2018).The changes in subtype specification might lead to changes in presynaptic properties via transsynaptic signaling, as shown for postnatal disruption of the transcription factor Pou4f1 that is specific for type I c and I b SGNs (Sherrill et al., 2019).In addition, we employed a new mouse mutant with disrupted IHC exocytosis due to mutations in the Otof gene (Otof TDA/TDA ).Otof TDA/TDA mice harbor three Otof mutations that disrupt Ca 2+ binding to the C 2 E domain of otoferlin and show abolished Ca 2+ influx-triggered exocytosis despite near normal abundance and distribution of otoferlin as well as maintained synapses at the age of 3 weeks.
Analyzing ribbon size and Ca 2+ influx at individual presynaptic AZs of Vglut3 −/− and Otof TDA/TDA IHCs, we found the spatial gradients for the amplitude and voltage dependence of Ca 2+ influx as well as the gradient of the ribbon size to be preserved.Despite the intact gradients, the variability of the voltage of half maximal activation among the AZs of Otof TDA/TDA IHCs was significantly lower compared to that of the WT IHCs.

Animals
All the experiments described in the study were performed in P21-P28 mice of either sex.Vglut3 −/− mice have been described before (Ruel et al., 2008) and the generation of Otof TDA/TDA will be described in a manuscript (Chen et al.) that is currently under peer review elsewhere.In brief, a CRISPR/Cas9 approach was employed and ribonucleoprotein particles were injected into C57Bl6N oocytes.Correct editing of Otof was validated in founder (F0) mice, which were then crossed with C57Bl6N mice.Germline transmission was confirmed by heterozygosity for the edited allele.F2 mice were born at a Mendelian ratio, and heterozygous breeding was used to generate Otof TDA/TDA and WT littermates for experiments.Otof TDA/TDA were found to be deaf by recordings of auditory brainstem responses but seemed otherwise fine according to routine observation.Breeding was done in compliance with the German national animal care guidelines and approved by the local animal welfare committee of the University Medical Center Göttingen and the Max Planck Institute for Multidisciplinary Sciences, as well as the animal welfare office of the state of Lower Saxony, Germany (LAVES, AZ 19/3134).
Data were acquired with an EPC-10 amplifier controlled by PatchMaster software (HEKA Elektronik).The holding potential of 10.3389/fnmol.2023.1248941 the cell was set to −87 mV.Whole cell Ca 2+ influx was triggered by applying either voltage step or voltage ramp depolarizations to the cell.Voltage step depolarizations were applied from −82 to 63 mV with 5 mV increments and voltage ramp depolarizations ranged from −87 to 63 mV over the course of 150 ms (1 mV/ms).Recordings were leak corrected using P/n protocol.All voltages were corrected offline for voltage drops across series resistance (R s ) and liquid junction potential, which was calculated to be 17 mV.Recordings were discarded from analysis in case they displayed leak currents beyond −50 pA at the holding potential (−87 mV), R s above 14 MOhm during the first 3 min after rupturing the cell, or Ca 2+ current rundown above 25% by the end of the experiment.
Z-stacks during the live imaging were acquired using a fast piezoelectric system (Piezosystem).To obtain the morphology of the IHC and identify the positions of the AZs, we first acquired a Z-stack of the cell through imaging TAMRA fluorescence using a 591 nm laser (Cobolt AB).Each plane was exposed for 0.5 s and the step size for Z-scanning was 0.5 µm.Ca 2+ imaging was performed by exciting Fluo-4FF with 491 nm laser (Cobolt AB).Ca 2+ imaging was restricted to the planes which contained ribbons.Hotspots of Fluo-4FF fluorescence increases were evoked by voltage ramp depolarizations and simultaneously imaged at a frame rate of 100 Hz at each plane.For each plane, two different voltage ramps were applied, one being 5 ms shifted relative to the other one.This served the purpose of increasing the voltage resolution of Ca 2+ imaging when later concatenating the two fluorescence traces evoked by the two different voltage ramps.

Data analysis 2.4.1. Patch-clamp and imaging
Data were analyzed with Igor pro 6.3 software (Wavemetrics) using custom written procedures.Whole-cell current-voltage relationships (IV curves) were analyzed by plotting the evoked Ca 2+ currents averaged over the 5-10 ms interval after the start of stimulation against the depolarization voltages.
The analysis of Ca 2+ imaging was performed as described previously (Cantu-Guerra et al., 2023).Briefly, Ca 2+ hotspots were visualized by subtracting the average of 10 resting frames from the average of 5 frames during stimulation.To obtain the intensity profile of Fluo4-FF fluorescence over time the intensity of the central pixel of the hotspot was averaged with the 8 surrounding pixels at all-time points.Two intensity profiles corresponding to the shifted voltage ramp stimuli were concatenated and plotted against voltage (FV curve).The FV curves were fitted using modified Boltzmann function: where F is the fluorescence intensity at a given voltage, F 0 is the fluorescence at the resting state, V rev is the reversal potential calculated from the IV recordings of the whole cell patch-clamp data, V m is the cell membrane voltage, V h is the voltage of halfmaximal Ca 2+ influx, and k is the slope.
Fractional activation curves of the Ca 2+ channels at single active zones were calculated by dividing FV curves by the lines fitted to the fluorescence decay of the FV curves at depolarized voltages (G max ).Resulted curves were fitted with the Boltzmann function (Figure 3A).
To analyze the position dependency of Ca 2+ influx properties at individual IHC AZs, the coordinates of the ribbons (visualized by TAMRA-conjugated dimeric Ctbp2 binding peptide) were transferred from the canonical Cartesian coordinate system into a self-defined polar coordinate system (Ohn et al., 2016;Cantu-Guerra et al., 2023).Briefly, we performed 3D reconstruction of the cells, whereby the symmetry vector of the cell (V sym , orthogonal vector to the plane of symmetry) as well as the central vector of the cell (V z , connecting the centers of mass of the bottom-most plane and the largest plane) were calculated.Afterward we obtained the vector defining modiolar-pillar axis (V pm ) of the cell by multiplying V sym and V z .Finally, the polar coordinates of the ribbons were calculated and plotted in polar charts.The two orthogonal axes of the polar charts represent apical-basal (referring to the tonotopic axis) and pillar-modiolar axes of the cell.

Analysis of confocal and STED imaging data
For the position-dependent analysis of ribbon size, we used a customized MATLAB plugin for the Imaris software, as it was described before (Jean et al., 2019;Sherrill et al., 2019).Briefly, Ctbp2 immunofluorescent spots were automatically detected on Imaris, and the quality of detection was adjusted subjectively.The intensities of the ribbons were calculated as the sum of pixel intensities of the 7x7x5 region around the center of mass of the immunofluorescent spots.Based on the cytosolic and nuclear staining, the central vector of the cell was assigned by connecting the base of the cell to the center of the nucleus of the cell and was further adjusted XY and YZ planes.The Cartesian coordinates of the ribbons, which were defined as the coordinates of the center of the masses of the 3D Gaussian fits were transformed to the polar coordinate system.Ribbon immunofluorescence intensities of each sample were normalized to the median fluorescence of the modiolar ribbons.
2D STED images of the ribbons and Ca v 1.3 line clusters were analyzed with Igor Pro software and were fitted with 2D Gaussian function to obtain full with at half maxima (FWHM) of long and short axes using genetic fit algorithm (Sanchez del Rio and Pareschi, 2001).Confocal and STED images were adjusted for visualization purposes using Fiji software.

Statistical analysis
Data were analyzed using Igor Pro software.Data is presented as mean ± standard error of the mean (SEM), and the standard deviation (SD) is shown for each data set.The number of the animals is indicated as N.For two sample comparison, normality of the distributions (Jarque-Bera test) and equality of variances (F-test) were tested.This was followed by Student's t-test or Mann-Whitney-Wilcoxon test in case the normality of distributions and/or equality of variances were not met.Levene's test was used to test the equality of variances between the distributions of voltage of half maximal activations of Ca 2+ influx in WT and Otof TDA/TDA IHCs.Significant differences are presented as * p < 0.05, * * p < 0.01, * * * p < 0.001.

Results
3.1.Heterogeneity of Ca 2+ influx at the AZs of Vglut3 −/− IHCs Transcriptomic studies of single SGNs in Vglut3 −/− mice revealed an altered molecular subtype specification of SGNs: 80% of the neurons gained type I a identity, while the proportion of type I b and I c neurons was down to 20% (Shrestha et al., 2018;Sun et al., 2018).This has been taken to indicate that postnatal glutamatergic transmission at the afferent IHC synapse is required to maintain the molecular identity of type I b and I c SGNs.Here, we asked the question if changes in synaptic transmission and/or altered transsynaptic signaling from SGNs affect the presynaptic heterogeneity of IHCs.To do so, we assessed the functional heterogeneity of IHC AZs by combining IHC patch-clamp and Ca 2+ imaging at IHC AZs in Vglut3 −/− mice at 3 weeks of age.Whole cell Ca 2+ currents tended to be slightly larger in Vglut3-deficient IHCs without reaching significance (Vglut3 +/+ : −161 ± 7.87 pA, SD = 40.12pA, n = 27, N = 16 vs.Vglut3 −/− : −170 ± 9.79 pA, SD = 48.96pA, n = 25, N = 12; Student's t-test, p = 0.49, Figures 1A, C), while a previous study showed a significantly increased Ca 2+ influx in Vglut3-deficient IHCs (Ruel et al., 2008).A potential explanation for this discrepancy is the age difference of mice.While an increase in the Ca 2+ current amplitude was previously reported for p12-p18 mice, we used 3-week-old mice in our study, at which stage a reduced synapse number might mask the increased Ca 2+ current.The voltage dependence of Ca 2+ channel activation and its voltage sensitivity, approximated as the slope of the Boltzmann function fit to the fractional activation curve, were not significantly altered, but there was a trend toward activation at lower potentials for Vglut3-deficient IHCs (Figures 1B, D, E).
To test whether the difference in F/F 0max between pillar and modiolar AZs in Vglut3 −/− IHCs was dominated by modiolar AZs with high fluorescence ("winner" AZ, Ohn et al., 2016), we analyzed modiolar-pillar gradient of maximum Ca 2+ influx without the "winners".As done before (Ohn et al., 2016), we defined "winner" AZs to have F/F 0max values at least 2.5 higher than the average F/F 0max of the rest (Supplementary Figures 1A,  B).Interestingly, the modiolar-pillar gradient of maximum Ca 2+ influx in Vglut3 −/− IHCs remained without the "winner" spots (Supplementary Figures 1C, D).

Heterogeneity of Ca 2+ influx at the
AZs of Otof TDA/TDA IHCs Our analysis of Vglut3 −/− mice showed intact modiolarpillar gradients of maximal synaptic Ca 2+ influx and ribbon size in IHCs.Moreover, despite a mild overall hyperpolarized shift of the voltage of half-maximal Ca 2+ channel activation, its pillar-modiolar gradient was preserved in Vglut3 −/− IHCs.This argues against a strict requirement of glutamatergic signaling for establishing or maintaining IHC synaptic heterogeneity.To test the impact of evoked exocytosis on the AZ heterogeneity and the spatial gradients of AZ properties, we chose to study a novel Otof mutant mouse.These mice harbor a triple aspartate to alanine mutation in the C 2 E domain of otoferlin Otof TDA/TDA -a protein that has been shown to be vital for IHC vesicle fusion (Roux et al., 2006;Pangrsic et al., 2010;Michalski et al., 2017).Exocytosis in IHCs of Otof TDA/TDA is largely abolished, but, unlike in other Otof mutants (e.g.50% synapse loss in IHCs of Otof −/− mice, Roux et al., 2006;Stalmann et al., 2021) the number of the IHC afferent synapses was preserved in 2-3-week-old mice (Chen et al.).

Discussion
Heterogeneity of IHC afferent synapses is one of the candidate mechanisms contributing to the functional diversity of type I SGNs in the cochlea.In this study we aimed to understand the role of synaptic transmission in the establishment of AZ heterogeneity in IHCs.For this reason, we employed two mouse models: Vglut3 −/− mice, which lack glutamatergic signaling at IHC afferent synapses, but maintain vesicle fusion, and Otof TDA/TDA mice, IHCs of which display largely abolished evoked exocytosis.By Ca 2+ imaging, we  analyzed the amplitude of synaptic Ca 2+ influx and its voltage dependence of activation at single AZs of IHCs in Vglut3 −/− and Otof TDA/TDA mutant mice.We then related those properties to the positions of the AZs along the modiolar-pillar axis of mutant IHCs and did not observe any striking changes of the previously described gradients (Ohn et al., 2016).Additionally, the modiolar-pillar gradient of ribbon size was retained in IHCs of Vglut3 −/− mice.We conclude that synaptic transmission at IHC afferent synapses is dispensable for the formation of presynaptic AZ heterogeneity.However, we observed an overall hyperpolarized shift of Ca 2+ channel activation in IHCs of both mutants and a lower variance of the voltage dependence of Ca 2+ channel activation in Otof TDA/TDA IHCs.Spiral ganglion neuron molecular subtypes in mice are established already at birth (Petitpré et al., 2018).Glutamatergic synaptic transmission during cochlear development is crucial for maintaining proper type I SGN molecular subtype specification (Shrestha et al., 2018;Sun et al., 2018).Specifically, transcriptomic studies have shown that disrupting glutamatergic transmission in Vglut3 −/− mice reduces the number of type I b and I c SGNs, and the majority of neurons gain a type I a fate.RNAscope results from Vglut3 −/− mouse tissues do not show obvious deviations from the WT mice at postnatal day P3, and the alterations in SGN molecular subtypes start between postnatal day 3 and 8 (Shrestha et al., 2018).In return, postsynaptic SGNs seem to regulate AZ properties by transsynaptic signaling.For example, expression of the transcription factor Pou4f1 is characteristic for type I c and I b SGNs and contributes to shaping AZ properties.Conditional deletion of Pou4f1 disrupted the modiolar-pillar gradient of the maximal amplitude of synaptic Ca 2+ influx in IHCs (Sherrill et al., 2019).Similarly, deletion of the Runx1 transcription factor that is selectively expressed in type I c and I b SGNs in a recent study resulted in a collapse of the modiolar-pillar gradient of ribbon size in IHCs (Shrestha et al., 2023).We hypothesized similar changes in Vglut3 −/− mice considering the dramatic reduction of type I b and I c neurons.Interestingly, we observed intact gradients of maximal Ca 2+ influx, voltage dependence of Ca 2+ channels, and ribbon size of AZs along the modiolar-pillar axis.This might suggest a limited impact of transsynaptic signaling for IHC AZ heterogeneity.Alternatively, transsynaptic signaling might be maintained despite the altered molecular identity or largely restricted to a certain time window during the early postnatal development of the animal.The latter might explain the discrepancy observed upon postnatal disruption of SGN subtype specification in Vglut3 −/− mice to the findings upon perinatal conditional Pou4f1 deletion.Interestingly, we also observed an intact modiolar-pillar gradient of maximal Ca 2+ influx for exocytosis-deficient AZs of Otof mutant mice for which the molecular subtype specification of SGN remains to be studied.Otof mutation would be expected to spare early synaptic signaling as IHC exocytosis in mice is independent of otoferlin during the first 3 postnatal days (Beurg et al., 2010).Finally, we observed an intact modiolar-pillar gradient of maximal Ca 2+ influx in dfcr mutants (Ohn et al., 2016) with disrupted function of the Usher protein harmonin that is required for normal mechanoelectrical transduction (Grillet et al., 2009;Michalski et al., 2009) and also regulates synaptic Ca 2+ influx (Gregory et al., 2011).All four manipulations, i.e., disruption of the function of harmonin, otoferlin, Vglut3, and Pou4F1, resulted in a mild hyperpolarized shift of Ca 2+ channel activation at IHC AZs.Considering the reduced fraction of SGNs with type I b/c identity in Pou4F1 and Vglut3 mutant mice one might speculate on a longer-term requirement of type I b and I c -specific transsynaptic signaling at IHC afferent synapses.However, other mechanisms might contribute to the change in Ca 2+ channel activation.This seems likely at least for disruption of the function of harmonin and otoferlin, which both interact with Ca v 1.3 channels (Ramakrishnan et al., 2009;Gregory et al., 2011).

Limitations of the present study
We note that all the experiments in this study have been performed at the apical turn of the organ of Corti around the 6-8 kHz region.Therefore, we cannot exclude the possibility that larger effects of impaired transsynaptic signaling could be observed at other tonotopic positions.Furthermore, despite the preserved gradients of IHC AZ properties, Vglut3 −/− animals display abnormal enlargement of synaptic ribbons.These results are inconsistent with the findings in Pou4f1 and Runx1 KOs, where deletion of type I b and I c SGN molecular markers led to reduction of average ribbon size.Thus, there is the possibility that direct presynaptic alterations upon Vglut3 deletion mask or overrule potential effects of the altered SGN molecular subtype specification.
3 clusters were line-(around 80%) or spot-like (over 10%) in both WT and Vglut3 −/− IHCs (Figure 4G).By fitting the 2D Gaussian function to the population of line-like Ca v 1.3 clusters of WT and Vglut3 −/− IHCs, we saw a reduction in FWHM of both long and short axes of IHC Ca v 1.3 clusters in Vglut −/− mice (Figures 10.3389/fnmol.2023.1248941