The Presynaptic Ribbon Maintains Vesicle Populations at the Hair Cell Afferent Fiber

1 The presynaptic ribbon maintains vesicle populations at the <a href="/tags/Hair_cell/" rel="tag">hair cell</a> afferent fiber 2 synapse 3 Lars Becker1, Michael E. Schnee1, Mamiko Niwa1*, Willy Sun2, Stephan Maxeiner3**, 4 Sara Talaei1, Bechara Kachar2, Mark A. Rutherford4, Anthony J. Ricci1,3 5 1 Department of Otolaryngology Stanford University, United States, 2 National Institute 6 of Deafness and Communicative Disorders, United States, 3 Molecular and Cellular 7 Physiology, Stanford University, United States, 4 Department of Otolaryngology, 8 Washington University in St. Louis, United States, 9 *Present address: Department of Otolaryngology Johns Hopkins University, United 10 States 11 ** Present address: Department of Neuroanatomy, Institute for Anatomy and Cell 12 Biology, Medical School Saarland University, Germany 13 14 15 16 17 18 19 1 20 Abstract: The ribbon is the structural hallmark of cochlear inner hair cell (IHC) afferent 21 synapses, yet its role in information transfer to spiral ganglion <a href="/tags/Neuron/" rel="tag">neurons</a> (SGNs) remains 22 unclear. We investigated the ribbon’s contribution to IHC synapse formation and 23 function using KO mice lacking RIBEYE. Despite loss of the entire ribbon structure, 24 synapses retained their spatiotemporal development and KO mice had a mild hearing 25 deficit. IHCs of KO had fewer synaptic vesicles and reduced exocytosis in response to 26 brief depolarization; high stimulus level rescued exocytosis in KO. SGNs exhibited a 27 lack of sustained excitatory postsynaptic currents (EPSCs). We observed larger 28 postsynaptic glutamate receptor plaques, potentially in compensation for the reduced 29 EPSC rate in KO. Surprisingly, large amplitude EPSCs were maintained in KO, while a 30 small population of low amplitude slower EPSCs was increased in number. The ribbon 31 facilitates signal transduction at physiological stimulus levels by retaining a larger 32 residency pool of synaptic vesicles. 33 34 35 Key words: auditory hair cell, synaptic transmission, RIBEYE, glutamate receptor, 36 multivesicular release 37 38 39 40 2 41 Introduction 42 The synaptic ribbon is an electron dense structure associated with presynaptic active 43 zones at sensory synapses in the retina, lateral line and inner ear organs. RIBEYE, the 44 major protein associated with the ribbon (Zanazzi and Matthews 2009), consists of a 45 unique amino-terminal A domain that forms the ribbon scaffold and a carboxy-terminal B 46 domain with lysophosphatidic acid acyltransferase activity which is almost identical to 47 CtBP2, a transcriptional co-repressor (Schmitz, Konigstorfer et al. 2000). The ribbon is 48 anchored to the presynaptic membrane by bassoon protein in mammalian IHCs 49 (Khimich, Nouvian et al. 2005) as well as retinal photoreceptors and bipolar cells (dick 50 2001). Although it is comprised predominantly of RIBEYE (Zenisek, Horst et al. 2004), 51 the ribbon complex may associate with over 30 different synaptic proteins (Uthaiah and 52 Hudspeth 2010, Kantardzhieva, Peppi et al. 2012). 53 IHCs respond to sound with graded receptor potentials that drive rapid and precise 54 synaptic release, encoding intensity and timing information (Fuchs 2005, Matthews and 55 Fuchs 2010). The ribbon is thought to sustain continuous encoding by tethering a pool 56 of vesicles that replenishes the local pool of docked and primed vesicles upon 57 depletion. Other functions ascribed to the ribbon include: regulating vesicle trafficking 58 from the cytoplasm to the <a href="/tags/Active_zone/" rel="tag">active zone</a>, serving as a vesicle trap to bind freely diffusing 59 vesicles, a location for vesicle formation, a physical barrier for Ca2+ ensuring a high 60 local Ca2+ signal, and as a primary structural component of the synapse anchoring Ca2+ 61 channels close to synaptic vesicle release sites (Parsons 1994, Frank, Rutherford et al. 62 2010, Graydon, Cho et al. 2011, Kantardzhieva, Peppi et al. 2012). 3 63 Postsynaptic EPSCs at ribbon synapses range in amplitude by up to 20X. The smallest 64 and largest EPSCs have similar kinetics, which together suggested the existence of a 65 mechanism to synchronize the release of multiple quanta (multivesicular release, MVR; 66 (Glowatzki and Fuchs 2002, Li, Keen et al. 2009, Schnee, Castellano-Munoz et al. 67 2013, Rudolph, Tsai et al. 2015). By holding vesicles and voltage-gated Ca2+ channels 68 at the active zone the ribbon may support MVR, occurring through various mechanisms 69 including: i) coordinated fusion of multiple single vesicles, ii) release of large pre-fused 70 vesicles, iii) rapid sequential fusion in which the 1st fusion event triggers fusion of 71 additional vesicles, or some combination of these mechanisms (Matthews and Fuchs 72 2010). 73 Previous attempts to study ribbon function used genetic disruption of the anchoring 74 protein Bassoon or acute destruction of the ribbon by fluorophore assisted light 75 inactivation (FALI). These manipulations resulted in smaller vesicle pool size, calcium 76 channel mislocalization, increased auditory thresholds, asynchrony in neural responses 77 and reductions in both tonic and phasic responses (Snellman, Mehta et al. 2011, Jing, 78 Rutherford et al. 2013). However, neither of those manipulations were selective for the 79 ribbon and so might be manifestations of accessory protein denaturing. 80 Recently, manipulation of the gene encoding CtBP2/RIBEYE allowed for deletion of 81 RIBEYE by removing the A-domain of ctbp2 to eliminate ribbons in mice, enabling a 82 more precise examination of ribbon function. This mouse will be referred to as KO and 83 the littermate controls as WT throughout the manuscript. Elimination of ribbons in the 84 retina reduced phasic and tonic transmission and rendered release sites sensitive to the 85 slow calcium buffer EGTA (Maxeiner, Luo et al. 2016). Here we present analysis of 4 86 cochlear synaptic anatomy, hair cell and afferent fiber physiology and hearing function 87 in the RIBEYE knockout mouse. Results are distinct from those in retina and from a 88 RIBEYE deletion in zebrafish (Lv, Stewart et al. 2016) , suggesting a more subtle 89 function for the ribbon than previously assumed. 90 Results: 91 IHC-Spiral Ganglia <a href="/tags/Neuron/" rel="tag">Neuron</a> (SGN) synapses are formed and maintained despite 92 absence of the ribbon: The inner ear structure of the KO was assessed first for the 93 presence of synaptic ribbons and synapses (Maxeiner, Luo et al. 2016). Validating loss 94 of hair cell ribbons was accomplished using immunohistochemistry for the RIBEYE B-95 domain (CtBP2). Anti-CtBP2 labelled the ribbons in wildtype (WT) but not in the KO 96 animals (postnatal day 21, P21)(Fig. 1A). Nuclear CtBP2 was still observed in both WT 97 and KO (not shown) because the KO is specific to the RIBEYE A-domain. The nuclear 98 staining served as a usful control for antibody function. 99 Using antibodies to Homer for postsynaptic labeling and Bassoon for presynaptic 100 labeling (Fig. 1B,C) we quantified the number of puncta and whether puncta were 101 juxtaposed as synaptic pairs (Fig. 1I,J). Bassoon and Homer puncta were similar in 102 number between WT and KO (Bassoon: 40 ± 15 in WT, vs. 43 ± 12 in KO, p = 0.76; 103 Homer: 17 ± 3 for both WT and KO; WT: n = 20 mice, KO: n = 19 mice, p = 0.64). The 104 number of paired Homer and Bassoon puncta (Fig. 1D) were similarly comparable: 105 14 ± 2 (n = 20) for WT and 13 ± 2 (n = 19, p = 0.18) for KO IHCs, suggesting that 106 synapses were formed and maintained in comparable numbers regardless of the 107 presence of ribbons. The higher number of Bassoon puncta likely reflects its localization 108 at efferent synapses. Figure 1- Supplemental Fig. 1 presents a lower magnification 5 109 image that illustrates the multiple roles of Bassoon at the auditory periphery. Bassoon is 110 a major component of the presynaptic elements at the auditory periphery including the 111 efferent fibers and the hair cell afferent synapses. Thus it is not surprising that paired 112 Bassoon puncta are much less than total puncta because Bassoon serves multiple 113 synapses. Similarly, when interpreting Bassoon KO data it is not surprising that its 114 phenotype may be more dramatic than simply the RIBEYE effect alone due to number 115 of synapses involved and schaffolding function of Bassoon that leads to structural 116 changes at the synapse. 117 RIBEYE KO in zebrafish revealed a non osmiophilic structure resembling a ribbon that 118 maintained organization of synaptic vesicles in the absence of the RIBEYE protein in 119 hair cells of the lateral line neuromasts (Lv, Stewart et al. 2016). We examined whether 120 such a structure existed in the mammalian cochlea in the IHCs of the KO using 121 transmission electron microscopy (TEM) and electron-tomography. Representative 122 examples of conventional TEM (Fig. 1E, 70 nm sections) and tomographic 123 reconstructions (Fig. 1F,G at 10 nm tomographic sections) are presented. The KO 124 presynaptic synapses consistently showed a small osmiophilic density in contact with 125 the pre-synaptic membrane at the center of the synapse that resembled the arciform 126 density seen at ribbon synapses in the retina. This density may be the Bassoon 127 enriched linker between the membrane and the ribbon. Interestingly, this density is not 128 observed in the Bassoon KO mouse (Frank et al., 2010). Tomographic reconstructions, 129 (Fig. 1G, right) clearly show the presynaptic density, which is smaller than the ribbon 130 and is present also at ribbon synapses in WT IHCs (Fig. 1G, left). We found no spatial 131 organization of vesicles near active zones in KO IHCs, suggestive of the ‘ghost’ ribbons 6 132 observed in the zebrafish lateral-line hair cells (Lv, Stewart et al. 2016). Thus, in the KO 133 mouse, IHCs lack synaptic ribbons and have no obvious compensatory structures. We 134 surmise that the differences observed in the zebrafish model are a function of the KO 135 being incomplete or there being a compensation mechanism in zebrafish, not found in 136 mammal. 137 KO IHCs may upregulate a similar protein to replace RIBEYE loss. CtBP1 is a likely 138 candidate because it is similar to the RIBEYE-B domain, is expressed in hair cells and 139 localizes to the synaptic region (Kantardzhieva, Liberman et al. 2013). Antibody labeling 140 (Fig. 1H,K,L) demonstrates the presence of CtBP1 in both WT and KO animals which 141 overlapped with Homer in WT. A reduction in puncta number was identified in the KO 142 animal due to loss of localization at the afferent synapses (Fig. 1K,L). Thus, the CtBP1 143 localization at the synapse requires the presence of RIBEYE, and CtBP1 does not 144 substitute for RIBEYE in the KO animal. 145 Synaptic vesicles are normal in size but fewer in number: Vesicle diameters 146 (distance between the outer edges of the bilayer in each vesicle) were measured in the 147 reconstructed tomograms. We measured in both X and Y directions with no differences 148 observed. In the WT the average vesicle diameter was 33.6 ± 3 nm (n = 21) and in the 149 KO it was 33.8 ± 3 nm (n = 21). 150 Synaptic vesicles were conspicuously fewer in number the KO. Vesicles were counted 151 in volumes around the center of the reconstructed synapses (Fig. 1G), where one 152 boundary was the presynaptic membrane. The volumes had XY dimensions of 453 ± 6 153 nm for WT (n = 3) and 569 ± 117 nm for KO (n = 11), and Z dimensions of 88 ± 3 and 154 95 ± 10 nm for WT and KO, respectively. Total vesicle numbers were 20 ± 2 for WT and 7 155 7 ± 3 for KO, significantly greater for WT compared with KO synapses (p < 0.001, Welch 156 correction for unequal variance). Our TEM data is qualitatively similar to that reported in 157 the companion paper and supports the conclusion that ribbons are not present in the 158 KO. 159 Auditory nerve activity is reduced in absence of synaptic ribbons: Previous 160 investigation of ribbonless synapses in the KO mouse focused on the retina and did not 161 assess function at the systems level (Maxeiner et al., 2016). To determine how hearing 162 was affected, we measured ABRs and distortion product otoacoustic emissions 163 (DPOAEs). DPOAEs are a direct measure of outer hair cell function and cochlear 164 amplification. DPOAEs are not expected to be affected by absence of RIBEYE and 165 serve as a control for developmental acquisition of mature cochlear mechanics. DPOAE 166 threshold measurements were not different across frequencies from 4 - 46 kHz (Fig. 167 2A). Data are presented in decibels (dB) as referenced to sound pressure level (SPL) 168 with lower values indicating enhanced sensitivity in the mid-cochlea. Normal DPOAEs 169 indicate that cochlear amplification was not effected mechanically by the loss of outer 170 hair cell ribbons, suggesting that sound-driven activation of IHCs was intact, and that 171 any differences in auditory nerve activity should result from changes at the IHC afferent 172 synapses. 173 Wave 1 of the ABR is a direct measurement of the synchronized firing of SGNs at 174 sound onset, triggered by synaptic output from cochlear IHCs. The subsequent waves 175 of the ABR reflect the ensuing activation of brainstem nuclei in the ascending auditory 176 pathway (Zheng, Henderson et al. 1999). ABR thresholds were elevated by 177 approximately 10 dB at all probe frequencies in KO mice (Fig. 2B), except for 48 kHz 8 178 where hearing threholds are already relatively high. The variance in our control data 179 ranged from 8-12dB depending on frequency; this dictated the sample size needed to 180 detect a 5% change as statistically different. ABR threshold was identified as the 181 stimulus intensity required to evoke a voltage response 5x the RMS noise floor for the 182 measurement. The companion paper found a similar elevation in threshold though it did 183 not reach statistical significance. The difference is likely due to sampling size where we 184 powered our sample sized based on variance found in control measurements. The ABR 185 waveform (Fig. 2C: 23 kHz, 60 dB) shows a large reduction in wave 1 amplitude with no 186 effect on the subsequent peaks. This was surprising and suggests considerable 187 plasticity in central processing of data translated from cochlea periphery. KO mice had 188 smaller wave 1 peak amplitudes, for all sound levels that evoked a response (Fig. 2D). 189 When wave 1 growth functions were plotted relative to the maximal response amplitude 190 within genotype, the amplitude versus level function for KO scaled up to overlap with 191 WT (Fig. 2E). Considering wave 1 amplitude as a fraction of the maximal amplitude, the 192 overlap of the normalized wave 1 growth curves suggested that loss of maximum output 193 could produce an elevation of threshold in the ABR measurement that does not 194 necessarily reflect a sound detection limit or a change in sensitivity of the auditory 195 system. Indeed, normal ABR amplitudes beyond wave 1 indicate compensation in the 196 cochlear nucleus. Future work should determine if KO mice have increased behavioral 197 thresholds. 198 In mice lacking Bassoon function, where IHCs lacked most of their membrane-anchored 199 ribbons, there was an increase in wave 1 latency (Khimich, Nouvian et al. 2005) and in 200 first-spike latency in recordings of SGNs in vivo (Buran, Strenzke et al. 2010). ABR 9 201 wave 1 latencies were not different between WT and KO at any stimulus intensity (Fig. 202 2F), suggesting a milder phenotype in the KO as compared to Bassoon. 203 As the ABR measurements are averaged responses of 260 stimulus repetitions, a 204 possible explanation for the reduced ABR wave 1 amplitude in KO is that the response 205 reduces over time. If the ribbon is providing a means of maintaining vesicles near the 206 synapse, the concomitant loss of vesicles at ribbonless synapses might reduce the 207 ongoing response to repetitive stimulation. Comparison of ABR amplitudes where the 208 number of repeats was varied from 260 to 1000 showed no difference in amplitudes, nor 209 did repeated measurements meant to evoke depletion.Thus, rundown alone is unlikely 210 to account for the decrease in ABR wave 1. 211 Characterization of molecular synaptic components indicates an increase in 212 postsynaptic glutamate receptor distribution: To begin identifying cellular 213 mechanisms that might underlie the measured reduction in ABR wave 1 amplitude we 214 investigated the distribution of presynaptic Ca2+ channels and postsynaptic glutamate 215 receptors as indicators of functional synapses. Figure 3 presents immunohistochemical 216 localizations of presynaptic ribbons with anti-CtBP2, presynaptic voltage-gated Ca2+ 217 channels with anti-CaV1.3, and postsynaptic AMPA-type glutamate receptors with anti-218 GluA3 in P35 mice organ of corti. There was no difference in the number of puncta 219 between WT and KO for CaV1.3 (20 ± 6, n = 9 vs. 18 ± 5, n = 13, p = 0.51) or GluA3 (15 220 ± 3, n = 9 vs. 15 ± 2, n = 13, p = 0.43) (Fig. 4A,B). There was also no difference in the 221 number of paired synaptic puncta between genotypes, with values of 14 ± 2 (n = 9) for 222 WT and 13 ± 2 (n = 13) for KO (p = 0.13). Thus, the presence of the ribbon did not 223 influence the number of apparently functional synapses per IHC. 10 224 Careful inspection of GluA3 immunoreactivity showed that the glutamate receptor 225 puncta were larger on average in the KO (Fig. 4D). Puncta volumes were measured for 226 presynaptic markers CaV1.3 and Bassoon as well as postsynaptic markers Homer and 227 GluA3 (Fig. 4C). Like GluA3, Homer puncta were significantly larger (KS-test, 228 p ≤ 0.0001) on average in the KO (Fig. 4D). The volumes of CaV1.3 and Bassoon 229 distributions were very similar among genotypes however the volume of Bassoon was 230 significantly higher in KO. Most WT and KO synapses exhibited the typical elongated 231 stripe-like appearance of CaV1.3 puncta. However, we noted a tendency for KO 232 synapses to have CaV1.3 clusters that were fragmented into smaller puncta (Fig. 3C, 233 arrow; see also Companion Paper). These anatomical data support the hypothesis that 234 the ribbon is not a major regulator of synapse formation or stability; however, it does 235 perhaps suggest that postsynaptic receptors are sensitive to activity and the increased 236 plaque size might reflect a compensation for reduced activity. 237 Basolateral IHC properties: We compared the properties of the IHC voltage-gated 238 Ca2+ current (Fig. 5), which drives exocytosis. We first blocked outward K+ currents 239 using Cs+ and tetraethylammonium (TEA) in the internal solution to isolate Ca2+ currents 240 in WT and KO IHCs (Fig. 5A,B). Current-voltage plots showed no difference in voltage 241 of half-activation (V1/2) or maximum current amplitude (Fig.5C-E). Half activation 242 voltages were -34 ± 0.3 mV (n = 18, WT) and -33.4 ± 0.3 mV (n = 26, KO) for animals 243 P10-P13. The companion paper found small changes in voltage dependent properties 244 of the calcium channels at older ages and in higher external calcium; differences that 245 might suggest compensatory effects of ribbon loss or later specializations being ribbon 246 dependent. Examples of current elicited with K+ as the major internal cation and without 11 247 TEA are presented in Fig. 5F,G. Recordings were made at P20-24. No differences were 248 observed in maximal current or in V1/2 (Fig. 5H). Zero-current potentials monitored in 249 current-clamp mode (Fig. 5I) were not different, with means of -63 ± 10 mV (n = 5, WT) 250 and -65 ± 8 mV (n = 8, KO). 251 Release is slower and smaller for naturalistic stimuli in absence of the ribbon: We 252 measured presynaptic exocytosis with the two-sine wave method for tracking vesicular 253 fusion by monitoring real time changes in cellular membrane capacitance (Cm) from 254 IHCs (Santos-Sacchi 2004, Schnee, Castellano-Munoz et al. 2011). The two-sine 255 method allows continuous monitoring of Cm during the depolarizing stimulus as 256 compared to a before and after monitoring of capacitance changes, which is particularly 257 helpful during smaller, longer depolarizations (Schnee, Santos-Sacchi et al. 2011). 258 Typically, small depolarizations lead to relatively slow, linear increases in Cm during 259 depolarization. In contrast, larger depolarizations induce an abrupt departure from 260 linearity termed the ‘superlinear’ response (Schnee et al. 2011). Both types of 261 responses were present in recordings from IHCs of WT and KO mice alike (Fig. 6A,B). 262 From Cm measurements, we estimated the sizes of synaptic vesicle pools, their rates of 263 release and the Ca2+-dependence of release. In response to strong depolarizations, we 264 found no difference in maximal release between genotypes for either the linear or 265 superlinear release components (Fig. 6C). The linear component was 52 ± 24 fF (n = 266 15, WT) compared to 42 ± 22 fF (n = 15, KO); and the superlinear component of release 267 was 223 ± 79 fF (n = 17, WT) compared to 253 ± 116 fF (n = 17, KO). Likewise, no 268 differences in release rates (Fig. 6D) were observed for the linear component: 101 ± 72 269 fF/s (n = 15, WT) compared to 75 ± 50 fF/s (n = 14, KO) or for the superlinear 12 270 component: 256 ± 164 fF/s (n = 17, WT) and 253 ± 117 fF/s (n = 18, KO). In addition, 271 the Ca2+-dependence of release for the linear component was not different (0.9 ± 0.03 272 fF/pC (n = 37, WT) as compared to 0.93 ± 0.03 fF/pC (n = 34, KO). The voltage at which 273 the superlinear component of release first appears was also not different between mice 274 (Fig. 6E). Fitting the data with a simple Gaussian function gave values of -40 ± 9 mV for 275 WT and -41 ± 3 mV for KO for the center values. However the width of the functions 276 were different, with values of 7.6 ± 2 for WT compared to 14.3 ± 3 for KO (values p< 277 0.002 level). Thus, although the variance in release properties increased for the KO 278 animal, there were no major differences in the ability of the synapses to respond to 279 strong stimuli. 280 In contrast to the comparable maximal responses, smaller depolarizations revealed a 281 reduction in release at early time points in the KO. The difference observed was 282 masked at longer time points or by stronger depolarization (Fig. 6F-J). Comparing 283 capacitance changes at short intermediate and longer time points in response to smaller 284 depolarizations showed that the KO consistently had reduced release as compared to 285 the WT data. Fitting the data in Fig. 6G,H with a simple exponential demonstrated that 286 although both genotyes reached the same values of release with strong depolarizations 287 the relationships varied in two ways. For the shortest time point release started at -40 288 mV as compared to -45 mV with all other variables remaining constant. Given that the 289 numbers for release are quite small and variable, conclusions should be conservative, 290 but support the argument that fewer vesicles are available for release at this early time 291 point and recruitment is minimal. For the longer time point Fig. 6H, the voltage-292 dependence (Cm/Vm) varied with the KO, 7.3 ± 3.6 for WT as compared to 12.7 ± 5.8 13 293 for KO. F-tests show these fits to be different at the p < 0.002 level. Longer time points 294 as depicted in Fig. 6I,J show no difference between groups. In addition, for smaller 295 depolarizations the delay to release onset was greater in the KO IHCs, consistent with a 296 reduction in vesicles immediately available for release (Fig 6K). For example, 297 depolarizations to -44 mV resulted in significantly shorter latency in WT (102 ± 98 ms, n 298 = 16) compared to KO (204 ± 166 ms, n = 17, p = 0.039). Stronger depolarizations (> -299 39 mV) resulted in no difference in release latency. The time constants of endocytosis 300 were not significantly different (6.0 ± 4s, n = 13, WT and 7 ± 5 s, n = 13, KO). Together 301 these data suggest that fewer vesicles were available for release at ribbonless 302 synapses, but that with stronger depolarizations or longer time courses of depolarization 303 the recruitment of additional vesicles masked this immediate difference in release 304 latency and amplitude. 305 The ribbon may be a barrier to Ca2+ diffusion, creating a restricted space for sharp Ca2+ 306 nanodomains beneath the ribbon (Roberts 1994, Graydon, Cho et al. 2011). 307 Intracellular Ca2+ buffering can alter release properties depending on the relative 308 distribution of Ca2+ channels with release sites. In retina the KO increases buffering 309 efficacy (Maxeiner et al. 2016), suggesting looser coupling between Ca2+ channels and 310 release sites. To investigate the effects of ribbon absence on stimulus-secretion 311 coupling in p11 to p13 IHCs, we compared between genotypes the effects on release 312 properties of changing concentration of the slow Ca2+ buffer EGTA (Fig. 7). Examples of 313 release obtained with either 0.1 or 5 mM EGTA are presented in Fig. 7A (control 314 experiments all use 1 mM EGTA). Although increasing buffer concentration reduced 315 release and lowering buffer increased release, the relative changes were comparable 14 316 between WT and KO mice for release measured at 1 sec (Fig. 7B). Data previously 317 demonstrated that increasing Ca2+ buffering increased the time to superlinear release 318 (Schnee, Santos-Sacchi et al. 2011). Fig. 7C demonstrates that this effect is retained in 319 the KO and that there are no major differences between WT and KO modulation by 320 Ca2+ buffering. These results contrast with findings from retinal bipolar cells and in the 321 zebrafish KO (Maxeiner, Luo et al. 2016); see Discussion). Similarly, release rates 322 measured for steps to -45 mV were not significantly different for the initial release 323 components (Fig. 7D). We further investigated the first component of release, typically 324 thought to represent vesicles in early release pools, not requiring trafficking. Comparing 325 release at two voltages again showed that the buffer greatly altered release properties 326 but not in a differential way between genotypes. However, it should be noted that the 327 difference in early release observed in Fig. 6 with 1 mM EGTA was lost with 0.1 mM 328 EGTA. Thus, it is possible that a sensitivity exists that is below a clear detection limit for 329 our capacitance measurements. 330 EPSCs are more heterogeneous in size and kinetics in the absence of the ribbon: 331 Cm measurements are a powerful tool for investigating whole-cell exocytosis but they 332 lack sensitivity (1 fF reflects fusion of ~20 vesicles), they reflect information from all 333 synapses and they can be quite variable. To obtain a more refined view of activity from 334 individual synapses we recorded postsynaptically, from the afferent fiber bouton. Hair 335 cells bathed in artificial perilymph lack the standing inward current through 336 mechanoelectric transduction channels in hair bundles, causing the resting membrane 337 potential to be relatively hyperpolarized (Farris, Wells et al. 2006, Johnson, Kennedy et 338 al. 2012). To depolarize hair cells and increase the rate of vesicle fusion, we applied a 15 339 high potassium (40 mM K+) solution to depolarize the hair cell. K+ application to the hair 340 cell in current-clamp mode depolarized hair cells by an average of 32 ± 5 mV (n = 8; 341 Fig. 8A). There was no difference in depolarization between genotype thus allowing 342 conclusions regarding postsynaptic responses to be ascribed to presynaptic effects. 343 Afferent boutons of type I auditory nerve fibers were recorded ex vivo at ages of P17-344 P21. Due to the approach vector of the recording pipette, recordings were made from 345 boutons located on the side of the IHC facing the ganglion (modiolar face) rather than 346 the pillar cell side. The start of the high K+ response was defined when the mean EPSC 347 rate exceeded the rate in normal external solution by 2 standard deviations. Examples 348 of postsynaptic responses to K+ applications are provided in Figure 8B-D. Two types of 349 responses were observed, sustained responses (Fig. 8B,C) and transient responses 350 (Fig. 8D). WT responses were mainly of the sustained type (7 of 8) while the KO 351 responses had a smaller proportion of sustained responses (7 of 11). EPSC rates are 352 plotted for transient (Fig. 8E) and sustained responses (Fig. 8F) for both WT and KO. 353 EPSC rates in 40 mM K+ perilymph were quantified over one second windows at 100 354 ms intervals and the means are displayed as thicker lines (Fig. 8F). The peak release 355 rates varied considerably across fiber populations (0-120 s-1) and there was a tendency 356 for the KO neurons to reach a lower peak EPSC rate on average: 57 ± 30 s-1, (n = 11, 357 WT) compared with 44 ± 31 s-1, (n = 18, KO) (Fig. 8G). 358 The time to peak EPSC rate was less variable than absolute rates (Fig. 8H). The KO 359 had one outlier that steadily increased throughout the K+ application, removal of this 360 point from the statistical analysis resulted in the KO reaching peak EPSC rate sooner (9 361 ± 10 sec, n = 10) than WT (21 ± 12 sec, n = 9; p = 0.037). Observing the sustained 16 362 release group shows that both WT and KO reduced EPSC rate over time but that the 363 KO reduced more completely than WT (Fig. 8G). 8/11 KO reduced to less than 5% of 364 peak as compared to 3/8 WT. Integrating the rates over time (Fig. 8I) confirmed an 365 increase in total EPSCs in WT (1.8 ± 1.1 x 103) compared to KO (0.81 ± 0.66 x 103, p = 366 0.021). 367 EPSC amplitude distributions were generated for each recorded fiber. Frequency 368 histograms of EPSC amplitudes could be fit with either a double Gaussian function or a 369 Gaussian and gamma function where the smallest peak was always fit by a Gaussian 370 function (e.g. Fig. 8J,K). The area under the smaller peak was calculated and plotted as 371 a percentage of the total in Fig. 8L, where the KO showed a statistically larger (p = 372 0.034) percentage of small events with 5.5 ± 5.6% (n = 12) as compared to 1 ± 0.7% (n 373 = 7). The increase in number of small events might suggest less coordination in release 374 because there are fewer vesicles filling release sites. It might also suggest that smaller 375 responses are visible because of an increased in receptor plaque size as well. Only 376 fibers with more than 100 EPSCs were included for this analysis. No difference was 377 found in the proportion of simple events for each unit, where a simple event is described 378 as having a single rise and decay phase (Fig. 8M). We also compared the median 379 amplitudes (Fig. 8N). Although there was a tendency for EPSCs in the KO to be greater 380 in amplitude 212 ± 46 pA (n = 13) compared to 173 ± 36 pA (n = 7), the values were not 381 statistically different. A difference here would support the argument of a postsynaptic 382 compensatory increase in glutamate receptor plaque size. The coefficient of variation 383 (CV) for the amplitude distributions were different on average (p = 0.017; Fig. 8O). The 384 control values are similar to those obtained by (Li, Keen et al. 2009, Grant, Yi et al. 17 385 2010). Recordings with a larger proportion of smaller events had larger CV. When small 386 events were removed from the histogram, the CVs were not statistically different 387 between KO and WT. The postsynaptic responses in KO also had a greater proportion 388 of EPSCs > 350 pA (Fig. 8J,K, 9D): 1.3 ± 1.3% in WT compared to 8.0 ± 10% for KO 389 EPSCs (t-test for unequal variances, p = 0.04). The change in amplitude was not 390 accompanied by a change in kinetics. The increased amplitude might reflect the 391 increase in postsynaptic glutamate receptor plaque (Fig. 3,4). 392 Examples of EPSCs are presented in Fig. 9A to show the variation in amplitudes and to 393 identify a smaller population of EPSCs with slower kinetics (see inset). As the ribbon is 394 proposed to be involved with multivesicular release (MVR), and the kinetic similarities 395 between large and small EPSCs are a signature of MVR, we investigated the decay of 396 EPSCs in auditory nerve fiber recordings from WT and KO animals. Fitting the EPSC 397 decay with single exponentials provided time constants (Fig. 9B). Calculating the CVs of 398 EPSC decay time constants (Fig. 9C) revealed more variability in KO but no significant 399 difference compared to WT. The skewness (Fig. 9F) of the decay time constant 400 distributions was significantly larger for the KO (0.46 ± 0.2, n = 13) compared to WT 401 (0.29 ± 0.07, n=7; p = 0.04); the KO mean EPSC decay time was greater than the 402 median decay time. As a population 0.44% of WT EPSCs were < 100 pA in amplitude 403 with decay time constants > 1 ms while 1.9% of KO events fell in that category. Plotting 404 the decay time constant against amplitude for each EPSC in all fibers (Fig. 9D) (WT: n = 405 7 recordings, 15,564 EPSCs; KO: n = 13 recordings, 17,210 EPSCs) identifies a 406 population of small slow EPSCs in the KO that are present in greater proportion than in 407 WT (upper left quadrant). The mean EPSC decay time constants plotted as a function of 18 408 EPSC amplitude for each recording (bin of 10 pA) illustrates that all the KO fibers had a 409 proportion of EPSCs with slow time courses while the WT have only 2 fibers with these 410 slower events (Fig. 9E). An example of the smaller, slower EPSC is provided in the 411 inset of Fig. 9A. The distributions of EPSC durations for all EPSCs < 100 pA (Fig. 9G,H) 412 demonstrates the shift toward slower longer duration EPSCs in KO fibers (p > 0.001, 413 using the Kolmogorov-Smirnov test). The cumulative probability functions of EPSC 414 duration for all fibers by genotype (thick red and blue lines) illustrates the larger 415 proportion of slow EPSCs in the KO fibers (Fig. 9H). These data may also support the 416 argument for a larger postsynaptic glutamate receptor plaque, where diffusion of 417 glutamate, coupled with the lower glutamate concentration might serve to slow the 418 decay kinetics. 419 Discussion: 420 The KO mouse provides an unprecedented opportunity to investigate the functional 421 relevance of synaptic ribbons in driving information transfer at the primary auditory 422 synapse. Synapse formation and stability appeared normal based on synapse counts 423 and protein localization, despite complete absence of the ribbon (Figs. 1, 3-4). No 424 evidence was found for partial or defective ribbons structures, nor did we detect 425 compensation by other proteins in the absence of RIBEYE (Fig. 1). Also, cochlear 426 mechanics appeared normal as assessed by DPOAE (Fig. 2). Thus, this mouse 427 provides an excellent model to investigate the ribbon’s role in synaptic transmission. 428 Whole animal function: ABR responses reported a 10 dB threshold shift and about a 429 50% reduction in ABR amplitude with no significant effect on the shape of the 430 waveforms or on the delay (Fig. 2). These data agree qualitatively with the companion 19 431 paper data, the lack of significance likely being a function of the sample size. The minor 432 auditory deficit may not be expected to produce a change in behavioral sound detection 433 threshold as the ensuing ABR peaks, representing brainstem nuclei, were unaffected. 434 Alternatively, central compensation might occur that mask peripheral deficits. These 435 data suggest that the ribbon is not essential for setting hearing threshold, and that its 436 role in auditory function is more nuanced than for example otoferlin which when absent 437 or altered leads to deafness with threshold elevations of more than 100 dB (Roux, 438 Safieddine et al. 2006, Pangrsic, Lasarow et al. 2010). Further work is needed to better 439 understand ribbon function at the systems level. Additional testing to better investigate 440 auditory processing such as sound localization experiments may provide additional 441 insight into how the ribbon might serve systems level function. 442 Synapse formation: Synapse formation is surprisingly normal with appropriate number 443 of synapses and synaptic markers present (Figs. 1,4). Little change in Ca2+ channel 444 distribution or biophysics were observed in the KO (Fig. 5) relative to Bassoon 445 disruption, suggesting that Ca2+ channel distribution or regulation does not depend 446 critically on the ribbon (Frank, Rutherford et al. 2010). Our companion paper identifies 447 minor changes in Ca2+ channel distribution and voltage sensitivity at later ages; whether 448 these are a function of ribbon absence or activity dependent modulation remains to be 449 explored. The lack of effect on calcium channel properties and distributions at early 450 ages supports the hypothesis of a compensatory mechanism occurring later during 451 synapse maturation. Given the reports of late development changes in ribbon size and 452 glutamate receptor plaque sizes, activity driven shaping of the synapses is a reasonable 453 expectation (Fernandez, Jeffers et al. 2015). Postsynaptically, both Homer and the 20 454 glutamate receptors had larger volumes in the KO animal than in WT (Fig. 4). This 455 change also may be an indication of activity dependent synaptic shaping where the 456 reduced activity in the KO animal results in a postsynaptic up regulation of receptors. 457 The increase in the population of larger amplitude EPSC’s in KO animals further 458 supports this conclusion (Figs 8I,J, 9D). 459 Comparison to other systems: In zebrafish a frameshift mutation was used to 460 eliminate ribeye from ribbon synapses. This work revealed ghost like ribbons where 461 vesicles were organized about invisible ribbon-like structures (Lv C et al.,2016). Neither 462 our investigations, nor the companion paper nor the original work investigating the 463 retina (Maxeiner et al., 2015) found comparable ghosts in this mouse. We would 464 conclude that either the frame shift approach did not account for all ribeye or there is a 465 strong compensatory mechanism in zebrafish that is not found or is much weaker in 466 mammal. We investigated CTBP1 as a potential compensatory molecule but as Fig. 1 467 shows there is no labeling near synapses in the KO mouse. The presence of ribbons in 468 photoreceptors of zebrafish support the idea that there are fundamental differences 469 between zebrafish and mammalian systems. Additionally, work in zebrafish suggests a 470 tight reciprocal relationship between calcium channel numbers, calcium current 471 amplitude and ribbon size (Lv, Stewart et al. 2016). As no changes in calcium channel 472 distribution or numbers were observed in the KO animal despite the loss of the ribbon, 473 this relationship does not appear to exist in mammal or is greatly reduced, again 474 supporting fundamental differences between model organisms. 475 Mechanism of ABR amplitude reduction: The decrease in ABR amplitude is likely a 476 function of the reduced frequency of EPSCs leading to fewer action potentials 21 477 generated (Fig. 8F). In addition, the increase in proportion of EPSCs with lower 478 amplitudes and longer durations (Fig. 9H) is expected to increase the likelihood of spike 479 generation failure (Rutherford, Chapochnikov et al. 2012). As larger EPSCs are better 480 phase-locked than smaller EPSCs (Li, Cho et al. 2014) and present works finds a 5-481 10% increase in smaller EPSCs one might predict a lower vector strength and reduced 482 phase locking. Our companion paper in fact does observe these differences in single 483 unit firing. Similarly the increased population of smaller EPSCs is reminiscent of 484 observations in young animals when compared to older animals and so might reflect a 485 developmental delay in the KO animal. Fewer fibers fire together at sound onset, 486 reducing auditory nerve synchrony and wave 1 amplitude (Fig 2C,D). Measurements of 487 exocytosis from IHCs in response to small depolarizations, clearly showed a reduction 488 in release and an increase in time to release in the KO (Fig. 6E,F). This suggests a 489 decrease in vesicles available at the synapse for immediate release (supported by TEM 490 counts). The reduction in available vesicles for release as well as the population of 491 reduced amplitude EPSCs might also predict a spike timing deficit as described in the 492 companion paper. However, the more rapid response in WT was masked by stronger 493 stimulation (Fig. 6F), indicating that vesicle replenishment remained robust despite the 494 absence of the ribbon-associated synaptic vesicle pool. Given the robust nature of 495 vesicle trafficking, the primary role for the ribbon maybe in ensuring a population of 496 vesicles is present for the timing of onset responses, whereas vesicle trafficking can 497 serve to replenish vesicle populations during sustained release. A similar result was 498 reported by (Maxeiner, Luo et al., 2016) in retina. 22 499 Postsynaptic recordings suggest that the KO reaches peak release sooner than WT and 500 to a similar peak rate, implying that vesicle numbers are comparable for immediate 501 release. In contrast, TEM suggests fewer vesicles available at the synapse and the 502 small increase in population of small EPSCs also suggests a reduction in filled release 503 sites. Thus, it is possible that the ribbon acts as a barrier for vesicle interaction with the 504 release site while simultaneously serving to increase the total vesicles number 505 available. Interestingly, this idea was first suggested by (Furukawa and Matsuura 1978) 506 and later supported by (Schnee, Castellano-Munoz et al. 2013) as a possible 507 mechanism for explaining neural adaptation. However, the companion paper suggests 508 modifications at the synapse that might also be in accord with the postsynaptic 509 measurements here as well, i.e. more vesicles at the membrane. If there is an increase 510 in presynaptic density area and more vesicles per active zone, it might be predicted that 511 faster immediate release occurs but sustainability might be reduced, EPSC data also 512 supports this idea. To resolve these possibililities we need a better understanding of the 513 molecular underpinnings of the active zone and number of available release sites. 514 Synaptic vesicle replenishment for sustained release: Our data would argue against 515 a role for the ribbon in vesicle formation and replenishment. Total exocytosis, from both 516 the linear and superlinear components, was not reduced in the KO, implying a similar 517 total number of vesicles that could be recruited for release (Figs. 6A,B and 7A). In 518 contrast to zebrafish lateral line (Lv, Stewart et al. 2016), we measured no change in 519 vesicle size between KO and WT, indirectly supporting our view that the ribbon is not 520 regulating or involved in vesicle formation, a result corroborated by the companion 23 521 paper. Thus, no change in vesicle size, or maximal release supports the conclusion that 522 the ribbon is not involved in vesicle formation. 523 Ca2+ influx and coupling to exocytosis: The ribbon may act as a barrier to Ca2+ 524 diffusion, creating a higher level of local [Ca2+] to synchronize vesicle fusion of multiple 525 vesicles (Graydon, Cho et al. 2011). In this case, the ribbon’s presence would reduce 526 the sensitivity of exocytosis to exogenous Ca2+ buffers (by elevating concentrations of 527 Ca2+). Likewise in retina from KO mice, EGTA reduced spontaneous release in the KO 528 but not in WT (Maxeiner, Luo et al., 2016). In contrast, our data shows that buffering 529 efficacy was not altered in the KO (Fig. 7), suggesting the ribbon does not play a major 530 role in regulation of intracellular Ca2+ concentration at IHC active zones. The similar 531 response between WT and KO might indicate a small effect though at a level below a 532 clear detection limit for capacitance measurements. It is possible that the geometric 533 differences and synaptic architecture between retina and cochlea alter the sensitivity to 534 Ca2+ buffers or that the effect noted in retina is indirect, for example EGTA-AM might 535 alter the presynaptic cell’s resting potential. Thus, our results are not fundamentally 536 different from (Maxeiner, Luo et al., 2016), both supporting the idea that the ribbon 537 serves to consolidate vesicles near to release sites as suggested by (von Gersdorff, 538 Vardi et al. 1996). 539 Ribbon and Multiquantal release: The underlying mechanism for multiquantal release 540 remains an open question but might be driven by simultaneous multiquantal or 541 multivesicular release (Glowatzki and Fuchs 2002). The majority of EPSCs measured in 542 the KO remain large and uniformly fast in onset and decay times and the EPSC 543 amplitude is not reduced suggesting that the ribbon does not play a pivotal role in 24 544 multivesicular release. Thus, mechanisms that involve sequential fusion of vesicles 545 along the ribbon and then release or rapid sequential vesicle release that piggyback 546 onto a release site from the ribbon are unlikely to be valid as these should be 547 dramatically altered by the loss of the ribbon. One remaining possibility is the 548 synchronous release of vesicles from multiple release sites. Although the ribbon is not 549 required for this mechanism, it may indirectly modulate this mechanism by influencing 550 the number of docked vesicles available for release. The increase in number of smaller 551 slower EPSCs likely arising from fewer docked vesicles available for synchronous 552 release may support a ribbon role; however, the small relative contribution of this 553 vesicle pool indicates a minor role. The phenomenon of large EPSC amplitude 554 variability may still be explained by a univesicular mechanism including a dynamic 555 fusion pore, which presumably would not require a ribbon (Chapochnikov, Takago et al. 556 2014). Where identified, the slow footlike response described by (Chapochnikov, 557 Takago et al. 2014) was not different between genotypes. 558 Comparison to Bassoon KO: In mice, the effects of KO are subtle compared with 559 disruption of Bassoon protein. Loss-of-function mutation in Bassoon led to a reduction in 560 the number of IHC ribbons associated with presynaptic elements, ABR threshold 561 elevation of 30 dB, longer ABR wave 1 latency and first-spike latency, and fewer Ca2+ 562 channels at active zones (Khimich, Nouvian et al. 2005, Buran, Strenzke et al. 2010, 563 Frank, Rutherford et al. 2010, Jing, Rutherford et al. 2013). Thus, the effects of loss of 564 Bassoon protein (and membrane-anchored ribbons) were greater than loss of RIBEYE 565 protein, consistent with a more critical role for Bassoon in scaffolding synaptic proteins. 566 Also given Bassoon’s presence at efferent synaptic endings in the organ of Corti, it is 25 567 not surprising that the functional loss of Bassoon has a greater phenotype than that of 568 RIBEYE (Supplemental Fig. 1). 569 Summary: A central tenet for ribbon function is maintaining a large pool of vesicles 570 near the synapse. The ribbon may serve as a conveyor belt to both maintain and to 571 traffic vesicles to release sites (Parsons 1994, Nouvian, Beutner et al. 2006). Our data 572 suggests the ribbon increases the time a vesicle remains in the synaptic region. Robust 573 vesicle trafficking and replenishment either via recycling or transporting new vesicles to 574 the synaptic zone provides vesicles to the synaptic zone, but the time vesicles remain 575 near to release sites may be shortened in the absence of the ribbon serving to maintain 576 them close to the synapses. A model for this basic idea was proposed (Graydon, Zhang 577 et al. 2014) that reproduced data from bipolar cell synapses. The addition of a more 578 robust trafficking mechanism may allow this model to also reproduce hair cell synapses. 579 The increase in variance, whether measured as CV, skewness or simply standard 580 deviation in measured parameters such as time to release, release rate and EPSC 581 amplitude was greater for KO. The increased variance may simply reflect a nonuniform 582 population of vesicles near the synapse in the absence of the ribbon. Thus, the ribbon 583 may serve as a biological buffer, maintaining a uniform population of vesicles near 584 release sites. 585 Materials and Methods 586 Animals 587 Mice of both sexes from a mixed genetic background (C57bl6, 129/Ola) were bred from 588 heterozygous breeding pairs and genotyped by PCR for the knockout of the A-domain 589 in Ctbp2 (Maxeiner et al., 2016) while wild-type littermates served as controls. The 26 590 Administrative Panel on Laboratory Animal Care at Stanford University (APLAC 591 #14345) and Animal Care and use Committee and Washington University in St. Louis 592 approved all animal procedures. Experiments were performed blinded to genotype. 593 Auditory measurements: We measured auditory brainstem responses (ABRs), 594 distortion products of optoacoustic emissions (DPOAEs) in mice of postnatal day 21 as 595 previously described (Oghalai, 2004). Mice were anesthetized by administering 596 100 mg/kg ketamine and 10 mg/kg xylazine intraperitoneally and their body temperature 597 held constantly at 37°C (FHC, DC-temperature controller and heating pad) until they 598 fully recovered. 599 Sound stimuli were generated digitally by a self-written software (MATLAB 2009b, 600 MathWorks), controlling a custom-built acoustic system using a digital-to-analog 601 converter (National Instruments, NI BNC-2090A), a sound amplifier (TDT, SA1), and 602 two high frequency speakers (TDT, MF1). The speakers were connected to an ear bar 603 and calibrated in the ear canal prior to each experiment using a probe-tip microphone 604 (microphone type 4182, NEXUS conditioning amplifier, Brüel and Kjær). 605 Auditory Brainstem Responses (ABRs) were recorded by placing three needle 606 electrodes subcutaneously at the vertex, below the left ear, and a ground electrode 607 close to the tail. The signals were amplified 10,000 times using a biological amplifier 608 (Warner Instruments, DP-311) digitized at 10 kHz, and digitally band-pass filtered from 609 300 to 3,000 Hz. The stimulus for eliciting the ABR was a 5 ms sine wave tone pip with 610 cos2 envelope rise and fall times of 0.5 ms. The repetition time was 50 ms, and 260 611 trials were averaged. 27 612 At each frequency, the peak to peak voltages of ABR signals at stimulus intensities 613 ranging from 10 to 80 dB sound pressure levels (SPL) were measured in 10 dB steps 614 and fitted and interpolated to find threshold five standard deviations above the noise 615 floor. For statistical purposes we defined threshold as 80 dB SPL, if no ABR was 616 detected. 617 Distortion Products of Otoacoustic Emission (DPOAE): For stimulation of DPOAEs 618 two sine wave tones of equal intensity (l1=l2) and 1-second duration were presented to 619 the ear. The tones ranged from 20 to 80 dB SPL attenuated in 10 dB increments and 620 the two frequencies (f2 = 1.2f1) ranged from f2 with 4 to 46 kHz. The acoustic signal was 621 detected by the microphone in the ear bar was sampled at 200 kHz and the magnitude 622 of the cubic distortion product (2f1-f2) determined by FFT. Averaging 20 adjacent 623 frequency bins surrounding the distortion product frequency determined the noise floor. 624 DPOAE thresholds were reached when the signal was greater than three standard 625 deviations above the noise floor. 626 Tissue Preparation, Staining and Confocal Microscopy: For staining of pre- and 627 postsynaptic markers we used antibodies against Homer (Synaptic Systems, 160003, 628 1:800 dilution), Bassoon (AbCam, ab82958, 1:500 dilution), CtBP1 (BD-Biosciences, 629 612042, 1:600 dilution), CtBP2/RIBEYE (Santa Cruz Biotechnology, sc-5966, 1:300 630 dilution; BD Transduction Lab, 612044, 1:400), CaV1.3 (Alomone Labs, ACC-005, 1:75), 631 GluA3 (Santa Cruz Biotechnology, sc-7612, 1:200 dilution). Cochlea of P22 or P35 mice 632 were isolated in HBSS solution or artificial perilymph, a small hole was introduced at the 633 apex, cochlea were gently perfused through round and oval windows with freshly 634 prepared 4 % (w/v) PFA (Electron Microscopy Sciences) in PBS until the fixative 28 635 became visible at the apex. Organs were incubated in the same fixative solution for 30 636 min at room temperature. Whole mount organs of Corti were micro dissected, the 637 tectorial membrane removed, the tissue permeabilized for 30 min in 0.5 % (v/v) 638 Triton X-100/PBS, blocked for 2 h in 4 % (w/v) BSA/PBS at room temperature. Staining 639 with all primary antibodies was performed overnight at 4°C in the same blocking buffer. 640 For staining with antibodies against GluA2 and CaV1.3 the protocol was slightly varied: 641 fixation was 20 min at 4°C, followed by decalcification in 10 % EDTA (Sigma) for 2 h, 642 permeabilization and blocking was carried out in a blocking buffer containing 16% 643 donkey serum and 0.3 % Triton X-100. After washing all species appropriate secondary 644 antibodies (Alexa Fluor 488, 546 or 555, 647: 1:600 dilution in 4 % (w/v) BSA/PBS) 645 were applied for 1 h at room temperature. After washing, the organs of Corti were 646 mounted hair bundles facing upward in ProLong Gold© Antifade (Life Technologies). 647 Samples were imaged using confocal microscopy (LSM700 or LSM880, Zeiss) on 1.4 648 NA oil immersion objectives for confocal imaging, and 1.2 NA water immersion objective 649 for Airyscan imaging. The optimal voxel size was adjusted to the green channel 650 (confocal x/y/z: 100/100/350 nm; Airyscan x/y/z: 33/33/222 nm). RIBEYE WT and KO 651 samples were prepared in parallel and images were collected with identical settings. 652 All images were taken in the apical region, between 200-1000 µm from the apex, of the 653 organ of Corti. 654 Image analysis 655 For three-dimensional analysis of synaptic punctum volume, intensity and 656 colocalization, we used the Imaris software package (Version 8.4, Bitplane). Puncta 657 were initially detected using the spot algorithm allowing for different spot sizes and 29 658 elongated point spread function in z-direction (initial seed size x-y/z: CtBP1, Ctbp2, 659 Homer, 0.44/1.13 µm; Bassoon 0.37/1.13 µm; GluA3 0.7/1.4 µm; CaV1.3 0.56/1.4 µm), 660 followed by co-localizing the spots with a threshold lower than 1. All counts were 661 normalized to the number of Inner hair cells in a region of interest. 662 To estimate the size of synaptic puncta we used the surface algorithm in Imaris. For all 663 stainings, the surface detail was set to 0.15 µm, background subtraction to 0.562 µm, 664 and touching object size was 0.750 µm (Bassoon puncta were split at 0.36 µm). 665 TEM and Tomography 666 Temporal bones of P21 mice (WT: n = 5, KO: n = 5) were isolated in HBSS, a small 667 hole was introduced at the apex of the cochlea, gently perfused through round and oval 668 windows with freshly prepared 4 % (w/v) PFA, 2 % (w/v) Glutaraldehyde (Electron 669 Microscopy Sciences) in a buffer containing (in mM) 135 NaCl, 2.4 KCl, 2.4 CaCl2, 4.8 670 MgCl2, 30 HEPES, pH 7.4 until the fixative became visible at the apex. Temporal bones 671 were soaked for 4 hours in the same buffer and shipped in PBS. Cochleae were 672 microdissected out from fixed adult mouse temporal bones and slowly equilibrated with 673 30% glycerol as cryoprotection. Tissues were then plunge frozen in liquid ethane 674 at -180C using a Leica Biosystems grid plunger. Frozen samples were freeze 675 substituted using Leica Biosystems AFS in 1.5% uranyl acetate in absolute methanol 676 at -90C for 2 days and infiltrated with HM20 Lowicryl resin (Electron Microscopy 677 Sciences) over 2 days at -45C. Polymerization of the resin was carried out under UV 678 light for 3 days at temperatures between -45C and 0C. Ultrathin sections at 100 nm 679 were cut using a Leica ultramicrotome and collected onto hexagonal 300-mesh Cu grids 680 (Electron Microscopy Sciences). 30 681 Samples were imaged using a 200kV JEOL 2100 with a Gatan Orius 832 CCD camera. 682 Single images were acquired using DigitalMicrograph (Gatan). Tilt series were acquired 683 using SerialEM (Mastronarde 2005) at 1 tilt increment from -60 to +60 and 684 tomographic reconstruction of tilt series and segmentation of tomograms was done with 685 IMOD software suite (Kremer, Mastronarde et al. 1996). Fiji was used to generate 686 projection images from tomograms and for cropping, rotating and adjusting the 687 brightness and contrast of images. 688 Hair cell recordings: 689 IHCs from the apical coil of the organ of Corti were dissected from p10-13 Ctbp2 690 littermates of either sex, placed on a plastic coverslip and secured with two strands of 691 dental floss. Cells were visualized with a water immersion lens (100x) attached to a 692 BX51 Olympus upright microscope. Patch-clamp recordings were obtained at room 693 temperature (21-23 C) in an external solution containing in (mM) NaCl 140, KCl, 5.4, 694 CaCl2, 2.8, MgCl2 1, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) 10, D-695 glucose 6, creatine, ascorbate, Na pyruvate 2, pH 7.4 adjusted with NaOH. 100 nM 696 apamin was included to block the sK K+ channel. Cesium at 3 mM was used externally 697 to reduce inward leak current. Pipette solution contained (mM) CsCl 90, TEA, 698 tetraethylammonium chloride 13, 3 NaATP, MgCl2 4, creatine phosphate 5, ethylene 699 glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), HEPES 10, pH 700 adjusted with CsOH to 7.2. 701 Two-Sine Method 702 Hair cells were voltage-clamped with an Axon Multiclamp 700B (Axon Instruments-703 Molecular Devices, Sunnyvale, CA.) and data recorded at 200-300 kHz with a National 31 704 Instrument USB-6356 D/A acquisition board and JClamp software (SciSoft). Vesicle 705 fusion was determined by monitoring membrane capacitance as a correlate of surface 706 area change. Capacitance was measured with a dual sinusoidal, FFT-based method 707 (Santos-Sacchi 2004). In this RC analysis method, two voltage frequencies, f1 and f2 708 (twice the f1 frequency) are summed and the real and imaginary components of the 709 current response are used to determine the magnitudes of the three model components. 710 The time resolution of the Cm measurement is the period of f1, which we varied from 711 0.16 to 0.32 ms, corresponding to 6250 to 3125 Hz. Stimulation protocols were 712 performed after 10 minutes in the whole-cell mode to allow Ca2+ current run up and full 713 exchange of the internal solution into the cell (Schnee, Castellano-Munoz et al. 2011). A 714 dual sinusoid of 40 mV amplitude was delivered superimposed to the desired voltage 715 step. The mean IHC Cm was 10 ± 1 pF N = 25 and uncompensated series resistance of 716 10 ± 3 M. All voltages were corrected for a 4 mV junction potential. 717 Postsynaptic Recordings 718 Whole-cell voltage-clamp recordings were made at terminal endings of SGNs from P17-719 21 animals (Grant, Yi et al. 2010). Cochlea were acutely dissected from the temporal 720 bone in chilled (~ 4°C) standard extracellular solution containing 1 mM curare. The 721 apical turn was excised after removing stria vascularis and tectorial membrane, and 722 placed under two dental floss fibers on a recording chamber filled with the standard 723 extracellular solution at room temperature. Recording pipettes were made from 1.5 mm, 724 thick-wall borosilicate glass (WPI). Pipettes were pulled using a multistep horizontal 725 puller (Sutter) and fire polished while applying a pressure to the back end of pipettes to 726 an impedance of 6-8 MΩ (Johnson, Forge et al. 2008). When whole-cell recording was 32 727 achieved from a bouton, 5 µM TTX was perfused into the chamber, and EPSCs were 728 recorded in the standard solution (5.8 mM K+) for at least 3 min, then in 40 mM KCl for 729 1-5 min (depending on the duration of synaptic activity on each nerve). Standard 730 external solution contained (in mM): 5.8 KCl, 140 NaCl, 0.9 MgCl2, 1.3 CaCl2, 0.7 731 NaH2PO4, 5.6 D-glucose, 10 HEPES, 2 Ascorbate, 2 Na-Pyruvate, 2 Creatine 732 Monohydrate, Osmolarity was 305 mosmls, pH = 7.4 (NaOH). Intracellular solutions 733 contained (in mM): 135 KCl, 3.5 MgCl2, 0.1 CaCl2, 5.0 EGTA, 5.0 HEPES, 2.5 Na2-ATP, 734 pH = 7.2 (KOH), osm ~290 mosmls. 735 Data Analysis: 736 Data were collected blinded to genotype for the experimentalist. Genotype was revealed 737 during analysis phase to ensure appropriate n values were obtained between groups. 738 Although gender was noted, no gender differences were observed between groups and 739 so data is presented combined. Data analysis was largely performed using unpaired 740 two-tailed t-tests unless otherwise stated. When non normal distributions were 741 observed, as in the EPSC histograms we selected different comparators as described 742 with the data. Similarly when large n values would bias comparisons, such as with 743 analyzing immunohistochemistry data, we moved to alternative more appropriate tests 744 as described in the text. Data is presented as mean ± standard deviation unless 745 otherwise stated. Sample size is presented in legends or in text. For physiology 746 experiments sample size reflects cell numbers which also indicates animal numbers as 747 we did not record from more than one cell in a preparation. For immunohistochemistry, 748 data is presented for animal numbers as well as image, cell and puncta numbers. 33 749 The voltage against capacitance plots were arbitrarily fit with an exponential equation of (-x(x-x )/t) 750 the form: Y = Y0 + A1 exp 0 where: Y0 is the baseline change in capacitance and is 751 near 0, A1 is the asymptote for the maximal change in capacitance, x0 is the voltage 752 where changes in capacitance measurements were first noted and t reflects the voltage-753 dependence of the release (Cm/Vm). The equation for the Gaussian fit used in Fig. (-2*((x-x )/w)^2) 754 6E is: Y = Y0 + (A/(w*√(π/2)))*exp c where Y0 is baseline, typically 0, A is the 755 area under the curve, w is the width and xc is the peak of the Gaussian. 756 Acknowledgments 757 This work was supported by National Institutes of Health, National Institute on Deafness 758 and Other Communication Disorders (NIDCD), Grants R01DC014712 to M.A.R.; M.A.R. 759 was also supported by an International Project Grant from Action on Hearing Loss. 760 NIDCD; F32 4F32 DC013721 grant from NIDCD to MN, Intramural Research Program 761 (Z01-DC000002) and NIDCD Advanced Imaging Core ZIC DC000081) to W.S. and 762 B.K.: RO1DC009913 to AJR, core grant P30 44992. We thank Autefeh Sajjadi for her 763 help with genotyping animals and basic husbandry. We thank Bill Roberts for comments 764 on the manuscript. 765 The authors declare no competing financial interests 766 References 767 Buran, B. N., N. Strenzke, A. Neef, E. D. Gundelfinger, T. 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Pujol, S. tom Dieck, A. Egner, E. D. Gundelfinger and T. 833 Moser (2005). "Hair cell synaptic ribbons are essential for synchronous auditory 834 signalling." Nature 434(7035): 889-894. 835 836 Kremer, J. R., D. N. Mastronarde and J. R. McIntosh (1996). "Computer visualization of 837 three-dimensional image data using IMOD." J Struct Biol 116(1): 71-76. 838 37 839 Li, G. L., S. Cho and H. von Gersdorff (2014). "Phase-locking precision is enhanced by 840 multiquantal release at an auditory hair cell ribbon synapse." Neuron 83(6): 1404-1417. 841 842 Li, G. L., E. Keen, D. Andor-Ardo, A. J. Hudspeth and H. von Gersdorff (2009). "The 843 unitary event underlying multiquantal EPSCs at a hair cell's ribbon synapse." J Neurosci 844 29(23): 7558-7568. 845 846 Lv, C., W. J. Stewart, O. Akanyeti, C. Frederick, J. Zhu, J. Santos-Sacchi, L. Sheets, J. 847 C. Liao and D. Zenisek (2016). 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Strenzke, N. Brose, U. Muller, E. Reisinger and T. Moser 866 (2010). "Hearing requires otoferlin-dependent efficient replenishment of synaptic 867 vesicles in hair cells." Nat Neurosci 13(7): 869-876. 868 869 Parsons, T. D. (1994). "Calcium-triggered exocytosis and endocytosis in an isolated 870 presynaptic cell: capacitance measurements in sacucular hair cells." Neuron 13: 8. 871 Roberts, W. M. (1994). "Localization of calcium signals by a mobile calcium buffer in 872 frog saccular hair cells." J Neurosci 14(5 Pt 2): 3246-3262. 873 874 Roux, I., S. Safieddine, R. Nouvian, M. Grati, M. C. Simmler, A. Bahloul, I. Perfettini, M. 875 Le Gall, P. Rostaing, G. Hamard, A. Triller, P. Avan, T. Moser and C. Petit (2006). 876 "Otoferlin, defective in a human deafness form, is essential for exocytosis at the 877 auditory ribbon synapse." Cell 127(2): 277-289. 878 879 Rudolph, S., M. C. Tsai, H. von Gersdorff and J. I. Wadiche (2015). "The ubiquitous 880 nature of multivesicular release." Trends Neurosci 38(7): 428-438. 881 39 882 Rutherford, M. A., N. M. Chapochnikov and T. Moser (2012). "Spike encoding of 883 neurotransmitter release timing by spiral ganglion neurons of the cochlea." J Neurosci 884 32(14): 4773-4789. 885 886 Santos-Sacchi, J. (2004). "Determination of cell capacitance using the exact empirical 887 solution of partial differential Y/partial differential Cm and its phase angle." Biophys J 888 87(1): 714-727. 889 890 Schmitz, F., A. Konigstorfer and T. C. Sudhof (2000). "RIBEYE, a component of 891 synaptic ribbons: a protein's journey through evolution provides insight into synaptic 892 ribbon function." Neuron 28(3): 857-872. 893 894 Schnee, M. E., M. Castellano-Munoz, J. H. Kong, J. Santos-Sacchi and A. J. Ricci 895 (2011). "Tracking vesicle fusion from hair cell ribbon synapses using a high frequency, 896 dual sine wave stimulus paradigm." Commun Integr Biol 4(6): 785-787. 897 898 Schnee, M. E., M. Castellano-Munoz and A. J. Ricci (2013). "Response properties from 899 turtle auditory hair cell afferent fibers suggest spike generation is driven by 900 synchronized release both between and within synapses." J Neurophysiol 110(1): 204-901 220. 902 40 903 Schnee, M. E., J. Santos-Sacchi, M. Castellano-Munoz, J. H. Kong and A. J. Ricci 904 (2011). "Calcium-dependent synaptic vesicle trafficking underlies indefatigable release 905 at the hair cell afferent fiber synapse." Neuron 70(2): 326-338. 906 907 Snellman, J., B. Mehta, N. Babai, T. M. Bartoletti, W. Akmentin, A. Francis, G. 908 Matthews, W. Thoreson and D. Zenisek (2011). "Acute destruction of the synaptic 909 ribbon reveals a role for the ribbon in vesicle priming." Nat Neurosci 14(9): 1135-1141. 910 911 Uthaiah, R. C. and A. J. Hudspeth (2010). "Molecular anatomy of the hair cell's ribbon 912 synapse." J Neurosci 30(37): 12387-12399. 913 914 von Gersdorff, H., E. Vardi, G. Matthews and P. Sterling (1996). "Evidence that vesicles 915 on the synaptic ribbon of retinal bipolar neurons can be rapidly released." Neuron 16(6): 916 1221-1227. 917 918 Zanazzi, G. and G. Matthews (2009). "The molecular architecture of ribbon presynaptic 919 terminals." Mol Neurobiol 39(2): 130-148. 920 921 Zenisek, D., N. K. Horst, C. Merrifield, P. Sterling and G. Matthews (2004). "Visualizing 922 synaptic ribbons in the living cell." J Neurosci 24(44): 9752-9759. 923 41 924 Zheng, X. Y., D. Henderson, S. L. McFadden, D. L. Ding and R. J. Salvi (1999). 925 "Auditory nerve fiber responses following chronic cochlear de-efferentation." J Comp 926 Neurol 406(1): 72-86. 927 928 Figure Legends 929 Figure 1. Number of synapses stays unaffected in ribbon less organs of corti. 930 A-D Max intensity projections of confocal z-sections of inner hair cells (IHCs) in apical 931 cochlea turns of P22 KO and wildtype littermates, immunolabeled for CtBP2/RIBEYE 932 (A), Homer (B), Bassoon (C) and Overlay (D). Virtually no punctated extra nuclear 933 CtBP2 staining can be seen in KO mice that is colocalized with other synaptic proteins, 934 while Bassoon and Homer appear generally unaffected. (E) 100 nm thick TEM sections 935 of pre- and postsynaptic densities in WT and KO mice at P28 confirm the absence of 936 electron dense ribbon structures in KO mice. (For E-G) Black arrows point to ribbon 937 structure, white arrows point to synaptic vesicles and white arrowheads point to the 938 remaining density resembling the arciform density. (F) Single slice from a 10 nm thick 939 tomographic section. (G) Reconstruction of complete synapses from data obtained used 940 to create E,F. Presynaptic density in cyan and postsynaptic density in green.Tethered 941 synaptic vesicles modeled in yellow, electron dense structure attributed to synaptic 942 ribbons in magenta. (H) Confocal max intensity projection of IHCs immunolabeled for 943 CtBP1 (green) and Homer (magenta). (I) Quantification of RIBEYE, Homer and 944 Bassoon puncta in the organ of corti normalized to the number of IHCs in analyzed 945 regions of interest, showing complete loss of synaptic RIBEYE staining in KO. The 42 946 amount of Homer and Bassoon staining stays unaffected (p > 0.05). WT in blue, n = 20 947 mice, quantified puncta: RIBEYE = 3796, Bassoon = 10215, Homer = 4340; KO in red, 948 n = 19 mice, n puncta: RIBEYE = 33, Bassoon = 10825, Homer = 4201. (J) Paired 949 synaptic puncta per IHC is lost in absence of RIBEYE staining (p ≤ 0.001) between WT 950 and KO. The number of synapses defined by colocated postsynaptic Homer and 951 presynaptic Bassoon puncta (p = 0.18) is unaffected in KO. (K) The number of extra 952 nuclear CtBP1 puncta is greatly reduced (p = 0.003) in KO mice. (WT in blue, n = 9 953 mice, n puncta: CtBP1 = 19929, Homer 4940; KO in red, n = 7 mice, n puncta: CtBP1 = 954 11960, Homer = 5033). (L) Remaining CtBP1 puncta show significantly reduced 955 (p ≤ 0.001) pairing with postsynaptic Homer staining. Scale Bars: A-D, H: 5 µm; E, F: 956 100 nm. Boxes represent standard deviations of the mean. Significance levels of two 957 tailed unpaired t-tests: n.s., not significant; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001. 958 959 Figure 1 Supplemental Figure 1: Immunohisotchemistry from WT (A) and KO (B) for 960 Bassoon (green) CTBP2 (red) and myosin VIIa (Blue). Low power images show 961 Bassoon is present in efferents of outer hair cells as well as presynaptically in IHCs. 962 Bassoon localization is much broader than RIBEYE’s and is minimally effected in the 963 mutant animal. 964 965 Figure 2 Hearing in RIBEYE knockout mice. A Distortion products of otoacoustic 966 emissions (DPOAE) show no significant differences in the tested frequency range (4-967 46 kHz in half octave increments) between KO (red, n = 30) and WT littermates (blue, 43 968 n = 22), suggesting normal outer hair cell function. (B) Auditory brainstem response 969 (ABR) measurements in the tested frequency range (4-46 kHz in half octave 970 increments) reveal a small (≈ 10 dB SPL), but significant threshold shift in KO (red, n = 971 28) over their WT littermates (blue, n = 22) at P21-P22. (C) Mean ABR waveforms to 972 5 ms tone pips at 23 kHz at 60 dB SPL from KO (red, n = 30) and wild type littermates 973 (blue, n = 24) reveal a decrease in amplitude of the first ABR wave in KO. (D) 974 Quantification of peak to peak amplitude (wave I: p1-n1) differences between KO (red) 975 and WT littermates (blue) at 23 kHz were significant over most sound pressure levels 976 (20 dB SPL: p > 0.05; 30 & 60 dB SPL: p ≤ 0.01; 40-60 dB SPL: p ≤ 0.001) (E) 977 Amplitudes of first peak amplitudes normalized to their respective values at 60 dB SPL. 978 Amplitudes scale with their maximum value in WT and KO. (F) First peak latency (p1) is 979 not significantly altered in KO mice. For wave I amplitude and latency (D-F) analysis the 980 n-number varied between sound pressure levels by a clearly detectable first wave and 981 speaker calibration, n of WT/KO: 20 dB, 11/7; 30 dB, 21/11; 40 dB, 23/22; 50 dB, 23/26; 982 60 dB, 23/28; 70 dB, 5/6. Boxes represent standard deviations of the mean; whiskers 983 and shaded line in (C) represent standard errors of the mean. Significance levels of two 984 tailed unpaired t-tests: n.s., not significant; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001. 985 986 Figure 3. Ribbonless synapses contain clusters of presynaptic voltage-gated Ca2+ 987 channels juxtaposed to postsynaptic glutamate receptors. A-D Max intensity 988 projections of airyscan z-sections of IHCs in apical cochlea turns of P35 KO and WT 989 littermates, immunolabeled for CtBP2/RIBEYE (A), GluA3 (B), CaV1.3 (C) and overlay 990 between calcium channels and glutamate receptors (D). Inner hair cell afferent 44 991 synapses from WT have a synaptic ribbon: a cytoplasmic presynaptic density comprised 992 mainly of RIBEYE protein, recognized by anti-CtBP2 (green). Co-labelled are the 2+ 993 closely associated presynaptic voltage-gated Ca channels (CaV1.3, gray) and 994 postsynaptic AMPA receptors (GluA3, magenta). Inner hair cell afferent synapses from 995 KO lacked synaptic ribbons. The AMPA receptors in KO mice exhibited a ring-like 996 morphology, and the CaV1.3 channels clustered in the form of round spots or elongated 997 stripes, both similar to WT synapses. Scale bar: 2 µm 998 Figure 4. Changed size distribution but unaffected synapse number and 999 organization in ribbonless synapses (A) Quantification of RIBEYE, GluA3 and 1000 CaV1.3 puncta in the organ of corti normalized to the number of IHCs in analyzed 1001 regions of interest. The number of GluA3 receptor and CaV1.3 puncta is unaffected (p > 1002 0.05). WT in blue, n = 9 images from 3 mice, 95 cells quantified puncta: RIBEYE = 1003 1456, GluA3 = 1459, CaV1.3 = 1897 KO in red, n = 13 images from 3 mice, 248 cells, n 1004 puncta: RIBEYE = 28, GluA3 = 2149, CaV1.3 = 2681. (B) Colocalization of synaptic 1005 puncta per IHC. In absence of RIBEYE, the number of synapses, defined by 1006 colocalization of postsynaptic GluA3 and presynaptic CaV1.3, is unaffected (p = 0.13) in 1007 KO. (C) Volume of presynaptic CaV1.3, Bassoon and postsynaptic Homer and GluA3 in 1008 WT (blue, n synapses: CaV1.3 = 1370; Bassoon = 1917; Homer = 5219; GluA3 = 1009 1611) and KO (red, n synapses: CaV1.3 = 2132; Bassoon = 1817; Homer = 4990; 1010 GluA3 = 2334) mice. (D) Normalized cumulative distributions of volumes show 1011 increased sizes of postsynaptic GluA3 and Homer immunoreactivity. (C) Boxes 1012 represent standard deviations of the mean. Significance levels of two tailed unpaired t-1013 tests (A/B) or two sample Kolmogorov-Smirnov test (C): D represents the maximum 45 1014 distance of the Kolmogorov-Smirnov distribution functions. n.s., not significant; * p ≤ 1015 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001. 1016 Figure 5: No differences were observed in calcium or potassium currents 1017 between KO and WT animals. Ca2+ currents were obtained from mice ranging in age 1018 from P10-P12 for WT (A, blue) and P10-13 KO (B, red), with time course of stimulus 1019 shown above, are comparable in all measured properties. Voltage-current plots, 1020 presented in (C) as mean ± SD show no difference (n provided in figure). Summaries of 1021 peak current (D) and voltage of half activation (V1/2, E) are not statistically different (n= 1022 18 and 26 for WT and KO, p = 0.13 for peak current and 0.326 for V1/2). Potassium 1023 currents, obtained from responses to 50 ms voltage pulses ranging from -124 mV to 16 1024 mV in 10 mV increments, from a holding potential of -84 mV are presented in (F) for WT 1025 and (G) for KO mice at P24. A summary of the half activation voltage as estimated from 1026 a Boltzmann fit to the tail current voltage-current (H) found no statistical difference (n = 1027 6 and 8 for WT and KO, respectively with p = 0.084). Current-clamp measurements 1028 found no difference in resting potential (I) (n = 5 and 8 for WT and KO, respectively, p = 1029 0.69). 1030 1031 Figure 6. Capacitance responses recorded from RIBEYE knockout mice show 1032 little difference between phenotypes. Example records of Cm responses (top), 1033 showing two release components and Ca2+ currents (below) recorded from a WT (blue) 1034 (A) and KO (red) (B) in response to 1s depolarizations from -84 mV to -49 and -44 mV. 1035 Dashed line indicates pre-stimulus baseline Cm. Black solid line indicates the 1st linear 1036 component of release and cyan line the 2nd, superlinear component. (C) Summary plot 46 1037 of 1st and 2nd component magnitudes. No statistical difference was found between 1038 groups. For the linear component n = 15 for WT and 15 for KO, p = 0.23. For 2nd, 1039 superlinear component n = 17 for both groups and p = 0.38. Filled stars indicate mean 1040 for this and all subsequent figures. (D) Summary plot of 1st and 2nd component rates 1041 obtained by linear fits to data points, as indicated by lines in (A,B). No statistical 1042 difference was found between groups where, n = 16 for WT amd 13 for KO, with p = 1043 0.266. For the second component rate, n = 17 for both, with p = 0.085. (D). (E) presents 1044 a population histogram for the voltage at which the superlinear response is first 1045 observed. Fits to Gaussian distributions show no change in the mean value but an 1046 increase in the width of the plot for the KO. (F) Example of delay in onset of capacitance 1047 change with small depolarizations for a KO cell. Plots of Cm verses voltage responses 1048 measured at 0.1 (G), 0.5 seconds (H) 1 sec (I) or 3 sec (J) show a significant difference 1049 for release at 0.1s and -44 mV (p=0.03), error bars represent SEM. Fitting the 1050 relationships with simple exponential functions reveal differences in the shapes of the 1051 curves but not in maximum release for the 0.1 and 0.5 plots but not the 1 and 3 sec time 1052 points. N provided in figure for each group. (K) Shows time to release is significantly 1053 longer for KO compared to WT for depolarizations eliciting less than 50% of the 1054 maximal calcium current; n = 32 for WT and 26 for KO, p = 0.0012. 1055 Figure 7. Changing internal Ca2+ buffering does not distinguish phenotypes. (A) 1056 Example records of Cm changes in response to 1s depolarizations from -49 mV in 0.1 1057 EGTA and -39 mV in 1 and 5 mM EGTA for WT (blue) and KO (red). (B) Plot of the 1058 change in Cm in response to 1s voltage steps of -49, -44 and -29 mV for 0.1 EGTA and 1059 -49,-44,-39 and -34 mV for 5 EGTA, n provided in figure. (C) The time to the 2nd 47 1060 component increases significantly with elevated Ca2+ buffer (p value indicated in figure) 1061 but is similar between phenotypes; for 0.1 EGTA n = 7 for WT and 6 KO , p = 0.93, 1062 steps to -29 mV for WT and KO, for 1 EGTA n = 15 for WT and13 for KO , with p = -1063 0.13, steps to -36 + 8 mV WT and -37 + 9 mV KO and for 5 EGTA n = 3 for WT and 4 1064 for KO, with p = 0.67, steps to -39 + 5 mV WT and -35 + 2 mV KO. (D) The rate of 1065 release to the -49 mV step declines equally between phenotypes with increasing 1066 [EGTA] for 0.1 EGTA, n = 7 for WT and 6 for KO, p = 0.323, for 1 EGTA n = 20 for WT 1067 and 22 for KO, p = 0.99 and for 5 EGTA n = 7 for WT and 6 for KO, p = 0.235. (E,F) 1068 Provide box plots of the first component of release for depolarizations to -49 mV (E) or -1069 44 mV (F). Effects of buffers were similar between the two groups. 1070 Figure 8: Subtle differences exist between WT and KO mice postsynaptic 1071 properties. Postsynaptic bouton recordings were obtained from WT or KO in control 1072 and during application of 40 mM K+ to the bath. Hair cell recordings demonstrate similar 1073 depolarizations in WT and KO, suggesting that any differences in response properties 1074 may be synapticaly mediated. (A). Cells rapidly recovered following washout of K+. The 1075 solid line below the trace indicates the duration of the application. 1076 Postsynaptic recordings from afferent boutons in response to 40 mM K+ external 1077 solution application are presented in (B) for WT and (C) for KO with a sustained 1078 response and (D) for KO with a transient response. Recordings were made in the 1079 presence of 5 µM tetrodotoxin (TTX). Frequency responses were generated using a 1 1080 second window sliding at 100 ms intervals (E,F). Populations were defined as transient 1081 or sustained by whether firing persisted after 20 seconds. Number of each response 1082 type is presented as fraction in each panel. Maximum frequency responses are 48 1083 presented in box plot format with mean value shown as start (G); n = 10 for WT and 18 1084 for KO, p = 0.313. The time to peak is presented in (H) where onset of K+ application 1085 response was defined as the time point when the frequency exceeded the spontaneous 1086 frequency +3x SD for two overlapping time windows (200 ms). The time to peak was 1087 statistically different when the one outlier was removed (time point at 181 sec); n = 8 for 1088 WT and 10 for KO. Time to peak only includes fibers whose peak frequency reached 30 1089 Hz because those lower had too few points to make an accurate measure. (I) 1090 Integrating the frequency response over 50 seconds shows that WT release in a more 1091 sustainable manner than the KO, n = 8 for WT and 10 for KO, p = 0.021. Representative 1092 probability density functions for EPSC amplitudes are presented for WT number of 1093 events = 6583 WT and 1551 KO in (J) and KO in (K). Solid lines represent fits to either 1094 double Gaussian functions or Gaussian and gamma functions (smaller peak always fit 1095 with Gaussian function). The curve fitting the small events were integrated to measure 1096 the % of events under the small peak (shown next to each example). Only nerves that 1097 had at least 100 events are included to ensure robustness of the fitting. (L) presents a 1098 box plot of the WT and KO percent of area contributed by the small peak. Comparisons 1099 using a t-test for unequal variance (Welch test) suggest a significant, p = 0.032, n = 7 1100 for WT and 13 for KO. (M) presents a box plot comparing the percentages of simple 1101 events, defined as single rise and decay times plots are not significantly different, n = 7 1102 for WT and 13 for KO, p = 0.3. (N) provides box plots of the median amplitude, where 1103 no significant difference was observed, n = 7 for WT and 13 for KO for each, p = 0.105, 1104 while (O) are box plots of the coefficient of variation for the distributions in (N). Plots 1105 were different, p = 0.017, when equal variance was not assumed. 49 1106 Figure 9: KO synapses have a larger population of small slow EPSCs. 7 WT 1107 including 15,564 events and 13 KO fibers including 17210 events were analyzed. (A) 1108 shows examples of EPSCs that range in amplitude and kinetics. The inset highlights 1109 one of the small slower EPSCs identified in KO fibers. Median decay times (B) and the 1110 Coefficient of Variation for the distribution in decay times (C) are presented as box plots. 1111 Neither population show statistical significance. (D) plots the skewness of the 1112 distributions of decay times and demonstrates that the KO decay time is significantly 1113 greater (away from 0), suggesting that the mean is larger than median, largely because 1114 they have a long tail of large decay time. (E), plots the EPSC decay time constant for 1115 every fiber (each fiber a different color) for both WT and KO. (F), summarizes the data 1116 from C as average decay time constant with EPSCs binned over 10 pA. (G), pooling 1117 fibers for WT and KO recordings allowed for the generation of a probability density 1118 function for event duration. These distributions are significantly different by KS test 1119 (p<10 -18). KS test (p<10-18). (H) is the cumulative probability function for the duration of 1120 EPSCs. Dashed lines are the individual fiber distributions. Solid red and Blue lines 1121 represent the population aggregate (summed responses) for KO and WT respectively. 1122 The cyan and magenta lines are the averages for WT and KO respectively. 1123 1124 1125 1126 1127 50 1128 1129 1130 1131 1132 1133 1134 1135 1136 1137 1138 1139 1140 1141 51

The Presynaptic Ribbon Maintains Vesicle Populations at the Hair Cell Afferent Fiber

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