Disruption of Otoferlin Alters the Mode of Exocytosis at the Mouse Inner Hair Cell Ribbon Synapse

Sound encoding relies on Ca2+-mediated exocytosis at the ribbon synapse between cochlear inner hair cells (IHCs) and type I spiral ganglion neurons (SGNs). Otoferlin, a multi-C2 domain protein, is proposed to regulate Ca2+-triggered exocytosis at this synapse, but the precise mechanisms of otoferlin function remain to be elucidated. Here, performing whole-cell voltage-clamp recordings of excitatory postsynaptic currents (EPSCs) from SGNs in otoferlin mutant mice, we investigated the impact of Otof disruption at individual synapses with single release event resolution. Otof deletion decreased the spontaneous release rate and abolished the stimulus-secretion coupling. This was evident from failure of potassium-induced IHC depolarization to stimulate release and supports the proposed role of otoferlin in Ca2+ sensing for fusion. A missense mutation in the Otof gene (pachanga), in which otoferlin level at the IHC plasma membrane was lowered without changing its Ca2+ binding, also reduced the spontaneous release rate but spared the stimulus-secretion coupling. The slowed stimulated release rate supports the hypothesis that a sufficient abundance of otoferlin at the plasma membrane is crucial for the vesicle supply. Large-sized monophasic EPSCs remained present upon Otof deletion despite the drastic reduction of the rate of exocytosis. However, EPSC amplitude, on average, was modestly decreased. Moreover, a reduced contribution of multiphasic EPSC was observed in both Otof mutants. We argue that the presence of large monophasic EPSCs despite the exocytic defect upon Otof deletion supports the uniquantal hypothesis of transmitter release at the IHC ribbon synapse. Based upon the reduced contribution of multiphasic EPSC, we propose a role of otoferlin in regulating the mode of exocytosis in IHCs.


INTRODUCTION
Ca 2+ influx and subsequent neurotransmitter release at inner hair cell (IHC) active zones govern sound encoding at the first auditory synapse. Excitatory postsynaptic currents (EPSCs) recorded from type I spiral ganglion neurons (SGNs) show remarkable variability in amplitude and shape. This EPSC heterogeneity led to the hypothesis of synchronized multiquantal release (MQR) at hair cell synapses (Glowatzki and Fuchs, 2002;Keen and Hudspeth, 2006;Goutman and Glowatzki, 2007;Neef et al., 2007;Li et al., 2009;Grant et al., 2010;Graydon et al., 2011;Schnee et al., 2013). MQR would cause monophasic (temporally compact) EPSCs when the exocytosis of vesicles comprising a MQR event is highly synchronized or multiphasic (temporally non-compact) EPSCs when synchronization of MQR is poor. However, whereas Ca 2+ influx by presynaptic depolarization increases the EPSC amplitude at the frog hair cell synapse (Li et al., 2009;Graydon et al., 2011), neither changes in voltage-gated Ca 2+ influx (Glowatzki and Fuchs, 2002;Grant et al., 2010) nor those in presynaptic Ca 2+ buffering (Goutman and Glowatzki, 2007) affects the EPSC amplitude distribution at the rat IHC ribbon synapse. Strikingly, even when the presynaptic Ca 2+ influx was abolished in mouse IHCs, EPSC size remained heterogeneous and the charge distribution of mono-and multiphasic EPSCs were unchanged (Chapochnikov et al., 2014). This, together with other experimental observations and modeling, led to the proposal that uniquantal release (UQR) is a candidate mechanism for exocytosis at the mammalian IHC ribbon synapse, whereby a combination of large clusters of postsynaptic AMPA receptors and presynaptic fusion pore dynamics would generate large and variably shaped EPSCs (Chapochnikov et al., 2014; for review, see Takago and Oshima-Takago, 2018). The UQR hypothesis of IHC exocytosis has received further support by recent studies manipulating presynaptic IHC Ca 2+ influx (Huang and Moser, 2018) or directly measuring membrane fusion steps via cellattached membrane capacitance recordings from IHCs (Grabner and Moser, 2018).
Disruption of OTOF, coding for otoferlin, was identified to cause hereditary deafness DFNB9 (Yasunaga et al., 1999), while missense mutations of OTOF can lead to less profound hearing impairment (Varga et al., 2003;Marlin et al., 2010;Vogl et al., 2016; for review, see Pangršič et al., 2012;Moser and Starr, 2016). As a multi-C 2 domain protein, otoferlin, in analogy to synaptotagmins, was initially proposed to serve as a Ca 2+ sensor for fusion at the IHC ribbon synapse based on a functional analysis of a mouse line with a null mutation in otoferlin (Otof −/− , Roux et al., 2006) and biochemical studies (Johnson and Chapman, 2010). Moreover, a role of otoferlin in efficient vesicle priming is proposed based upon an analysis of a mouse line called pachanga that carries a missense mutation in otoferlin C 2 F domain (Otof D1767G/D1767G or Otof Pga/Pga , Pangršič et al., 2010). Whereas such a mutation does not affect Ca 2+ binding, Otof Pga/Pga IHCs show reduced membrane-bound otoferlin level but unaltered fusion, thus suggesting that the hearing impairment arises from strongly reduced vesicle replenishment (Pangršič et al., 2010). This hypothesis is further supported by analyses of mouse lines carrying a missense mutation in otoferlin C 2 C domain that again lowers membrane-bound otoferlin level but unaltered fusion (Ofof I515T/I515T , Strenzke et al., 2016) and a null mutation in transmembrane recognition complex40 receptor tryptophan-rich basic protein that is essential for the insertion of otoferlin into the endoplasmic reticulum in IHCs . Thus, the abundance of otoferlin in IHCs is critical for maintaining the vesicle resupply to the ribbon-type active zone. Otoferlin's function in vesicle replenishment appears to involve the regulation of short filamentous tethering formation between synaptic vesicles and the active zone membrane (Vogl et al., 2015) and the facilitation of clearance of vesicular release sites from previously exocytosed membranes (exocytosisendocytosis coupling) via an interaction with the endocytic adaptor protein 2 (Duncker et al., 2013;Jung et al., 2015). On the other hand, a recent study utilizing mice with double missense mutations in otoferlin C 2 C domain that affect Ca 2+ sensing (Otof D515A,D517A/D515A,D517A , Michalski et al., 2017) has reported that fast and sustained components of release are reduced despite unaltered otoferlin level, probably due to dysfunctional Ca 2+ binding of otoferlin.
In order to further examine the otoferlin's function we performed postsynaptic voltage-clamp recordings from afferent boutons of type I SGNs in wild-type (Otof +/+ ), Otof Pga/Pga and Otof −/− mice and investigated exocytosis at the levels of single synapses and single release events. Analyzing the spontaneous and stimulated release rates as well as the amplitude and shape of EPSCs, we find evidence for roles of otoferlin in Ca 2+ -dependent fusion and replenishment of vesicles. Moreover, we propose an additional role of otoferlin in regulating the exocytic mode of IHCs to facilitate multiphasic EPSCs, potentially by controlling the vesicle fusion pore during uniquantal release at the IHC ribbon synapse.

Ethics Statement
All experiments complied with national animal care guidelines in Germany and Japan and were approved by the University of Göttingen board for animal welfare together with the animal welfare office of the state of Lower Saxony (Germany) as well as National Rehabilitation Center for Persons with Disabilities animal experimentation committee (Japan).

Chemicals and Equipment
All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, United States) except for tetrodotoxin (Tocris Bioscience, Bristol, United Kingdom or Wako Pure Chemical Industries, Ltd., Osaka, Japan). The EPC-10 amplifier controlled by Patchmaster software (HEKA Elektronik, Lambrecht, Germany) as well as an upright microscope with differential interference contrast optics (Axioskop FS2, Carl Zeiss, Oberkochen, Germany or BX51WI, Olympus, Tokyo, Japan) was used.

Data Analysis and Statistics
For detection and analysis of EPSCs, MiniAnalysis software (Synaptosoft, Decatur, GA, United States) was used with a detection threshold set at 3-5 times greater than the root mean square (rms) of the baseline noise. To classify EPSCs into monoor multiphasic, the methods introduced by Grant et al. (2010) as well as Chapochnikov et al. (2014) were employed. For plotting, IGOR Pro (Wavemetrics, Lake Oswego, OR, United States), Sigmaplot (Systat Software Inc., San Jose, CA, United States) were used. The passive membrane properties such as series resistance (R s, 40 ± 2 M for 13 Otof +/+ SGNs, 41 ± 3 M for 7 Otof Pga/Pga SGNs, 40 ± 2 M for 13 Otof −/− SGNs), membrane capacitance (C m , 1.9 ± 0.2 pF for Otof +/+ , 1.9 ± 0.1 pF for Otof Pga/Pga , 1.8 ± 0.2 pF for Otof −/− ) and membrane input resistance (R m, 498 ± 141 M for Otof +/+ , 831 ± 225 M for Otof Pga/Pga , 651 ± 106 M for Otof −/− ) were calculated as previously described (Chapochnikov et al., 2014). Recordings with R s > 50 M or less than 20 EPSCs were excluded from the EPSC amplitude/charge or kinetics analysis, but included in the EPSC frequency analysis (except for recordings with less than 5 EPSCs) not to ignore very low-frequency auditory nerve fibers. Data is shown as mean ± SEM. Statistical significance was evaluated by Student's t-test or one way ANOVA followed by post-hoc Tukey's test.

Missense Mutation of Otoferlin Decreases the Rate of Spontaneous Release, Loss of Otoferlin Abolishes Stimulus-Secretion Coupling in IHCs
EPSCs were recorded from afferent boutons of type I SGNs in P8-11 Otof +/+ , Otof Pga/Pga (missense mutation with reduced abundance of otoferlin) and Otof −/− mice. At rest (1.3 mM Ca 2+ and 5.8 mM K + in aPL), SGNs of all three genotypes Asterisks at the bottom (spontaneous rate) and top (stimulated rate) show significant differences ( * p < 0.05). Note that the vertical axes for this and the next panels are shown on logarithmic scale. In some recordings, where 40 mM KCl was not applied, data points for 5.8 mM [K + ] e alone were plotted. steadily exhibited spontaneous EPSCs ( Figure 1A), but their rate was reduced in SGNs of both mutants. The frequencies of spontaneous EPSCs were 0.71 ± 0.19 Hz for Otof +/+ (n = 13, Figure 1B), 0.18 ± 0.07 Hz for Otof Pga/Pga (n = 7, p = 0.048 compared to Otof +/+ ) and 0.14 ± 0.04 Hz for Otof −/− SGNs (n = 13, p = 0.010 and 0.985 compared to Otof +/+ and Otof Pga/Pga , respectively). Hence, unlike disruption of synaptotagmin I or II at the central synapses, which is thought to unclamp spontaneous release (Nishiki and Augustine, 2004;Pang et al., 2006;Xu et al., 2009;Kochubey and Schneggenburger, 2011;Lee et al., 2013;Liu et al., 2014), Otof deletion did not increase the spontaneous EPSC frequency. This argues against a clamping function of otoferlin for spontaneous release and highlights the importance of otoferlin in vesicle replenishment, since the spontaneous rate was reduced to a similar extent in Otof −/− and Otof Pga/Pga mice, which in a previous study showed intact vesicle fusion but impaired replenishment in IHCs (Pangršič et al., 2010).
Next, we examined the stimulus-secretion coupling in IHCs. High K + solution (40 mM in aPL) was bath-applied onto IHCs to increase the open probability of their Ca V 1.3 L-type Ca 2+ channels. The frequency of EPSCs in Otof +/+ SGNs was increased from 0.25 ± 0.11 (5.8 mM, control) to 9.67 ± 2.90 Hz (first 10 s during high K + stimulation, n = 6, p = 0.021, Student's paired t-test, Figures 1A,B). Here, the spontaneous EPSC frequency for this control (5.8 mM K + ) was undervalued due to our preferential application of high K + -containing aPL onto low-frequency synapses, since remaining more active synapses (1.10 ± 0.28 Hz, n = 7) provided a sufficient number of EPSCs for the analyses of EPSC size and shape even in the normal K + -containing aPL. In Otof Pga/Pga SGNs, high K + stimulation increased the frequency of EPSCs in 4 out of 5 Otof Pga/Pga SGNs from 0.20 ± 0.10 (control) to 1.86 ± 0.57 Hz (high K + , n = 5, p = 0.047), indicating that the missense Otof mutation in Otof Pga/Pga mice spares stimulus-secretion coupling in IHCs. The reduced rate of stimulated release in Otof Pga/Pga IHCs (p = 0.032 compared to Otof +/+ ) is compatible with impaired vesicle replenishment (Pangršič et al., 2010). In contrast, the high K + stimulation failed to increase the EPSC frequency in Otof −/− SGNs (control: 0.12 ± 0.06 Hz vs. high K + : 0.10 ± 0.05 Hz, n = 5, p = 0.302), indicating that Otof deletion abolishes stimulus-secretion coupling in IHCs. The difference in stimulated release rate between Otof +/+ and Otof −/− SGNs again showed significance (p = 0.010).

Otoferlin Disruption Decreases EPSC Amplitude and the Fraction of Multiphasic EPSCs
We studied the effects of Otof disruption on the size and shape of EPSCs. As reported in previous studies using the organ of Corti of rats (Glowatzki and Fuchs, 2002;Grant et al., 2010), high K + stimulation of mouse Otof +/+ organ of Corti increased the frequency of EPSCs without affecting the EPSC size (monophasic EPSC amplitude: 141 ± 31 pA for control vs. 157 ± 24 pA for high K + , multiphasic EPSC amplitude: 101 ± 13 pA for control vs. 109 ± 16 pA for high K + ) or the fraction of multiphasic EPSCs (41.9 ± 3.8 % for control vs. 41.1 ± 1.7 % for high K + ). Moreover, EPSC size and kinetics distributions were unaltered by high K + stimulation in each phenotype SGNs (Supplementary Figures S1, S2). Therefore, EPSCs recorded in control and high K + were pooled for subsequent analyses in this study.
Surprisingly, despite the reduction in release rate and the lack of stimulus-secretion coupling, Otof −/− synapses showed variable EPSC amplitudes from 11 up to several hundred pA (Figure 2). As shown in Figure 3 and Table 1, the average amplitudes of mono-and multiphasic EPSCs as well as the average charge transfer of mixed EPSCs were reduced in Otof −/− SGNs (p = 0.013 for monophasic EPSC amplitude, p = 0.015 for multiphasic EPSC amplitude, p = 0.011 for EPSC charge). There was a trend to smaller EPSC amplitudes also for Otof Pga/Pga SGNs, which, however, did not reach significance. EPSC kinetics (i.e., rise time and decay time for monophasic as well as time to peak and half width for multiphasic), on the other hand, was not different among those three groups (Figures 2, 4).

Roles of Otoferlin for Stimulus-Secretion Coupling and Vesicle Replenishment
In the present study, we recorded EPSCs from the postsynaptic afferent boutons of type I SGNs in wild-type and otoferlin mutant mice, and tested the function of otoferlin in exocytosis at the IHC ribbon synapse. We found a complete disruption of stimulussecretion coupling in Otof −/− SGNs. As Ca 2+ influx and vesicle availability on a morphological level are maintained at the AZs of Otof −/− IHCs (Roux et al., 2006), we argue that Ca 2+ no longer efficiently drives fusion in the absence of otoferlin. This supports the role of otoferlin as a Ca 2+ sensor of fusion (Roux et al., 2006;Johnson and Chapman, 2010;Michalski et al., 2017). But how is otoferlin-independent spontaneous release regulated? Possible mechanisms include (1) an additional high affinity Ca 2+ sensor whose capacity is saturated at resting [Ca 2+ ] i and (2) a Ca 2+independent release process. For (1), the activation range of the remaining Ca 2+ sensor should be below the resting [Ca 2+ ] i of a few tens of nanomolar (Beutner and Moser, 2001). However, to date, there has been no report about such a molecule that meets this requirement. The closest candidate Ca 2+ sensor for fusion is FIGURE 3 | Otoferlin deletion decreases EPSC amplitude and charge. (A-C) EPSC size for Otof +/+ (black), Otof Pga/Pga (dark gray) and Otof −/− (light gray) SGNs. Significant differences between Otof +/+ and Otof −/− SGNs ( * p < 0.05). Cumulative histograms of monophasic EPSC amplitude (A1), multiphasic EPSC amplitude (B1) and mono-and multiphasic (mixed) EPSC charge (C1) as well as individual (circles) and mean ± SEM (bars) values of monophasic EPSC amplitude (A2), multiphasic EPSC amplitude (B2) and mono-and multiphasic EPSC charge (C2) are shown. Note that a break line is inserted in the horizontal axis of panel C2 to clearly demonstrate the differences among groups. the C 2 domain protein Doc2b that promotes membrane fusion at submicromolar Ca 2+ concentrations (but higher than 100 nM) (Groffen et al., 2010).
Interestingly, the absence of otoferlin did not increase the spontaneous EPSC frequency, which is in contrast to the effect of ablation of synaptotagmin I (Xu et al., 2009) or II (Pang et al., 2006) in the mouse central neurons, further highlighting differences among otoferlin and synaptotagmin I (Reisinger et al., 2011). We note here that the "spontaneous" release from resting IHCs likely includes Ca 2+ -evoked release that is triggered by rare openings of Ca V 1.3 channels. We consider it likely that the reduction of spontaneous release reflects a combination of disrupted Ca 2+ -triggered spontaneous release of IHCs and reduced vesicle replenishment.
Previous studies showed that otoferlin abundance at the IHC plasma membrane correlates with the presynaptic function and sound encoding (Strenzke et al., 2016). Otof Pga/Pga mice, wherein otoferlin membrane abundance is attenuated down to 3% of Otof +/+ mice (Pangršič et al., 2010;Strenzke et al., 2016), displays lower EPSC frequency upon high K + -induced IHC depolarization. Although our combination of postsynaptic recordings and high K + stimulation cannot track fast stimulussecretion coupling, the robust EPSC rates in Otof Pga/Pga afferents ( Figure 1A) suggests that Ca 2+ -triggered membrane fusion is intact despite the mutation in the C 2 F domain (i.e., D1767G, Pangršič et al., 2010). On the other hand, the dual mutations in the C 2 C domain (i.e., D515A and D517A), which alter its Ca 2+ -binding (Johnson and Chapman, 2010) but preserves otoferlin level (Michalski et al., 2017), impairs both vesicle fusion and replenishment functions of otoferlin, suggesting that Ca 2+ -sensing of otoferlin is critical for both steps of exocytosis (Michalski et al., 2017). Thus, not only Ca 2+ -sensing capacity but also plasma membrane level of otoferlin is essential for IHC exocytosis.

Multiquantal Versus Uniquantal Release: Can Otoferlin Disruption Provide Insight?
The heterogeneity of EPSC amplitude and shape is a hallmark of ribbon synapses, but the underlying mechanisms are not well understood. Notably, the mammalian IHC ribbon synapse differs from other ribbon synapses such as the amphibian and reptile hair cell synapses (Li et al., 2009;Graydon et al., 2011;Schnee et al., 2013) as well as the mammalian retinal bipolar-AII cell synapse (Singer et al., 2004;Mehta et al., 2013), where low [Ca 2+ ] i conditions break EPSCs down into unitary events. In contrast, at the rodent IHC ribbon synapse the EPSC amplitude remains sizable despite the massive buffering of presynaptic Ca 2+ (Goutman and Glowatzki, 2007) or abolition of Ca 2+ influx (Chapochnikov et al., 2014). This and other findings have led us to consider an alternative hypothesis for explaining EPSC amplitude and shape heterogeneity at the IHC synapse: the large EPSC amplitude may reflect activation of a large number of postsynaptic AMPA receptors (Saito, 1990;Meyer et al., 2009) activated by glutamate liberated from a single synaptic vesicle. Post-fusion regulation of such uniquantal release by a dynamic fusion pore may explain multiphasic EPSCs (successive bouts of release through a flickering pore) and small monophasic EPSCs (incomplete release of a vesicle's glutamate content) (Chapochnikov et al., 2014;Huang and Moser, 2018; for review, see Moser and Vogl, 2016;Takago and Oshima-Takago, 2018).
Given the presence of large monophasic EPSCs at the Otof −/− IHC afferent synapse (Figure 2), i.e., under conditions of strongly reduced rate of release that seem not permissive for synchronized fusion of multiple vesicles, we favor the interpretation that uniquantal release prevails in IHCs. Interestingly, the fraction of multiphasic EPSCs was smaller when otoferlin was disrupted. Within the framework of the uniquantal release hypothesis of IHC exocytosis this can be explained as favoring full-collapse fusion and/or singular fusion pore openings. Accordingly, otoferlin would then enhance promote fusion pore flickering. Alternatively, within the framework of multiquantal release hypothesis, which the present study cannot exclude, our observation might be explained as highly synchronized fusion of a smaller number of vesicles upon Otof disruption. Besides, either hypothetic mechanism might be modulated by altered Ca 2+ signaling at the release site as might have resulted from the scaffolding function of Otoferlin for Ca 2+ channels (Hams et al., 2017) or attenuated fast-inactivating Ca 2+ currents in Otoferlin-lacking IHCs (Vincent et al., 2017). However, we note that we had previously found normal Ca 2+ signals at the individual IHC AZs in Otof Pga/Pga mice (Pangršič et al., 2010). Further testing these hypotheses and elucidating the underlying molecular events remain important goals for future studies.
A smaller fraction of multiphasic EPSCs was also found in Otof Pga/Pga SGNs ( Figure 5) and we suggest that the reduction of otoferlin levels at the plasma membrane suffices to alter the IHC exocytic mode. Tens of missense mutations have been described for patients with otoferlin-related deafness (for review, see Pangršič et al., 2012;Santarelli et al., 2015). It is noteworthy that even a missense mutation of otoferlin such as D1767G (pachanga) drastically decrease the heterogeneity of EPSC shape (this study). Although the functional significance of multiphasic EPSCs at the IHC ribbon synapse remains to be clarified, a wide range of spike jitter in the SGN afferent boutons caused by variable EPSP waveforms (Rutherford et al., 2012) may contribute to the heterogeneity of auditory nerve fiber responses in response to sound (Liberman, 1982;Grant et al., 2010). By promoting the shift in IHC exocytic mode, otoferlin may serve to endow the diversity in sound encoding at this ribbon-type synapse.

AUTHOR CONTRIBUTIONS
HT and TM designed the study. HT and TO-T performed experiments and analyzed the data. HT, TO-T, and TM wrote the manuscript. The experiments were performed at the University Medical Center Göttingen and Research Institute of National Rehabilitation Center for Persons with Disabilities.

FUNDING
This study was supported by a fellowship of MED-EL company (to HT) as well as grants from the German Research Foundation through the Collaborative Research Center 889 (Project A2 to TM) and JSPS KAKENHI (Grant Nos. 25670722 and 16K11204 to HT).

ACKNOWLEDGMENTS
We would like to thank Dr. Ulrich Müller and Dr. Ellen Reisinger for providing otoferlin mutant mice as well as Dr. Elisabeth Glowatzki and Dr. Darina Khimich for teaching postsynaptic patch clamp technique. We also would like to thank Drs. Masao Tachibana, Tomoyuki Takahashi, Takeshi Sakaba, Jeong-Seop Rhee, Nikolai Chapochnikov, Chao-Hua Huang, Yukihiro Nakamura, Yoshinori Sahara, Shinichi Iwasaki, Kensuke Watanabe, Ken Kitamura, Yuko Seko, and Koichi Mori for comments and helps.