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Articles in PresS. J Neurophysiol (October 28, 2015). doi:10.1152/jn.00559.2015 1 TITLE: Calcium-induced calcium release supports recruitment of synaptic vesicles in 2 auditory hair cells. 3 ABBREVIATED TITLE: Stored calcium promotes vesicle recruitment to ribbon synapses. 4 AUTHOR NAMES AND AFFILIATION: Manuel Castellano-Muñoz1,3, Michael E. 5 Schnee1 and Anthony Ricci1,2. 6 7 Department of Otolaryngology1 and Molecular and Cellular Physiology2. Stanford University School of Medicine. 300 Pasteur Drive. Stanford, CA 94305 (USA). 8 9 Current address: Institute of Bioengineering. Miguel Hernández University. Avenida de la Universidad, s/n 03202 Elche, Alicante (Spain). 3 10 11 12 AUTHOR CONTRIBUTIONS: M.C.-M, M.E.S. and A.J.R. performed research. M.C.-M and A.J.R. designed experiments, analyzed data and wrote the paper. 13 14 CONFLICT OF INTEREST: The authors declare that no competing interests exist. 15 Copyright © 2015 by the American Physiological Society. 16 Abstract 17 Hair cells from auditory and vestibular systems transmit continuous sound and balance 18 information to the central nervous system through the release of synaptic vesicles at ribbon 19 synapses. The high activity experienced by hair cells requires a unique mechanism to sustain 20 recruitment and replenishment of synaptic vesicles for continuous release. Using pre and 21 postsynaptic electrophysiological recordings, we explored the potential contribution of 22 calcium-induced calcium release (CICR) in modulating the recruitment of vesicles to 23 auditory hair cell ribbon synapses. Pharmacological manipulation of CICR with agents 24 targeting endoplasmic reticulum calcium stores reduced both spontaneous postsynaptic 25 multiunit activity and the frequency of excitatory postsynaptic currents (EPSCs). 26 Pharmacological treatments had no effect on hair cell resting potential or activation curves 27 for calcium and potassium channels. However, these drugs exerted a reduction in vesicle 28 release measured by dual-sine capacitance methods. In addition, calcium substitution by 29 barium reduced release efficacy by delaying release onset and diminishing vesicle 30 recruitment. Together these results demonstrate a role for calcium stores in hair cell ribbon 31 synaptic transmission and suggest a novel contribution of CICR in hair cell vesicle 32 recruitment. We hypothesize that calcium entry via calcium channels is tightly regulated to 33 control timing of vesicle fusion at the synapse whereas CICR is used to maintain a tonic 34 calcium signal to modulate vesicle trafficking. 35 Keywords 36 Hair cell, dual sine capacitance, CICR, intracellular stores, ribbon synapse, synaptic 37 transmission 38 39 Introduction 40 Hair cells, the sensory receptors in the auditory and vestibular systems, convert mechanical 41 information into synaptic activity through the release of neurotransmitter at ribbon synapses. 42 Each hair cell contains tens of synaptic ribbons (Schnee et al. 2005; Schnee et al. 2011; 43 Sneary 1988), presynaptic specializations surrounded by synaptic vesicles and associated to 44 active zones and L-type Ca2+ channels (Issa and Hudspeth 1994; Roberts et al. 1990; Tucker 45 and Fettiplace 1995). Similar to other sensory synapses, hair cell ribbon synapses operate in 46 a graded fashion, reaching high release rates and exhibiting little fatigue. Both of these 47 properties require rapid vesicle replenishment by a mechanism that is not well understood. 48 Calcium-induced Ca2+ release (CICR) is a mechanism by which the influx of Ca2+ 49 through Ca2+ channels in the plasma membrane activates Ca2+ release from intracellular 50 stores (Verkhratsky 2005). CICR is implicated in a number of neuronal functions such as 51 neuronal excitability, gene expression, synaptic plasticity and release (Bouchard et al. 2003). 52 In central synapses, both endoplasmic reticulum (ER) and mitochondria are well-known 53 intracellular Ca2+ stores and their Ca2+ homeostatic modulation alter synaptic transmission 54 pre- and postsynaptically (Llano et al., 2000; Emptage et al., 2001; Bardo et al., 2006). CICR 55 is also suggested to contribute to synaptic transmission at ribbon synapses (Babai et al. 2010; 56 Lelli et al. 2003). 57 Calcium imaging identified CICR in turtle auditory papilla hair cells (Tucker and 58 Fettiplace 1995), frog semicircular canal (Lelli et al. 2003), P6-11 mouse inner hair cells 59 (Iosub et al. 2015; Kennedy and Meech 2002) and rat and guinea pig outer hair cells (Evans 60 et al. 2000; Mammano et al. 1999). In mammalian outer hair cells, CICR is functionally 61 associated to subsynaptic Ca2+ stores in close proximity to efferent terminals (Lioudyno et al. 62 2004). In addition, Ca2+ can be released by inositol triphosphate-gated Ca2+ stores at the base 63 of the outer hair cell hair bundle (Mammano et al. 1999). Although pharmacological data 64 demonstrates the presence of intracellular stores in hair cells, its physiological role is 65 debatable. Intracellular Ca2+ stores have been functionally associated to the control of BK 66 channel activity in inner hair cells (Beurg et al. 2005; Marcotti et al. 2004) modulation of 67 outer hair cell electromotility (Dallos et al. 1997), homeostatic control of presynaptic Ca2+ 68 levels (Kennedy and Meech 2002; Tucker and Fettiplace 1995), time-dependent segregation 69 of afferent and efferent signaling (Im et al. 2014) and regulation of vesicular trafficking, 70 exocytosis and synaptic transmission (Hendricson and Guth 2002; Lelli et al. 2003). 71 Here, we performed auditory-nerve multiunit and single unit recordings as well as 72 hair-cell dual-sine capacitance experiments to study the potential contribution of CICR to 73 hair-cell synaptic transmission. Pharmacological and divalent cation substitution results are 74 consistent with a role for CICR in the recruitment of vesicles to support maintained release in 75 auditory hair cell ribbon synapses. 76 Materials & Methods 77 Tissue preparation 78 The auditory papilla of red-ear sliders (Trachemys scripta elegans) was dissected as 79 previously described (Schnee et al. 2005). All animal procedures were approved by the 80 Stanford IACUC committee and are in accord with NIH guidelines and standards. Turtle half- 81 head preparations were used for multiunit activity measurements from the eighth cranial 82 nerve. The turtle head was split in half and pinned in a sylgardTM dissection chamber with 83 either an external solution similar to the one used in patch clamp recordings or with 84 bicarbonate-buffered perilymph containing (in mM) NaCl 126, KCl 2.5, NaHCO3 13, 85 NaH2PO4 1.7, CaCl2 1.8, MgCl2 1 and glucose 5 (continuously bubbled with 95% O2 and 5% 86 CO2). The brain was removed and the auditory nerve exposed (Figure 1A), cutting the 87 connections to posterior ampulla and saccule. The ventral otic membrane was trimmed to 88 allow access to perfusion prior to mounting the preparation in the recording dish. A gravity- 89 controlled perfusion pipet was located approximately 5 mm above the otic capsule and was 90 connected to a perfusion system with a flow rate of roughly 1 ml/min. 91 For intracellular hair-cell recordings, the inner ear was dissected from the otic capsule 92 in external solution containing 128 mM NaCl, 0.5 mM KCl, 2.8 CaCl2, 2.2 mM MgCl2, 10 93 mM HEPES, 6 mM glucose, 2mM creatine monohydrate, 2mM ascorbate, 2 mM pyruvate 94 and the pH was adjusted to 7.6 and osmolality adjusted to 275 mosml/kg. The external 95 solution was supplemented with 20 µM curare to eliminate efferent activity and 100 nM 96 apamin was included in some of the experiments to block SK activity. We found no evidence 97 of remaining efferent activity when incubated with curare in both pre and postsynaptic 98 recordings. To disrupt mechanotransduction channels, after trimming extraneous tissue and 99 removing the otoconia, the papilla was incubated for 15 min in external solution and perfused 100 with 200 µl of 5 mM BAPTA before and after removing the tectorial membrane with a fine 101 insect pin. In some experiments where the tectorial membrane was left intact, cell 102 visualization was impaired but no obvious electrophysiological effects were observed. The 103 basilar papilla was transferred to the recording chamber and secured with single strands of 104 dental floss. Cells were imaged with an Axioskop 2 FS plus (Zeiss, Thornwood, NY) with 105 bright field optics using a 60x 0.9 NA water objective (LUMPlan Fl/IR, Olympus). Perfusion 106 of bath and drugs was delivered using a Minipuls 3 pump (Gilson, Midleton, WI). 107 Electrophysiology 108 For multiunit activity we used the turtle half-head preparation, where the auditory nerve was 109 inserted in an hourglass-shaped suction electrode with a micromanipulator (Narishige, East 110 Meadow, NY) and compound action potentials were recorded using a differential AC 111 preamplifier (Grass, P55 Asto-Med, Westwarwick, RI). One electrode was inserted into the 112 borosilicate suction pipet and the neutral electrode was in contact with the bath. The signal 113 was band-passed filtered (1Hz-1kHz) and amplified 1,000 times. 114 potentials were collected through a data acquisition interface (CED Micro 1401 mkII, 115 Cambridge Electronic Design, UK) and analyzed with Spike 2 software (Cambridge 116 Electronic Design, UK). Noise levels were identified by blocking afferent activity with 1 μM 117 tetrodoxin (TTX) or prolonged high potassium concentration and spike threshold was set to 118 3xSD of baseline noise. Drugs were applied via the local perfusion system after a baseline 119 firing rate was established. Control perfusion with the external solution (sans drug) was used 120 to confirm no mechanical artifacts and firing stability throughout recording (96 ± 2 % after 121 20 min, n = 3). 122 For hair-cell patch clamp experiments, thick-wall borosilicate electrodes of resistance 2.5-3.5 123 MΩ were used with internal solution containing 110 mM CsCl, 1 mM EGTA, 5 mM creatine 124 phosphate, 3 mM Na2ATP, 10 mM HEPES, 3 mM MgCl2, 2 mM ascorbate and pH adjusted 125 to 7.2 and osmolality at 255 mosml/kg. Stimulus protocols were performed starting 10 min 126 after whole-cell configuration to allow solution equilibration and run up stabilization of the 127 Ca2+ current (Schnee 2003). Hair cells were voltage clamped with an Axopatch 200B (Axon 128 Instruments-Molecular Devices, Sunnyvale, CA) or a VE-2 amplifier (Alembic Instruments, 129 Montreal, Canada). Data were collected at 100-200 kHz with an IOTech Daq/3000 130 acquisition board (MC Measurement Computing, Norton, MA) driven by jClamp software 131 (Scisoft). Voltage was intentionally not corrected for junction potential or series resistance to 132 match the values used in two-sine capacitance protocols (Schnee et al. 2011). Dual sinusoidal 133 stimulation was performed to compensate in-cell stray capacitance at different frequencies 134 before running capacitance protocols. A dual sinusoid with amplitudes and frequencies of 20- 135 30 mV at 3.1-6.2 kHz and 6.2-9.4 kHz was delivered superimposed to the desired voltage Compound action 136 step. Capacitance measurements were low-pass filtered at 40 Hz and the onset-offset gating 137 capacitative transients were removed offline. 138 Afferent fiber patch recordings were done using similar solutions as described for hair cell 139 recordings (Schnee et al. 2013). Patch electrodes were smaller tipped and had resistances of 140 9-11 MΩ. Series resistance was compensated to 70% resulting in uncompensated SR of 11 + 141 3 MΩ (n = 5). 142 Data analysis 143 In hair-cell patch-clamp experiments, the initial capacitance of cells located at the center of 144 the papilla was 12.4 ± 1.4 pF (n=47) 1 min after establishment of whole cell configuration. 145 Cells were discarded when the uncompensated series resistance was larger than 12 MΩ due 146 to the difficulty in the neutralization of in-cell stray capacitance. Cells were also discarded 147 when the leak current was larger than 50 pA after 9 min of recording to avoid calcium 148 channel inactivation due to slow calcium loading of the cells. Data and graphics show mean ± 149 SD and number of experiments (n) unless noted. Statistical analyses were performed using a 150 two-tailed Student’s t-test assuming normal distribution. Box plots are presented with raw 151 data [symbols], mean [star] and standard deviation of the mean [box]. In the capacitance 152 experiments where multiple release protocols were evoked, normalized percentage values are 153 related to the value evoked in the first pulse. In figures, the y-axis represents the amount of 154 release in a second stimulation protocol normalized by a first stimulation protocol (2nd-1st 155 pulse/ 1st pulse); therefore values below 0 represent a reduction in the 2nd pulse, whereas 156 values near 1 represent twice as much release in the 2nd pulse. 157 Results 158 Pharmacological manipulation of calcium stores reduces auditory nerve activity 159 The ability of neuronal endoplasmic reticulum (ER) and mitochondria to store and release 160 Ca2+ has been extensively characterized (Nicholls 2009; Tang and Zucker 1997; Verkhratsky 161 2005; Wan et al. 2012). To study the potential contribution of these Ca2+ stores in hair-cell 162 synaptic release, we first tested the pharmacological effect of Ca2+ store modulators on the 163 auditory nerve firing rate using an extracellular multiunit preparation (Figure 1A). Spike 164 activity in control experiments was abolished by TTX (1 μM), kynurenic acid (2 mM) or 165 DNQX (1 μM) (Figure 1C), confirming the glutamatergic nature of the hair-cell ribbon 166 synapse and that neural activity was driven by synaptic activity. Caffeine (10-20 mM), a non- 167 specific ER modulator that keeps ryanodine receptors (RyRs) in a semi-opened state (Zucchi 168 and Ronca-Testoni 1997), reduced the spike rate to 28 ± 21% of its initial rate (n = 8, p= 169 0.0001) (Figure 1B-C). BHQ, a blocker of the SERCA pump in the ER, reduced the spike 170 activity to 44 ± 31% of its initial rate (n = 6, p = 0.001). Similarly, ryanodine, which blocks 171 RyRs at high concentrations (60 μM), reduced multiunit activity to 73 ± 26% of control (n = 172 6, p= n.s.). Ruthenium red (40 μM), a non specific inhibitor of RyR, also reduced spike 173 activity (n=1, data not shown). Incubation with drugs known to reduce mitochondrial Ca2+ 174 buffering 175 tetraphenylphosphonium (TPP+) and antimycin A, reduced spike activity to a lesser extent. 176 Spiking was reduced by TPP+ (100 μM) to 74 ± 4% of control (n = 5, p = 0.002) and by 177 antimycin A (10 μM) to 77 ± 22% of control (n = 4, p = n.s.). Although pharmacological 178 manipulation suggested a potential contribution of both mitochondria and CICR Ca2+ stores 179 to synaptic activity, we concentrated our attention in the ER, which provided more robust 180 effects. by interfering with mitochondrial membrane potential, such as 181 The contribution of CICR to hair-cell synaptic transmission was first observed using 182 vestibular nerve recordings (Hendricson and Guth 2002; Lelli et al. 2003; Rossi et al. 2006). 183 In our experiments, application of caffeine reduced the spontaneous spiking rate, consistent 184 with the ER depletion effect reported in other systems (Albrecht et al. 2002; Alonso et al. 185 1999; Hongpaisan et al. 2001; Pozzo-Miller et al. 1997). Additional application of 100 nM 186 apamin, a blocker of the Ca2+-dependent SK channel, did not vary the spontaneous activity 187 reduction obtained with caffeine (data not shown), thus discarding a potential contribution of 188 SK-evoked hyperpolarization due to Ca2+ release from ER. Reduction during prolonged 189 caffeine application could alternatively be explained by eventual Ca2+ depletion in the ER 190 and a consequent impairment of a potential CICR mechanism. 191 Caffeine reduces postsynaptic activity in auditory fibers 192 The pharmacological effects observed in our multiunit preparation cannot, however, 193 distinguish between a pre or postsynaptic contribution of stores to auditory synaptic 194 transmission (Fitzjohn and Collingridge 2002). To obtain further evidence of a potential role 195 for CICR in synaptic activity, we tested the effect of caffeine on excitatory postsynaptic 196 currents (EPSCs) measured from individual afferent fibers from the auditory nerve (Schnee et 197 al. 2013). Postsynaptic afferent patch clamp recordings were made and spontaneous activity 198 recorded from the neurons (Figure 2). Application of caffeine (10 mM) resulted in a net 199 decrease in EPSC frequency that could be recovered upon washout (Figure 2A). Change in 200 frequency for 9 fibers is presented in Figure 2C. Of these, 5 are whole cell recordings and 4 201 are cell attached recordings where spike rate could be monitored. In all cases the frequency of 202 release was reduced. Frequency histograms for EPSC amplitudes were also generated; an 203 example is presented in Figure 2B. The mean EPSC amplitude tended to be reduced, maybe 204 due to a loss of synchrony (Schnee et al. 2013); however, this reduction was not statistically 205 significant (Figure 2D). In three of four fiber recordings there was a transient increase in 206 EPSC frequency followed by a decrease. Additionally, upon washout in three of four cells 207 there was an initial overshoot in EPSC frequency (data no shown), indicative of an increased 208 permeability to calcium, maybe due to the activation of store-operated calcium channels 209 (Lukyanenko et al. 2001). Together these data support the conclusion that there is a 210 presynaptic role for CICR in regulating synaptic vesicle release. 211 Both multiunit and single postsynaptic bouton recordings suggest that 212 pharmacological manipulations of CICR modulates action potential rate by reducing EPSC 213 frequency. The frequency of EPSCs is driven presynaptically as the rate of vesicle fusion. In 214 turn, fusion can be modulated at multiple levels that are direct or indirect. For example, 215 hyperpolarizing the hair cell will reduce release, inactivating Ca2+ channels might reduce 216 release directly, reduction in the Ca2+ current might also alter release properties or vesicle 217 trafficking and recycling (Grant and Fuchs 2008; Johnson et al. 2008; Lee et al. 2007; 218 Magistretti et al. 2015). The following experiments systematically evaluate each potential 219 mechanism for reducing synaptic vesicle fusion. 220 Caffeine has no effect on presynaptic electrical properties 221 A presynaptic effect is postulated to underlie the reduction in postsynaptic activity 222 after pharmacological manipulation of Ca2+ stores (Bouchard et al. 2003). The observed 223 reduction 224 hyperpolarization. Caffeine could trigger the release of Ca2+ from intracellular stores and 225 activate SK potassium channels, thus hyperpolarizing the hair cell and reducing release 226 probability. To examine this possibility, we tested the effect of caffeine perfusion on the 227 electrical properties of hair cells (Figure 3). The responses of hair cells recorded in current- 228 clamp to 10 pA current injects are presented in Figure 3A in the absence and presence of 229 caffeine. We found no change in resting potential and a variable effect on the electrical 230 resonance response. A summary of the resting potential data is presented in the box plots of 231 Figure 3B, where no consistent change was observed. In several cells the quality of the in EPSC frequency could be explained by caffeine-driven hair-cell 232 resonance was reduced, but this was not statistically different, nor did it typically recover 233 upon washing. 234 It is also possible that the reduced frequency of release obtained with caffeine is due 235 to the efflux of calcium from calcium stores, leading to Ca2+ channel inactivation at the 236 synapse (Schnee and Ricci, 2003). We compared hair cell Ca2+ current amplitudes in 237 response to a depolarizing pulse before and during drug application (caffeine or ryanodine) in 238 the external solution (Figure 3C). The same protocol (two 3-s depolarizing pulses separated 239 by 5 min) was performed using drugs in the internal solution (8Br-cAMP or thapsigargin) 240 (Figure 3D). Data summarized in Figure 3C,D suggest that the Ca2+ current amplitude was 241 not significantly altered by drug application. Thus, the most parsimonious explanation for the 242 reduction in vesicle release is a synaptically-driven mechanism. 243 We also investigated the macroscopic currents elicited in voltage-clamp (Figure 3E- 244 H). Macroscopic currents elicited from both hyperpolarizing and depolarizing voltage steps 245 did not vary significantly in the presence of caffeine. Current-voltage plots elicited from tail 246 currents at the time point indicated by the blue line and plotted in Figure 3G were also not 247 significantly different between control and caffeine treated. A summary of the voltages of 248 half activation shown in Figure 3H were also not significantly different. Therefore, these data 249 indicate that the changes recorded postsynaptically during caffeine perfusion are likely to be 250 a direct effect on synaptic properties within the hair cell. 251 Time dependence of superlinear release 252 The two sine technique (Schnee et al., 2011) was first used to monitor vesicle release during 253 cell depolarization to avoid the intercellular variability found in hair cells, particularly with 254 repeated measures (Levic et al. 2011; Patel et al. 2012; Quinones et al. 2012; Rutherford and 255 Roberts 2006). Two components of release were observed, an initial linear component, 256 previously demonstrated to be accounted for by vesicles within 0.7 µms of the synapses, and 257 a larger superlinear component that requires recruitment of additional vesicles (Figure 4A) 258 (Schnee et al. 2005; Schnee et al. 2011). The first linear release component is proportional to 259 the Ca2+ load (integral of calcium current) and correlates with the vesicle population near to 260 the synapses (Schnee et.al., 2005, 2011). In our hands the traditional pool descriptions of 261 rapidly and readily released are less valuable in that pool size as measured by depletion varies 262 within a given cell quite substantially and is often difficult to observe, suggesting a very 263 dynamic population of vesicles with robust recruitment (Schnee et.al., 2005, 2011). The first 264 component includes these pools but likely additional vesicles from immediately near the 265 synapse and potentially some vesicles that are recruited during depolarization. The second 266 superlinear component presents a higher release rate and is independent of Ca2+ load. Similar 267 superlinear release components are observed in other secretory cells (Andersson et al. 2011; 268 Seward et al. 1996) as well as photoreceptors and hair cells by applying trains of short 269 stimulation pulses using single sine capacitance techniques (Duncker et al. 2013; Innocenti 270 and Heidelberger 2008; Moser and Beutner 2000). At present, we consider the superlinear 271 release to represent the hair cell´s exocytotic activity when release sites are maximally filled 272 and releasing. This release component requires significant recruitment of vesicles (Schnee et. 273 al., 2005, 2011). This interpretation remains a hypothesis requiring further investigation. 274 Calcium imaging experiments also showed a non-linear rise in the intracellular Ca2+ levels 275 accompanying the superlinear capacitance change (Schnee et al. 2011). One possible 276 interpretation of this nonlinear signaling is a potential second internal source of Ca2+, maybe 277 supporting CICR. 278 Our postsynaptic results point to a potential role of hair cell ER Ca2+ homeostasis in 279 the ability to sustain ribbon synaptic transmission. To test whether ER Ca2+ homeostasis 280 modulates vesicle release, we used the dual sine capacitance technique to monitor release 281 properties under control conditions (Figure 4) and during pharmacological treatment (Figure 282 5-7). Previous experiments using single sine stimuli have demonstrated a presynaptic 283 facilitation effect that results in variation in responses to repeated stimuli which could make 284 interpretation of pharmacological manipulations requiring repeated measures difficult (Cho et 285 al. 2011; Schnee et al. 2011). Further, since repeated stimulation using long depolarizing 286 pulses has not been described, we first characterized release properties of control hair cells in 287 order to establish a comparator for subsequent pharmacological experiments. Control cells 288 were voltage-clamped to -85 mV and consecutive 3-s stimulation pulses to 50% of peak Ca2+ 289 current (as estimated from IV plots generated for each cell) were tested. These relatively long 290 protocols were selected to ensure observation and separation of the two release components, 291 while at the same time not overtaxing the cell so that it could replenish for multiple 292 stimulations (Schnee et al. 2005). Also, these stimulations are closer to physiological than 293 traditional depolarizations where potentials are stepped to elicit peak calcium currents. 294 Figure 4B shows an example in which hair cell calcium current and capacitance were 295 measured sequentially in response to a stimulation that elicited a linear and superlinear 296 component. In this example, multiple depolarizing pulses separated by 1 min lead to a slight 297 release reduction, consistent with vesicle depletion. Conversely, release was unexpectedly 298 enhanced when pulses were separated by 3 min (Fig. 4B). The amount of release was not 299 significantly modified when two consecutive pulses were separated by 1 min (Figure 4D). 300 However, longer interpulse intervals (IPI) lead to a significant release increase in all cells 301 studied. Changes in total release as well as linear and superlinear release were tested by two 302 consecutive pulses using different IPIs (1, 3 and 5 min) in a total of 19 cells. The linear 303 component was quantified by measuring the increase in membrane capacitance from the 304 onset of depolarization until the appearance of the superlinear component. Since we observed 305 variability in the onset of the superlinear component between pulses (see below), linear 306 release was measured in both pulses until the same time point, that is, until the superlinear 307 onset of the second pulse. For pulses separated by 1 min, release was the same as the initial 308 value (n= 5 cells) (Fig. 4D). Both components showed non-significant differences in the 309 second pulse: superlinear release reduction of 2 ± 17% and linear release reduction of 4 ± 7% 310 (Figure 4E-F). Similarly, the superlinear release onset was constant in the second pulse (0.02 311 ± 0.1s, n.s.) (Figure 4G). However, when pulses were separated by 3 min, consecutive pulses 312 unexpectedly lead to total release enhancement (38 ± 25% increase, n = 5 cells, p= 0.02) (Fig. 313 4D). Enhancement was also observed for 5-min interpulse interval (IPI) (52 ± 32%, n = 9 314 cells, p= 0.0001). Release enhancement was not accompanied by an increase in Ca2+ load (1 315 ± 2% increase for 3-min IPI, 5 ± 8% for 5-min IPI). The increase in release resulted from an 316 increase in the superlinear component for IPIs of 3-min (110 ± 42% increase, p=0.007) and 5- 317 min (115 ± 39%, p= 0.0001, for 5 min)(Fig. 4F), whereas the linear component did not 318 change significantly (5 ± 11% reduction for 3 min, 4 ± 10% increase for 5 min)(compare y- 319 axis in Figure 4E and 4F). The observed release enhancement was originated by a shortening 320 in the superlinear release onset time for IPIs of 3-min (0.6 ± 0.3s, p= 0.01) as well as 5-min 321 (0.5 ± 0.2s, p = 0.0001) (Fig. 4C and G) and not by a change in the rate (slope) of the 322 superlinear release. These data show that 1 min is enough for hair cell buffering mechanisms 323 to reach a steady state in cytoplasmic calcium levels after the long depolarizing pulse. 324 Therefore the observed increase in release after 3-5 minutes cannot be accounted for by an 325 increase in baseline calcium levels due to buffer saturation. Release enhancement, however, 326 may be consistent with an increase in ER Ca2+ reuptake leading to an increase in luminal Ca2+ 327 levels for subsequent stimulation pulses. Alternatively, it may underlie the activation of Ca2+- 328 dependent second messengers that could modulate Ca2+-stores, sensitivity to Ca2+ influx or 329 activate store-dependent expression of synaptic proteins (Alkon et al. 1998; Benech et al. 330 1999; Sutton et al. 1999). Although we cannot categorically interpret the physiological 331 relevance of this release shift, the control data are needed for the pharmacological 332 experiments described below. As these data demonstrate that repeated stimulations alter the 333 response, it is critical to characterize the change in order to accurately assess pharmacological 334 manipulations that require repeated measures. 335 To study whether the shift in the superlinear release onset was Ca2+-dependent and 336 release-specific or could also affect other Ca2+-dependent processes taking place distant to the 337 synaptic ribbon, we tested the threshold for SK channel activation, a channel located near 338 efferent terminals (Lioudyno et al. 2004). Hair cells were stimulated and SK was monitored 339 by removing apamin from the external solution (see examples in Figure 5A). In 120 of 124 340 cells superlinear release was preceded 0.7 ± 0.9 s by SK activation. Superlinear release onset 341 correlated with SK onset (R2 = 0.93) demonstrating that Ca2+ levels reached intracellular 342 locations not necessarily associated to the vicinity of the ribbon. In mammalian outer hair 343 cells, an ER-like cistern opposing every efferent contact is proposed to act as a Ca2+ store to 344 amplify Ca2+ levels for SK activation evoked by nicotinic acetylcholine receptor (nAChR) 345 opening (Fuchs 2014; Lioudyno et al. 2004). In these studies, ryanodine and other store 346 modulators alter SK currents, a result that contrasts with our experiments, where such an 347 obvious effect was not observed during continuous application of curare, an inhibitor of 348 nAChRs. The activation of SK channels could be directly triggered by the influx of Ca2+ 349 through L-type Ca2+ channels, thus circumventing the need for stored Ca2+ efflux to activate 350 SK channels during depolarization. Moreover, the sensitivity to Ca2+ might be biochemically 351 modulated and thus underlie changes in timing as has been described in neurons (Adelman et 352 al. 2012). 353 Pharmacological manipulation of calcium stores reduces hair-cell release 354 The effect of Ca2+ store modulators in hair-cell release was tested using the dual sine 355 capacitance technique (Figure 5,6). Figure 5A shows the release enhancement observed in 356 consecutive control stimulations, mainly due to the early onset of the superlinear component. 357 Total release increased by an average of 52% when comparing two pulses separated by 5 min 358 (Figure 6A). Data are normalized to account for the facilitation observed in control 359 conditions; thus, if there were no facilitation the baseline for control would be at 0 (as in the 360 linear response) (Figure 6). Linear release component increased 4% whereas superlinear 361 release increased by 115% (Figure 6B-C). The increases seen in response to a second pulse 362 are used as the comparator for the pharmacological manipulations such that the value for total 363 release in control is 152%, whereas the linear release was 104% and superlinear release 215% 364 of first pulse. The same depolarization protocol was delivered before and after continuous 365 extracellular application of 10 mM caffeine (Figure 5B) or 10 μM ryanodine. Both pulses 366 were separated in time by 5-10 min to assure drug access through the papilla. In the presence 367 of caffeine, mean total release in the second pulse was 37% of first pulse release (Figure 6A), 368 where linear release reached 65% and superlinear release 40% of the control pulse. 369 Ryanodine reduced total release to 58% of the initial pulse, where mean linear release 370 reached 85% and superlinear release just 41% of control values. Similarly, when 30 μM 8- 371 Br-cADPR, a ryanodine receptor antagonist, was included in the internal solution, total 372 release was 46% of control (Figure 5C). Addition of 0.5-2 μM thapsigargin, a SERCA 373 inhibitor, to the internal solution reduced release to 65% of control with the linear component 374 maintaining 89% of control and the superlinear reducing to 56% of control. Inclusion of 1 375 μM Xestospongin C, an IP3R blocker, had minimal effects on total release at 85% of control, 376 while 4 μM cyclic-ADP-ribose (cADPR), a RyR agonist, also showed limited efficacy 377 reaching 95% of control release values. The shift in the superlinear release onset observed in 378 controls was only significantly reduced by ryanodine application (Fig. 6D). 379 Total release was significantly reduced by 4 of the 6 compounds tested, with those 380 altering ryanodine receptors being more effective (Fig. 6A). Separating effects into release 381 components demonstrate that the largest effects were on the superlinear component. Figure 382 6B shows that only 3 compounds significantly affected the linear component of release. 383 Overall linear release in the second pulse was reduced to 84 ± 26% of control while the 384 superlinear component was reduced to 53 ± 29% of control. 19/29 pharmacologically-treated 385 cells showed a reduction from baseline in superlinear release (Figure 6B,C) as compared to 386 0/9 control cells. Thus the magnitude of the pharmacologically-driven release reduction was 387 more robust in the superlinear response (compare y-axis in Figure 6B and C). Furthermore, 388 the drug effects are likely underestimated when compounds are used in the patch electrode 389 because their effect likely begins prior to the initial measurement. Supporting this contention 390 is the reduction in maximal release rate for the first response in cells where drugs are 391 intracellularly perfused (0.5 ± 0.3pF/s, n = 19) as compared to control values (0.8 ± 0.6 pF/s, 392 n = 19). 393 The superlinear component of release could be reduced in several ways, first the onset 394 of superlinear release could be lengthened, secondly the maximal response could be reduced 395 (saturation), and third the rate of release (slope) could be reduced. Figure 6D demonstrates 396 that the onset of the superlinear response was only affected minimally and only statistically 397 significant for ryanodine. Also saturation of release did not appear to be the limiting factor as 398 depicted in the examples of Figure 5. As also seen in Figure 5, the maximal release rate 399 (slope) was reduced in the presence of drugs that inhibited CICR. In pharmacologically- 400 treated cells where total release reduction was significant, the mean release rate change (rate 401 2nd pulse - rate 1st pulse) was reduced by 40% ± 37% (reduction observed in 18 of 23 cells). 402 In controls, the mean release rate change was reduced by 9% ± 44% (reduction observed in 4 403 of 9 cells). A reduction in the rate of release can be interpreted as a reduction in vesicle 404 trafficking where trafficking is Ca2+ dependent. 405 Using dual sine capacitance recordings, our pharmacological data confirm previous 406 reports of a potential role of intracellular Ca2+ stores in hair cell synaptic physiology (Beurg 407 et al. 2005; Evans et al. 2000; Hendricson and Guth 2002; Kennedy and Meech 2002; Lelli et 408 al. 2003; Marcotti et al. 2004). As opposed to the release enhancement observed in controls, 409 hair cell pharmacological treatment lead to a reduction in both linear and superlinear release 410 after repeated stimulation, with the superlinear release component most clearly affected. This 411 release reduction was not accompanied by a reduction in the peak Ca2+ current and, therefore, 412 cannot be accounted for by a pharmacological effect of intracellular Ca2+ release on Ca2+ 413 channel inactivation (Lee et al. 2007). Altogether, these results suggest that the recruitment of 414 vesicles for release during prolonged stimulation might be physiologically linked to a CICR 415 mechanism. 416 Calcium triggers linear and superlinear release more efficiently than barium. 417 Barium (Ba2+) activates CICR with lower efficacy than Ca2+; it slows down exocytosis and 418 reduces the number of vesicles available for release (Neves et al. 2001; Proks and Ashcroft 419 1995; Przywara et al. 1993; Seward et al. 1996; von Ruden et al. 1993). Moreover, Ba2+ is not 420 reuptaken into the store through SERCA pumps (Kwan and Putney 1990; Przywara et al. 421 1993). To study whether Ba2+ alters linear or superlinear capacitance changes we replaced 422 external Ca2+ with equimolar Ba2+ (Figure 7). After evoking superlinear release with a 3-s 423 depolarization to 50% peak current, extracellular Ca2+ was substituted by equimolar Ba2+, and 424 release was tested again using the same protocol. As expected, Ba2+ abolished SK activation, 425 increased peak currents and reduced Ca2+ channel inactivation (Figure 7A)(Schnee and Ricci 426 2003). Barium exerted two different effects on release: reduced total release and delayed the 427 onset of the initial linear release component (Figure 7A). Box plots of total release for each 428 stimulus obtained with external Ca2+ or Ba2+ are plotted in Figure 7B. The response in Ca2+ 429 shows release enhancement in the 2nd and 3rd responses whereas the responses in Ba2+ were 430 unchanged or reduced, in spite of the larger currents observed (487 ± 185 pA in Ba2+ vs. Ca2+ 431 408 ± 173 pA, p = 0.01). Barium reduced total release in a second stimulation (72% of 432 control) and even further in a third pulse (46% of control). Normalizing release output more 433 clearly shows the enhancement observed in Ca2+ that is absent in the presence of Ba2+ (Figure 434 7C). The time to initial release was reduced in the presence of Ba2+ (Figure 7D) and the 435 delay was further increased for a third stimulation. The delay may be a reflection of Ba2+ 436 being less effective at driving release mechanisms (Bhalla et al. 2005). Additionally, Ba2+ 437 application promoted a merging of the two components. The linearization of the capacitance 438 response may reflect a single source of divalent ions driving the process as CICR is disabled 439 with Ba2+ application. 440 The maximum slope of the superlinear release component for a third stimulation was 441 reduced by 36 ± 30 % during Ba2+ application, as opposed to controls, which increased by 442 141 ± 119 % (p = 0.01) of the initial maximum slope in the first pulse (Figure 7F). Unlike 443 the superlinear decrease in release, the first component showed no change in release 444 following the delay. To make this comparison we measured the first component of release in 445 both Ca2+ and Ba2+ 500 ms after the onset of the capacitance change, thus bypassing the delay 446 induced by Ba2+ (Figure 7E). Eliminating the onset delays demonstrates that the first release 447 component was not significantly altered in amplitude by the divalent replacement. The delay 448 in the onset of release could be partially explained by a poor sensitivity for Ba2+ in the 449 presynaptic Ca2+ sensors for vesicle release and recruitment as suggested for SNARE- 450 mediated exocytosis (Bhalla et al. 2005). However, the role of SNARE proteins in vesicle 451 fusion at hair cell ribbon synapses remains controversial (Nouvian et al. 2011). The 452 intensified effect observed with multiple stimulation protocols, together with the marked 453 effect of Ba2+ on the superlinear release component (Figure 7F) are consistent with the 454 existence of a CICR mechanism governing the recruitment of new vesicles for release. 455 Discussion 456 The existence of Ca2+-induced Ca2+ release (CICR) has long been observed in conventional 457 as well as ribbon synapses (Bouchard et al. 2003; Castellano-Munoz and Ricci 2014). Yet the 458 functional significance and cellular mechanisms underlying CICR in these cell types are 459 unclear. Here, we studied the potential contribution of CICR at hair cell ribbon synapses and 460 obtained the following results. First, spontaneous postsynaptic multiunit activity, along with 461 EPSC frequency, was reduced by pharmacological manipulations that depleted Ca2+ stores 462 through ryanodine receptors. Second, no presynaptic changes in excitability, like a change in 463 resting potential or sensitivity could account for the reduction in postsynaptic response. 464 Third, pharmacological presynaptic effects on CICR resulted in a reduction of the superlinear 465 capacitance change that was larger and occurred earlier than changes to the first component 466 of release, consistent with the hypothesis that CICR is important for modulating vesicle 467 trafficking. Fourth, Ca2+ substitution by Ba2+ delayed the onset of release, likely due to its 468 ability to drive synaptic machinery. It also reduced superlinear release more than the first 469 component of release and this effect was greater for larger stimulations. 470 Our data suggest a role for ryanodine receptors regulating CICR from ER. 471 Identification of the location of the ER responsible for controlling synaptic vesicle 472 populations remains under investigation. Although mitochondria are highly localized to the 473 synaptic region (Graydon et al. 2011; Schnee et al. 2005), the role of the mitochondria as a 474 Ca2+ sink remains suspect, as our pharmacological data show some mild effect but one that 475 cannot easily be separated from the energy generation role typically ascribed to these 476 organelles. Likely the role of mitochondria at hair cell ribbons is similar to the Ca2+ buffering 477 role described at ribbon synapses in the visual pathway, in which mitochondria provide ATP 478 for Ca2+ pumps and also sequester Ca2+ from cytoplasm (Babai et al. 2010; Cadetti et al. 479 2006; Krizaj et al. 2003; Suryanarayanan and Slaughter 2006; Wan et al. 2012; Zenisek and 480 Matthews 2000). 481 The clear distinction between linear and superlinear release components can only be 482 experimentally obtained by depolarizing to potentials that allow less than maximal calcium 483 currents from hyperpolarized holding potentials (Schnee et al. 2011). In turtle hair cells, 484 prepulses to physiological membrane potentials augment release and trigger the convergence 485 of linear and superlinear release components (Schnee et al. 2011). Similarly, in postnatal rat 486 IHCs, depolarizing pulses preceded by holding the cell at physiological resting potentials 487 produced an increase in exocytosis and synaptic strength and a shortening of synaptic latency 488 at hair cell ribbon synapses as compared to holding the cell at more hyperpolarized potentials 489 (Goutman and Glowatzki 2011). We hypothesize that the superlinear response represents all 490 synaptic release sites being filled and fusion happening at maximal rates due to the enhanced 491 recruitment of synaptic vesicles. We further hypothesize that changes in release rates for the 492 linear component of release may in part represent release from multiple release sites where 493 the number of filled sites increases with Ca2+ load. This hypothesis also can explain the 494 variability in obtaining capacitance responses that show depletion (Schnee et al., 2005, 2011) 495 because if the calcium entry is low enough not to trigger recruitment then the pool size will 496 be set by vesicles present at any given moment. 497 It remains a question as to whether superlinear capacitance changes reflect synaptic 498 vesicle fusion, extrasynaptic vesicle fusion or even fusion of endosomes (Coggins et al. 2007; 499 Zenisek et al. 2000). Our pharmacological approach showed a postsynaptic reduction in spike 500 and EPSC rates that correlates with a presynaptic release reduction. Whereas hair cell linear 501 release was mildly reduced by drugs interfering with CICR, superlinear release was strongly 502 reduced. Similarly, substitution of Ca2+ by Ba2+ exerted a stronger effect on the superlinear 503 component of release. These results demonstrate that both release components are Ca2+ 504 dependent and likely interrelated, suggesting that superlinear release might have a 505 physiological role on synaptic transmission. Whether the release is synaptic or extrasynaptic 506 requires better resolution than what we have at the moment (Chen et al. 2014). However, 507 distributions of glutamate receptors as well as a lack of vesicles at nonsynaptic release sites in 508 hair cells would also support a synaptic role (Lenzi et al. 1999; Liberman et al. 2011; Schnee 509 et al. 2005). Also given that the postsynaptic response was a dramatic reduction in firing rate 510 and the major presynaptic response was a decrease in the superlinear capacitance response, it 511 is plausible that the superlinear response is a reflection of robust vesicle trafficking to the 512 synapse. 513 In neurons, Ca2+ regulates exocytosis of synaptic vesicles as well as the supply of new 514 vesicles to release sites (Dittman and Regehr 1998; Gomis et al. 1999; Stevens and Wesseling 515 1998; Wang and Kaczmarek 1998). Moreover, the intracellular release of Ca2+ by CICR has a 516 presynaptic role in neuronal synaptic transmission (Llano et al. 2000; Unni et al. 2004). CICR 517 is observed in hair cells of different animals in both auditory and vestibular organs, (Beurg et 518 al. 2005; Evans et al. 2000; Hendricson and Guth 2002; Kennedy and Meech 2002; Lelli et 519 al. 2003; Marcotti et al. 2004; Tucker and Fettiplace 1995). What could the physiological 520 significance of CICR in hair cells be? Hair cells must maintain a tight regulation between 521 Ca2+ influx through L-type Ca2+ channels and vesicle release at ribbon synapses to allow 522 precise control of release timing. This regulation is achieved in the vicinity of the ribbon by 523 controlling Ca2+ levels through buffering and extrusion mechanisms that locally rapidly 524 remove Ca2+. Given that the recruitment of vesicles for release during prolonged stimulation 525 is Ca2+-dependent, how do hair cells manage to bypass the strong Ca2+ regulation near the 526 ribbon to achieve Ca2+-dependent vesicle recruitment? One possibility is that the Kd of 527 trafficking is much lower than that for release. Alternatively, CICR may serve to amplify and 528 filter the synaptic signal (Figure 8). Thus, during continuous stimulation, the opening of L- 529 type Ca2+ channels could tightly modulate the fusion of vesicles near the ribbon while 530 amplification of this signal away from the synapse through CICR may trigger vesicle 531 recruitment. These calcium-dependent processes may be additionally modulated by the 532 effects of efferent activity on hair cell synaptic calcium levels (Im et al. 2014). We 533 hypothesize that CICR could have a functional role in the recruitment and replenishment of 534 synaptic vesicles to guarantee the availability of vesicles for release during protracted 535 stimulation. 536 Another puzzling finding is the within cell variability observed with repeated 537 stimulations. This was observed in turtle using the single sine technique with paired 538 stimulation (Schnee et al., 2011) and often appears as a facilitation of release with repeated 539 measures, particularly when interpulse intervals are short. Here we also identify a slower 540 facilitation that enhances release by shortening the onset time to superlinear with repeated 541 stimulations that have longer (minute) interpulse intervals. Together these data question 542 measurements where signal averaging is used or where multiple data points need to be 543 obtained via repetitive stimuli. More importantly though, is the question as to the 544 physiological relevance of the variability. The variability appears to be biological in that no 545 other measured biophysical parameters are changing over this time frame. This enhancement 546 could simply be due to changes in calcium levels or reflect a modulation in the sensitivity 547 triggered by a biochemical modification. In our experiments the onset of SK channel 548 activation paralleled superlinear release onset. Both phenomena are unaffected by CICR 549 pharmacological treatment, thus ruling out the possibility that release enhancement is simply 550 due to calcium baseline modulation. Moreover, the calcium sensitivity of SK2 and SK3 551 channels depends on the phosphorylation state of SK-bound calmodulin (Adelman et al. 552 2012), pointing to a phosphorylation modulation as the origin of the release and SK onset 553 shift. 554 One possibility consistent with present data is that the Ca2+ stores are incompletely 555 filled under our recording conditions and initial stimulations serve to fill this pool, which can 556 then be more efficient at driving vesicles to release sites. The stores might be considered in a 557 dynamic equilibrium with cytosolic Ca2+ where it can act as both sink and source depending 558 on excitation level. Thus holding a cell at -80 mV and dialyzing with ATP and a Ca2+ buffer 559 moves the equilibrium toward store depletion so that Ca2+ is driven into stores upon 560 stimulation. Thus this Ca2+ is available for release upon further stimulation. Consistent with 561 this idea, is that using holding potentials more depolarized leads to more robust release 562 (Schnee et al. 2011). 563 Another confusing component to these data is the variability in responsiveness to the 564 drug applications, even those where drugs were included within the patch pipette. Not all 565 cells responded and those that did showed a larger than expected variance. Again the 566 variation appears to be biologically driven and not a function of the biophysical status of the 567 hair cells. One possibility relates to the previous discussion that drug efficacy will be directly 568 determined by the state of the store (how filled it is) at the time of drug administration. 569 Another confounding point is that turtle hair cells have large Ca2+ currents and it is likely that 570 large depolarizations that lead to major influx via Ca2+ channels can mask the effects of Ca2+ 571 stores. Alternatively, there may be additional biochemical modifications that we are not 572 controlling, for example CAM kinase activity. 573 In summary, data presented identify a presynaptic role for CICR to modulate hair cell 574 vesicle release. Both pre and postsynaptic recordings support the argument that CICR may 575 regulate vesicle trafficking. Data demonstrate the involvement of Ryanodine receptors in this 576 pathway. Capacitance measurements target the major effect to a reduction in the superlinear 577 component of release, previously argued to require vesicle recruitment to the synapses. These 578 data indirectly support the contention that both linear and superlinear capacitance changes 579 might be synaptic in nature. Further a novel hypothesis is put forth suggesting that CICR is 580 needed to amplify and filter the tightly controlled synaptic Ca2+ signal to ensure continuous 581 and reliable vesicle trafficking required to maintain high and sustainable afferent firing rates. 582 Acknowledgemenets 583 We thank the members of our research group for providing helpful comments on the project 584 and the manuscript. This work was funded by NIDCD grant DC009913 to A.J.R. and by core 585 grant P30 44992. M.C.-M. was supported by a Dean’s Postdoctoral Fellowship from Stanford 586 School of Medicine and a Cajamadrid Foundation Fellowship. 587 588 References 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 Adelman JP, Maylie J, and Sah P. Small-conductance Ca2+-activated K+ channels: form and function. Annu Rev Physiol 74: 245-269, 2012. 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Zenisek D, and Matthews G. The role of mitochondria in presynaptic calcium handling at a ribbon synapse. Neuron 25: 229-237, 2000. Zenisek D, Steyer JA, and Almers W. Transport, capture and exocytosis of single synaptic vesicles at active zones. Nature 406: 849-854, 2000. Zucchi R, and Ronca-Testoni S. The sarcoplasmic reticulum Ca2+ channel/ryanodine receptor: modulation by endogenous effectors, drugs and disease states. Pharmacol Rev 49: 1-51, 1997. 776 777 778 779 780 781 782 783 784 785 Figure 1: Pharmacological disruption of intracellular calcium stores reduces extracellular spike activity in the 8th cranial nerve. A- presents low power view of half head preparation with the recording suction electrode and drug application pipette labeled. B- Upper panel shows the effect of caffeine application in spontaneous nerve activity. Lower panel presents spontaneous spike rate. Asterisk shows the time point of maximum effect after drug delivery selected for rate quantification. C- Box plots illustrating spike rate reduction by drugs that interfere with ER calcium homeostasis (10 mM caffeine, 50-100 μM BHQ and 60 μM ryanodine). Pharmacological modulation of mitochondrial calcium homeostasis (100 μM Tetraphenylphosphonium (TPP+) and 10 μM antimycin A) reduced spontaneous rate to a lesser extent. Kynurenic acid (KynA) and DNQX, two glutamatergic receptor antagonists, were used as controls. Paired t-tests show a significant difference at p<0.01 level (**). 786 787 788 789 790 791 792 793 794 795 Figure 2: Postsynaptic patch recordings from afferent terminals show a reduction in EPSC frequency and firing rate with caffeine application. A- presents spontaneous EPSCs from a whole cell recording of an afferent terminal. The red traces are in the presence of 10 mM caffeine and the blue traces are activity during washout of the drug. B- presents frequency histograms for EPSC amplitudes in the absence (black) and presence (red) of caffeine, where the solid lines are Gaussian fits to the histograms. C- plots the EPSC frequency (or the extracellular spike frequency) before and after caffeine administration. Paired t-tests show a significant difference at p<0.001 level. D presents bar graph of the mean EPSC amplitude as measured from the fits to the Gaussian curves in (B). Although a trend toward smaller mean amplitudes was found, there was no statistical difference observed. 796 797 798 799 800 801 802 Figure 3: Presynaptic excitability is not responsible for the reduction in vesicle release. A- presents membrane potential responses from a hair cell elicited by a 10 pA current injection about the cell’s resting potential, where the upper trace is control and the lower trace is in the presence of 10 mM caffeine. The slight reduction in quality of resonance is not significantly different. B- provides box plots of the zero current potential in the absence (black) and presence (red) of caffeine. No statistical difference is noted. C- plots the calcium current elicited in paired stimulus for cells in the 803 804 805 806 807 808 809 810 811 absence and presence of drug (color coded where red is caffeine, blue is ryanodine and black is control). No change is identified. D- summarizes calcium currents elicited from the first stimulus (darker colors) and the second stimulus (lighter shades) for drugs that were applied internally. No significant differences were found. E- provides voltage clamp responses for step voltages changes between -104 and +60 in 20 mV increments from a holding potential of -84 mV for control and caffeine treated (F). Tail current voltage plots generated at the time point indicated by the blue line in E are presented in G, where black is control and red caffeine. No difference was found between plots. H- presents box plots for the half-activating voltages obtained from the data in G. No significant difference was observed. 812 813 814 815 816 817 818 819 820 821 822 Figure 4: Long interpulse intervals result in an increase in hair cell superlinear release. Arepresentative example showing linear and superlinear components of release using two-sine capacitance method. B- Consecutive 3s-depolarizing pulses to 50% of peak current were delivered in whole cell. Upper trace shows release and lower trace presents calcium currents. Note the increase in release for 3 min interpulse interval (IPI) (scale: 100 fF/10 pA, 1 s). C- Traces from A superimposed. Note that superlinear release onset (arrowheads) started earlier after longer interpulse intervals. DNormalized data showing that release was enhanced only for 3-5 min interpulse intervals (n=19 cells). E-F-Boxplots showing that superlinear release increased only for 3-5 min intervals whereas linear release was unchanged. G- Superlinear onset shortened only for 3-5 min intervals. Paired ttests show a significant difference at p<0.01 level (**). 823 824 825 826 827 828 829 830 831 832 Figure 5: Pharmacological disruption of ryanodine receptors reduces hair cell release. A- Three control 3-s depolarizing pulses were delivered 5 min apart and current and Cm were monitored. Consecutive protocols showed release enhancement (dotted line). SK and superliner release onsets were triggered earlier in successive stimulations. Arrowheads show SK activation and arrows show initial linear release onset. Baseline was subtracted in order to compare the three pulses. BExtracellular caffeine application (10 mM) reduced release whereas SK current behaved as in control experiments. Caffeine additionally reduced peak current in 4 of 6 cells. C - Intracellular application of 8-Br-cADPR (30 μM) reduced release with no effect on the calcium load. Arrowhead points superlinear onset. SK current was blocked in this cell by apamin in the external solution. 833 834 835 836 837 838 839 840 841 842 Figure 6: Pharmacological disruption of intracellular calcium stores reduces hair cell release. ABoxplots showing that caffeine, 8-Br-cADPR, ryanodine and thapsigargin significantly reduced release. See dotted line for comparison with control. B-C- Linear and superlinear release was reduced by drugs that interfere with ER RyRs (10 mM caffeine, 30 μM 8-Br-cADPR, 10 μM ryanodine). Reduction was more pronounced in the superlinear component of release (note y-axis values in B vs. C). Superlinear release was also reduced significantly by 0.5-2 μM thapsigargin and 1 μM Xestospongin. cADPR (4 μM), an IP3R agonist, had no obvious effects on release. D- Only ryanodine showed a significant effect on superlinear onset. Unpaired t-tests show a significant difference: p<0.05 (*), p<0.01 (**). 843 844 845 846 847 848 849 850 851 852 853 854 Figure 7: Barium delays release and reduces superlinear release component. A- After substituting calcium by barium in the extracellular solution, linear release component onset was delayed and maximal release slopes reduced. Distinction between linear and superlinear components was blurred with subsequent depolarization protocols. Baseline was subtracted in order to compare the three pulses. B- Boxplot showing release values elicited by pulses delivered 5-10 min apart in control conditions and after substituting calcium by barium in the external solution. C- In controls, release enhancement was magnified by subsequent stimulation (black circles). Conversely, barium substitution reduced release (triangles). D- Barium substitution delayed initial release. E- Initial release measured 500 ms after release onset was not significantly altered by barium. F- Maximal release slope increased with successive stimulations in controls but was reduced by barium substitution. Unpaired t-tests show a significant difference: p<0.05 (*), p<0.01 (**). 855 856 857 858 859 860 861 862 863 864 865 866 867 Figure 8: CICR might sustain the recruitment of synaptic vesicles to the ribbon. Left panels represent a depolarizing pulse triggering an increase in capacitance in response to a calcium current. Right panels represent a hair cell presynaptic terminal and the simultaneous postsynaptic responses (EPSCs). A- At hyperpolarized membrane potentials, a small calcium influx triggers the fusion of vesicles near the synaptic ribbon. Prolonged depolarization leads to vesicle depletion and a reduction in EPSC frequency. B- Larger calcium influx triggers the fusion of vesicles near the synaptic ribbon as well as CICR from intracellular stores, allowing vesicle recruitment and an increase in EPSC frequency. The fusion of new recruited vesicles due to CICR is experimentally observed as a superlinear component of release. Note that under physiological conditions hair cells are maintained at depolarized membrane potentials, a situation in which both linear and superlinear components of release might merge. A Drug Delivery Recording Pipette Neck Nose B 0.4 mV 0.2 0 caffeine -0.2 spikes/s 50 * 25 0 0 1000 time (s) 2000 (spike rate-spike rateini) / spike rateini C 1 ** ** ** 0.5 ** ** 0 A e l e tro ynA NQX fein HQ odin PP+ ycin n f B an T tim co K D ca ry an B Events A 25 20 15 10 5 0 0.0 caffeine wash 100 ms 0.4 0.6 EPSCs (nA) D 40 30 EPSC Peak (nA) 200 pA Frequency (Hz) C 0.2 20 10 0 Control Caffeine 0.8 Control 10 mM caffeine 0.4 0.3 0.2 0.1 0.0 control A B -30 Control 10 mM Caffeine -40 -50 Vz (mV) caffeine 5 mV -60 -70 -80 5 ms C D 0.0 -0.1 Control -0.2 -0.2 -0.3 -0.4 -0.5 -0.5 E 8 F 8 6 6 control 4 4 2 2 0 0 -2 caffeine -2 100 125 150 175 100 120 140 160 180 Time (ms) Time (ms) G H 0 -10 1.6 -20 1.2 V1/2 I (nA) thapsigargin -0.3 -0.4 IK (nA) 8Br-cAMP -0.1 ryanodine Control ICa (nA) I Ca (nA) 0.0 caffeine 0.8 -30 -40 -50 0.4 -60 -100 -80 -60 -40 -20 Potential (mV) 0 20 Control caffeine A -30 mV -80 mV linear 1 pF superlinear Cm I Ca2+ 100 pA 1s B 1 1 3 C 1 D min total release pulse 2 (pF) 1.5 1 1 min 3 min 5 min 0.5 0 100 fF 0 0.5 1s E G 0 -0.2 ** 2 ** 2nd-1st (s) n.s. 0.2 1 0 -1 -0.4 1 3 5 IPI (min) ' superlinear onset superlinear release (2nd-1st)/1st (2nd-1st)/1st pulse 1 (pF) F linear release 0.4 1.5 1 1 3 5 IPI (min) 0.5 0 -0.5 -1.0 -1.5 ** 1 ** 3 5 IPI (min) Im (nA) A control 0.4 0.2 0 -0.2 -0.4 Cm (pF) min 10 min 15 min 20 1 0 0 2 4 time (s) 6 0 2 4 time (s) B 0 wash (min 25) ICa2+ (nA) caffeine (min 17) 0 -0.3 6 0 -0.1 -0.2 -0.3 8-Br-cADPR (min 10) 2 min 24 min 13 0.8 min 32 0.4 Cm (pF) -0.6 Cm (pF) 2 4 time (s) C 0.3 Im (nA) 6 1 0 0 8-Br-cADPR (min 15) 1000 0 2 4 time (s) 6 0 2 4 6 0 time (s) 2 4 6 time (s) 2000 3000 4000 5000 0 1 2 3 4 5 time (s) A B linear release total release * ** ** * (2nd-1st/1st) (2nd-1st/1st) ** ** ** l ro ine PR ine PR rgin p C t d n s D a D fe co caf rcA ano cA sig sto e y ap x 8B r th l ro ine PR ine PR rgin p C t d n s D a D fe co caf rcA ano cA sig sto e y ap x 8B r th C D superlinear release ** * ** 2nd-1st (2nd-1st/1st) ** ** ** superlinear onset l ro ine PR ine PR rgin p C t d n s D a D fe co caf rcA ano cA sig sto y ap xe 8B r th l ro ine PR ine PR rgin p C t d n s D a D fe co caf rcA ano cA sig sto y ap xe 8B r th B A total release (s) Im (nA) 0.2 1 Ca 2+ 2.0 2+ -0.2 2 Ba 3 Ba2+ Cm (pF) -0.4 1 2 3 1 total release (pF) 0 control Ba2+ ** 1.5 1.0 0.5 0 0 2 4 6 1st pulse 2nd pulse 3rd pulse Time (s) C D normalized total release Ba2+ control Ba2+ E 3rd pulse 2nd pulse (3rd-1st)/1st (2nd-1st)/1st ** ** ** control time to release (s) Ba2+ control Ba2+ control F normalized max release slope ** * 150 100 50 (3rd-1st)/1st (2nd-1st)/1st initial release (pF) initial release 0 1st pulse 2nd pulse 3rd pulse control Ba2+ control Ba2+ A Vm 1 2 Ca2+ Ca2+ Ca Ca2+ a a2+ 2+ Ca2++ Ca2 EPSCs I Ca2+ Cm linear release B 2 1 superlinear release 2 1 linear release 1 2 Ca2+ Ca2+ Ca Ca2+ a2+ a2+ Ca2+ Ca2 C 2 2++