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J Neurophysiol 115: 226 –239, 2016. First published October 28, 2015; doi:10.1152/jn.00559.2015. Calcium-induced calcium release supports recruitment of synaptic vesicles in auditory hair cells X Manuel Castellano-Muñoz,1 Michael E. Schnee,1 and Anthony J. Ricci1,2 1 Department of Otolaryngology, Stanford University School of Medicine, Stanford, California; and 2Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California Submitted 8 June 2015; accepted in final form 23 October 2015 hair cell; dual-sine capacitance; Ca2⫹-induced Ca2⫹ release; intracellular stores; ribbon synapse; synaptic transmission HAIR CELLS, the sensory receptors in the auditory and vestibular systems, convert mechanical information into synaptic activity through the release of neurotransmitter at ribbon synapses. Each hair cell contains tens of synaptic ribbons (Schnee et al. 2005, 2011; Sneary 1988), presynaptic specializations surrounded by synaptic vesicles and associated to active zones and L-type Ca2⫹ channels (Issa and Hudspeth 1994; Roberts et al. 1990; Tucker and Fettiplace 1995). Similar to other sensory synapses, hair cell ribbon synapses operate in a graded fashion, reaching high release rates and exhibiting little fatigue. Both of these properties require rapid vesicle replenishment by a mechanism that is not well understood. Ca2⫹-induced Ca2⫹ release (CICR) is a mechanism by which the influx of Ca2⫹ through Ca2⫹ channels in the plasma membrane activates Ca2⫹ release from intracellular stores (Verkhratsky 2005). CICR is implicated in a number of neu- Address for reprint requests and other correspondence: M. CastellanoMuñoz, Inst. of Bioengineering, Miguel Hernández Univ., Avenida de la Universidad, s/n 03202 Elche, Alicante, Spain (e-mail: [email protected]). 226 ronal functions such as neuronal excitability, gene expression, and synaptic plasticity and release (Bouchard et al. 2003). In central synapses both endoplasmic reticulum (ER) and mitochondria are well-known intracellular Ca2⫹ stores, and their Ca2⫹ homeostatic modulation alters synaptic transmission preand postsynaptically (Bardo et al. 2006; Emptage et al. 2001; Llano et al. 2000). CICR is also suggested to contribute to synaptic transmission at ribbon synapses (Babai et al. 2010; Lelli et al. 2003). Calcium imaging identified CICR in turtle auditory papilla hair cells (Tucker and Fettiplace 1995), frog semicircular canal (Lelli et al. 2003), P6 –P11 mouse inner hair cells (Iosub et al. 2015; Kennedy and Meech 2002), and rat and guinea pig outer hair cells (Evans et al. 2000; Mammano et al. 1999). In mammalian outer hair cells, CICR is functionally associated to subsynaptic Ca2⫹ stores in close proximity to efferent terminals (Lioudyno et al. 2004). In addition, Ca2⫹ can be released by inositol triphosphate-gated Ca2⫹ stores at the base of the outer hair cell hair bundle (Mammano et al. 1999). Although pharmacological data demonstrate the presence of intracellular stores in hair cells, their physiological role is debatable. Intracellular Ca2⫹ stores have been functionally associated with the control of BK channel activity in inner hair cells (Beurg et al. 2005; Marcotti et al. 2004), modulation of outer hair cell electromotility (Dallos et al. 1997), homeostatic control of presynaptic Ca2⫹ levels (Kennedy and Meech 2002; Tucker and Fettiplace 1995), time-dependent segregation of afferent and efferent signaling (Im et al. 2014), and regulation of vesicular trafficking, exocytosis, and synaptic transmission (Hendricson and Guth 2002; Lelli et al. 2003). Here we performed auditory nerve multiunit and single-unit recordings as well as hair cell dual-sine capacitance experiments to study the potential contribution of CICR to hair cell synaptic transmission. Pharmacological and divalent cation substitution results are consistent with a role for CICR in the recruitment of vesicles to support maintained release in auditory hair cell ribbon synapses. MATERIALS AND METHODS Tissue preparation. The auditory papilla of red-eared sliders (Trachemys scripta elegans) was dissected as previously described (Schnee et al. 2005). All animal procedures were approved by the Stanford Institutional Animal Care and Use Committee (IACUC) and are in accord with National Institutes of Health guidelines and standards. Turtle half-head preparations were used for multiunit activity measurements from the eighth cranial nerve. The turtle head was split in half and pinned in a Sylgard dissection chamber either with an external solution similar to that used in patch-clamp recordings or with bicarbonate-buffered perilymph containing (in mM) 126 NaCl, 0022-3077/16 Copyright © 2016 the American Physiological Society www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.5 on June 18, 2017 Castellano-Muñoz M, Schnee ME, Ricci AJ. Calcium-induced calcium release supports recruitment of synaptic vesicles in auditory hair cells. J Neurophysiol 115: 226 –239, 2016. First published October 28, 2015; doi:10.1152/jn.00559.2015.—Hair cells from auditory and vestibular systems transmit continuous sound and balance information to the central nervous system through the release of synaptic vesicles at ribbon synapses. The high activity experienced by hair cells requires a unique mechanism to sustain recruitment and replenishment of synaptic vesicles for continuous release. Using pre- and postsynaptic electrophysiological recordings, we explored the potential contribution of calcium-induced calcium release (CICR) in modulating the recruitment of vesicles to auditory hair cell ribbon synapses. Pharmacological manipulation of CICR with agents targeting endoplasmic reticulum calcium stores reduced both spontaneous postsynaptic multiunit activity and the frequency of excitatory postsynaptic currents (EPSCs). Pharmacological treatments had no effect on hair cell resting potential or activation curves for calcium and potassium channels. However, these drugs exerted a reduction in vesicle release measured by dual-sine capacitance methods. In addition, calcium substitution by barium reduced release efficacy by delaying release onset and diminishing vesicle recruitment. Together these results demonstrate a role for calcium stores in hair cell ribbon synaptic transmission and suggest a novel contribution of CICR in hair cell vesicle recruitment. We hypothesize that calcium entry via calcium channels is tightly regulated to control timing of vesicle fusion at the synapse, whereas CICR is used to maintain a tonic calcium signal to modulate vesicle trafficking. STORED CALCIUM PROMOTES VESICLE RECRUITMENT TO RIBBON SYNAPSES 2.5 KCl, 13 NaHCO3, 1.7 NaH2PO4, 1.8 CaCl2, 1 MgCl2, and 5 glucose (continuously bubbled with 95% O2-5% CO2). The brain was removed and the auditory nerve exposed (Fig. 1A), cutting the connections to posterior ampulla and saccule. The ventral otic membrane was trimmed to allow access to perfusion prior to mounting of the A Drug Delivery Recording Pipette Neck mV 0.4 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 ol A X ne Q ine + inA ntr Kyn NQ ffei BH nod TPP myc o D ca c ti rya an Fig. 1. Pharmacological disruption of intracellular calcium stores reduces extracellular spike activity in the 8th cranial nerve. A: low-power view of half-head preparation with the recording suction electrode and drug application pipette labeled. B, top: effect of caffeine application on spontaneous nerve activity. Bottom: 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 endoplasmic reticulum (ER) calcium homeostasis (10 mM caffeine, 50 –100 M BHQ, and 60 M ryanodine). In this and subsequent figures, box plots present raw data (symbols), mean (star), and SD (box). Pharmacological modulation of mitochondrial calcium homeostasis [100 M tetraphenylphosphonium (TPP⫹) and 10 M antimycin A] reduced spontaneous rate to a lesser extent. Kynurenic acid (KynA) and DNQX, 2 glutamatergic receptor antagonists, were used as controls. **Paired t-tests show a significant difference at P ⬍ 0.01 level. preparation in the recording dish. A gravity-controlled perfusion pipette was located ⬃5 mm above the otic capsule and was connected to a perfusion system with a flow rate of roughly 1 ml/min. For intracellular hair cell recordings, the inner ear was dissected from the otic capsule in external solution containing (in mM) 128 NaCl, 0.5 KCl, 2.8 CaCl2, 2.2 MgCl2, 10 HEPES, 6 glucose, 2 creatine monohydrate, 2 ascorbate, and 2 pyruvate and pH was adjusted to 7.6 and osmolality to 275 mosmol/kg. The external solution was supplemented with 20 M curare to eliminate efferent activity, and 100 nM apamin was included in some of the experiments to block SK activity. We found no evidence of remaining efferent activity with incubation with curare in both pre- and postsynaptic recordings. To disrupt mechanotransduction channels, after extraneous tissue was trimmed and the otoconia removed the papilla was incubated for 15 min in external solution and perfused with 200 l of 5 mM BAPTA before and after removal of the tectorial membrane with a fine insect pin. In some experiments where the tectorial membrane was left intact, cell visualization was impaired but no obvious electrophysiological effects were observed. The basilar papilla was transferred to the recording chamber and secured with single strands of dental floss. Cells were imaged with an Axioskop 2 FS plus (Zeiss, Thornwood, NY) with bright field optics using a ⫻60 0.9 NA water objective (LUMPlan Fl/IR, Olympus). Perfusion of bath and drugs was delivered with a Minipuls 3 pump (Gilson, Middleton, WI). Electrophysiology. For multiunit activity we used the turtle halfhead preparation, in which the auditory nerve was inserted into an hourglass-shaped suction electrode with a micromanipulator (Narishige, East Meadow, NY) and compound action potentials were recorded with a differential AC preamplifier (Grass, P55 Astro-Med, West Warwick, RI). One electrode was inserted into the borosilicate suction pipette, and the neutral electrode was in contact with the bath. The signal was band-pass filtered (1 Hz–1 kHz) and amplified 1,000 times. Compound action potentials were collected through a data acquisition interface (CED Micro 1401 mkII, Cambridge Electronic Design) and analyzed with Spike2 software (Cambridge Electronic Design). Noise levels were identified by blocking afferent activity with 1 M tetrodotoxin (TTX) or prolonged high potassium concentration, and spike threshold was set to 3 ⫻ SD of baseline noise. Drugs were applied via the local perfusion system after a baseline firing rate was established. Control perfusion with the external solution (sans drug) was used to confirm absence of mechanical artifacts and firing stability throughout recording (96 ⫾ 2% after 20 min, n ⫽ 3). For hair cell patch-clamp experiments, thick-walled borosilicate electrodes of resistance 2.5–3.5 M⍀ were used with internal solution containing (in mM) 110 CsCl, 1 EGTA, 5 creatine phosphate, 3 Na2ATP, 10 HEPES, 3 MgCl2, and 2 ascorbate, pH adjusted to 7.2 and osmolality at 255 mosmol/kg. Stimulus protocols were performed starting 10 min after whole cell configuration to allow solution equilibration and run up stabilization of the Ca2⫹ current (Schnee and Ricci 2003). Hair cells were voltage clamped with an Axopatch 200B (Axon Instruments-Molecular Devices, Sunnyvale, CA) or a VE-2 amplifier (Alembic Instruments, Montreal, ON, Canada). Data were collected at 100 –200 kHz with an IOTech Daq/3000 acquisition board (MC Measurement Computing, Norton, MA) driven by jClamp software (SciSoft). Voltage was intentionally not corrected for junction potential or series resistance to match the values used in two-sine capacitance protocols (Schnee et al. 2011). Dual sinusoidal stimulation was performed to compensate in-cell stray capacitance at different frequencies before capacitance protocols were run. A dual sinusoid with amplitudes and frequencies of 20 –30 mV at 3.1– 6.2 kHz and 6.2–9.4 kHz was delivered superimposed to the desired voltage step. Capacitance measurements were low-pass filtered at 40 Hz, and the onset-offset gating capacitative transients were removed off-line. Afferent fiber patch recordings were done with solutions similar to those described for hair cell recordings (Schnee et al. 2013). Patch electrodes were smaller tipped and had resistances of 9 –11 M⍀. J Neurophysiol • doi:10.1152/jn.00559.2015 • www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.5 on June 18, 2017 B Nose 227 228 STORED CALCIUM PROMOTES VESICLE RECRUITMENT TO RIBBON SYNAPSES RESULTS Pharmacological manipulation of calcium stores reduces auditory nerve activity. The ability of neuronal ER and mitochondria to store and release Ca2⫹ has been extensively characterized (Nicholls 2009; Tang and Zucker 1997; Verkhratsky 2005; Wan et al. 2012). To study the potential contribution of these Ca2⫹ stores in hair cell synaptic release, we first tested the pharmacological effect of Ca2⫹ store modulators on the auditory nerve firing rate, using an extracellular multiunit preparation (Fig. 1A). Spike activity in control experiments was abolished by TTX (1 M), kynurenic acid (2 mM), or DNQX (1 M) (Fig. 1C), confirming the glutamatergic nature of the hair cell ribbon synapse and that neural activity was driven by synaptic activity. Caffeine (10 –20 mM), a nonspecific ER modulator that keeps ryanodine receptors (RyRs) in a semiopened state (Zucchi and Ronca-Testoni 1997), reduced the spike rate to 28 ⫾ 21% of its initial rate (n ⫽ 8, P ⫽ 0.0001) (Fig. 1, B and C). BHQ, a blocker of the SERCA pump in the ER, reduced the spike activity to 44 ⫾ 31% of its initial rate (n ⫽ 6, P ⫽ 0.001). Similarly, ryanodine, which blocks RyRs at high concentrations (60 M), reduced multiunit activity to 73 ⫾ 26% of control [n ⫽ 6, P ⫽ not significant (n.s.)]. Ruthenium red (40 M), a nonspecific inhibitor of RyR, also reduced spike activity (n ⫽ 1, data not shown). Incubation with drugs known to reduce mitochondrial Ca2⫹ buffering by interfering with mitochondrial membrane potential, such as tetraphenylphosphonium (TPP⫹) and antimycin A, reduced spike activity to a lesser extent. Spiking was reduced by TPP⫹ (100 M) to 74 ⫾ 4% of control (n ⫽ 5, P ⫽ 0.002) and by antimycin A (10 M) to 77 ⫾ 22% of control (n ⫽ 4, P ⫽ n.s.). Although pharmacological manipulation suggested a potential contribution of both mitochondrial and CICR Ca2⫹ stores to synaptic activity, we concentrated our attention on the ER, which provided more robust effects. The contribution of CICR to hair cell synaptic transmission was first observed with vestibular nerve recordings (Hendricson and Guth 2002; Lelli et al. 2003; Rossi et al. 2006). In our experiments, application of caffeine reduced the spontaneous spiking rate, consistent with the ER depletion effect reported in other systems (Albrecht et al. 2002; Alonso et al. 1999; Hongpaisan et al. 2001; Pozzo-Miller et al. 1997). Additional application of 100 nM apamin, a blocker of the Ca2⫹-depen- dent SK channel, did not vary the spontaneous activity reduction obtained with caffeine (data not shown), thus ruling out a potential contribution of SK-evoked hyperpolarization due to Ca2⫹ release from ER. Reduction during prolonged caffeine application could alternatively be explained by eventual Ca2⫹ depletion in the ER and a consequent impairment of a potential CICR mechanism. Caffeine reduces postsynaptic activity in auditory fibers. The pharmacological effects observed in our multiunit preparation cannot, however, distinguish between a presynaptic and a postsynaptic contribution of stores to auditory synaptic transmission (Fitzjohn and Collingridge 2002). To obtain further evidence of a potential role for CICR in synaptic activity, we tested the effect of caffeine on excitatory postsynaptic currents (EPSCs) measured from individual afferent fibers from the auditory nerve (Schnee et al. 2013). Postsynaptic afferent patch-clamp recordings were made and spontaneous activity recorded from the neurons (Fig. 2). Application of caffeine (10 mM) resulted in a net decrease in EPSC frequency that could be recovered upon washout (Fig. 2A). Change in frequency for nine fibers is presented in Fig. 2C. Of these, five are whole cell recordings and four are cell-attached recordings where spike rate could be monitored. In all cases the frequency of release was reduced. Frequency histograms for EPSC amplitudes were also generated; an example is presented in Fig. 2B. The mean EPSC amplitude tended to be reduced, perhaps because of a loss of synchrony (Schnee et al. 2013); however, this reduction was not statistically significant (Fig. 2D). In three of four fiber recordings there was a transient increase in EPSC frequency followed by a decrease. Additionally, upon washout in three of four cells there was an initial overshoot in EPSC frequency (data not shown), indicative of an increased permeability to calcium, perhaps due to the activation of store-operated calcium channels (Lukyanenko et al. 2001). Together these data support the conclusion that there is a presynaptic role for CICR in regulating synaptic vesicle release. Both multiunit and single postsynaptic bouton recordings suggest that pharmacological manipulations of CICR modulate action potential rate by reducing EPSC frequency. The frequency of EPSCs is driven presynaptically as the rate of vesicle fusion. In turn, fusion can be modulated at multiple levels that are direct or indirect. For example, hyperpolarizing the hair cell will reduce release, inactivating Ca2⫹ channels might reduce release directly, and reduction in the Ca2⫹ current might also alter release properties or vesicle trafficking and recycling (Grant and Fuchs 2008; Johnson et al. 2008; Lee et al. 2007; Magistretti et al. 2015). The following experiments systematically evaluate each potential mechanism for reducing synaptic vesicle fusion. Caffeine has no effect on presynaptic electrical properties. A presynaptic effect is postulated to underlie the reduction in postsynaptic activity after pharmacological manipulation of Ca2⫹ stores (Bouchard et al. 2003). The observed reduction in EPSC frequency could be explained by caffeine-driven hair cell hyperpolarization. Caffeine could trigger the release of Ca2⫹ from intracellular stores and activate SK potassium channels, thus hyperpolarizing the hair cell and reducing release probability. To examine this possibility, we tested the effect of caffeine perfusion on the electrical properties of hair cells (Fig. 3). The responses of hair cells recorded in current clamp to 10-pA current injections are presented in Fig. 3A in J Neurophysiol • doi:10.1152/jn.00559.2015 • www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.5 on June 18, 2017 Series resistance was compensated to 70%, resulting in uncompensated series resistance of 11 ⫾ 3 M⍀ (n ⫽ 5). Data analysis. In hair cell patch-clamp experiments, the initial capacitance of cells located at the center of the papilla was 12.4 ⫾ 1.4 pF (n ⫽ 47) 1 min after establishment of whole cell configuration. Cells were discarded when the uncompensated series resistance was ⬎12 M⍀ because of the difficulty in the neutralization of in-cell stray capacitance. Cells were also discarded when the leak current was ⬎50 pA after 9 min of recording to avoid calcium channel inactivation due to slow calcium loading of the cells. Data and graphics show means ⫾ SD and number of experiments (n) unless noted. Statistical analyses were performed with a two-tailed Student’s t-test assuming normal distribution. In the capacitance experiments where multiple release protocols were evoked, normalized percentage values are related to the value evoked in the first pulse. In figures, the y-axis represents the amount of release in a second stimulation protocol normalized by a first stimulation protocol (2nd ⫺ 1st pulse/1st pulse); therefore values below 0 represent a reduction in the second pulse, whereas values near 1 represent twice as much release in the second pulse. STORED CALCIUM PROMOTES VESICLE RECRUITMENT TO RIBBON SYNAPSES B Events A 25 20 15 10 5 0 0.0 C 0.4 0.6 EPSCs (nA) D 30 20 10 0 Control 0.8 Control 0.4 0.3 0.2 0.1 0.0 Fig. 2. Postsynaptic patch recordings from afferent terminals show a reduction in excitatory postsynaptic current (EPSC) frequency and firing rate with caffeine application. A: spontaneous EPSCs from a whole cell recording of an afferent terminal. Red traces are in the presence of 10 mM caffeine, and blue traces are activity during washout of the drug. B: frequency histograms for EPSC amplitudes in the absence (black) and presence (red) of caffeine, where solid lines are Gaussian fits to the histograms. C: EPSC frequency (or extracellular spike frequency) before and after caffeine administration. Paired t-tests show a significant difference at P ⬍ 0.001 level. D: mean EPSC amplitude as measured from the fits to the Gaussian curves in B. Although a trend toward smaller mean amplitudes was found, no statistical difference was observed. the absence and presence of caffeine. We found no change in resting potential and a variable effect on the electrical resonance response. A summary of the resting potential data is presented in the box plots of Fig. 3B, where no consistent change was observed. In several cells the quality of the resonance was reduced, but this was not statistically different, nor did it typically recover upon washout. It is also possible that the reduced frequency of release obtained with caffeine is due to the efflux of calcium from calcium stores, leading to Ca2⫹ channel inactivation at the synapse (Schnee and Ricci 2003). We compared hair cell Ca2⫹ current amplitudes in response to a depolarizing pulse before and during drug application (caffeine or ryanodine) in the external solution (Fig. 3C). The same protocol (two 3-s depolarizing pulses separated by 5 min) was performed with drugs in the internal solution (8-BrcAMP or thapsigargin) (Fig. 3D). Data summarized in Fig. 3, C and D, suggest that Ca2⫹ current amplitude was not significantly altered by drug application. Thus the most parsimonious explanation for the reduction in vesicle release is a synaptically driven mechanism. We also investigated the macroscopic currents elicited in voltage clamp (Fig. 3, E–H). Macroscopic currents elicited from both hyperpolarizing and depolarizing voltage steps did not vary significantly in the presence of caffeine. Currentvoltage plots elicited from tail currents at the time point indicated in Fig. 3E and plotted in Fig. 3G were also not significantly different between control and caffeine treated. The voltages of half-activation summarized in Fig. 3H were also not significantly different. Therefore, these data indicate that the changes recorded postsynaptically during caffeine perfusion are likely to be a direct effect on synaptic properties within the hair cell. Time dependence of superlinear release. The two-sine technique (Schnee et al. 2011) was first used to monitor vesicle release during cell depolarization to avoid the intercellular variability found in hair cells, particularly with repeated measures (Levic et al. 2011; Patel et al. 2012; Quinones et al. 2012; Rutherford and Roberts 2006). Two components of release were observed, an initial linear component, previously demonstrated to be accounted for by vesicles within 0.7 m of the synapses, and a larger superlinear component that requires recruitment of additional vesicles (Fig. 4A) (Schnee et al. 2005, 2011). The first linear release component is proportional to the Ca2⫹ load (integral of calcium current) and correlates with the vesicle population near the synapses (Schnee et al. 2005, 2011). In our hands the traditional pool descriptions of rapidly and readily released are less valuable in that pool size as measured by depletion varies within a given cell quite substantially and is often difficult to observe, suggesting a very dynamic population of vesicles with robust recruitment (Schnee et al. 2005, 2011). The first component includes these pools but likely additional vesicles from immediately near the synapse and potentially some vesicles that are recruited during depolarization. The second superlinear component presents a higher release rate and is independent of Ca2⫹ load. Similar superlinear release components are observed in other secretory cells (Andersson et al. 2011; Seward et al. 1996) as well as photoreceptors and hair cells by application of trains of short stimulation pulses with single-sine capacitance techniques (Duncker et al. 2013; Innocenti and Heidelberger 2008; Moser and Beutner 2000). At present, we consider the superlinear release to represent the hair cell’s exocytotic activity when release sites are maximally filled and releasing. This release component requires significant recruitment of vesicles (Schnee et al. 2005, 2011). This interpretation remains a hypothesis requiring further investigation. Calcium imaging experiments also showed a nonlinear rise in the intracellular Ca2⫹ levels accompanying the superlinear capacitance change (Schnee et al. 2011). One possible interpretation of this nonlinear signal- J Neurophysiol • doi:10.1152/jn.00559.2015 • www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.5 on June 18, 2017 EPSC Peak (nA) wash 100 ms 0.2 40 Frequency (Hz) 200 pA 229 230 STORED CALCIUM PROMOTES VESICLE RECRUITMENT TO RIBBON SYNAPSES A B control Control -30 10 mM Caffeine -40 -50 Vz (mV) caffeine 5 mV -60 -70 -80 5 ms C 0.0 0.0 -0.2 -0.3 -0.5 -0.5 F IK (nA) 6 8 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 8 Control -0.2 -0.4 E 8Br-cAMP -0.1 ryanodine Control ICa (nA) ICa (nA) -0.1 D caffeine 0.8 -30 -40 -50 0.4 -60 -100 -80 -60 -40 -20 Potential (mV) ing is a potential second internal source of Ca2⫹, perhaps supporting CICR. Our postsynaptic results point to a potential role of hair cell ER Ca2⫹ homeostasis in the ability to sustain ribbon synaptic transmission. To test whether ER Ca2⫹ homeostasis modulates vesicle release, we used the dual-sine capacitance technique to monitor release properties under control conditions (Fig. 4) and during pharmacological treatment (Figs. 5–7). Previous experiments using single-sine stimuli have demonstrated a presynaptic facilitation effect that results in variation in responses to repeated stimuli, which could make interpretation of pharmacological manipulations requiring repeated measures difficult (Cho et al. 2011; Schnee et al. 2011). Furthermore, since repeated stimulation using long depolarizing pulses has not been described, we first characterized release properties of 0 20 Control caffeine control hair cells in order to establish a comparator for subsequent pharmacological experiments. Control cells were voltage-clamped to ⫺85 mV, and consecutive 3-s stimulation pulses to 50% of peak Ca2⫹ current (as estimated from currentvoltage plots generated for each cell) were tested. These relatively long protocols were selected to ensure observation and separation of the two release components, while at the same time not overtaxing the cell so that it could replenish for multiple stimulations (Schnee et al. 2005). Also, these stimulations are closer to physiological than traditional depolarizations where potentials are stepped to elicit peak calcium currents. Figure 4B shows an example in which hair cell calcium current and capacitance were measured sequentially in response to a stimulation that elicited a linear and a superlinear J Neurophysiol • doi:10.1152/jn.00559.2015 • www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.5 on June 18, 2017 Fig. 3. Presynaptic excitability is not responsible for the reduction in vesicle release. A: membrane potential responses from a hair cell elicited by a 10-pA current injection about the cell’s resting potential: control (top) and in the presence of 10 mM caffeine (bottom). The slight reduction in quality of resonance is not significantly different. B: zero-current potential (Vz) in the absence (black) and presence (red) of caffeine. No statistical difference is noted. C: calcium current (ICa) elicited in paired stimulus for cells in the absence and presence of drug (red, caffeine; blue, ryanodine; black, control). No change is identified. D: summary of calcium currents elicited from 1st (darker colors) and 2nd (lighter shades) stimuli for drugs that were applied internally. No significant differences were found. E and F: voltage-clamp responses for step voltage changes between ⫺104 and ⫹60 in 20-mV increments from a holding potential of ⫺84 mV for control (E) and caffeine (F). G: tail current voltage plots generated at the time point indicated by dashed line in E. Black, control; red, caffeine. No difference was found between plots. H: half-activating voltages (V1/2) obtained from data in G. No significant difference was observed. STORED CALCIUM PROMOTES VESICLE RECRUITMENT TO RIBBON SYNAPSES A 231 -30 mV -80 mV linear 1 pF superlinear Cm I Ca2+ 100 pA 1s B 1 1 3 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 0.4 n.s. 0.2 0 -0.2 F superlinear release ** 2 G 1 3 5 IPI (min) 1 0 1 Δ superlinear onset ** -1 -0.4 1.5 1 pulse 1 (pF) 2nd-1st (s) linear release (2nd-1st)/1st (2nd-1st)/1st E Fig. 4. Long interpulse intervals (IPIs) result in an increase in hair cell superlinear release. A: representative example showing linear and superlinear components of release using 2-sine capacitance method. Cm, membrane capacitance. B: consecutive 3-s depolarizing pulses to 50% of peak current were delivered in whole cell. Top: release. Bottom: calcium currents. Note the increase in release for 3-min IPI (scale: 100 fF/10 pA, 1 s). C: traces from A superimposed. Note that superlinear release onset (arrowheads) started earlier after longer IPIs. D: normalized data showing that release was enhanced only for 3- to 5-min IPIs (n ⫽ 19 cells). E and F: box plots showing that superlinear release increased only for 3- to 5-min intervals whereas linear release was unchanged. G: superlinear onset shortened only for 3- to 5-min intervals. **Paired t-tests show a significant difference at P ⬍ 0.01 level. 3 5 IPI (min) component. In this example, multiple depolarizing pulses separated by 1 min led to a slight release reduction, consistent with vesicle depletion. Conversely, release was unexpectedly enhanced when pulses were separated by 3 min (Fig. 4B). The amount of release was not significantly modified when two consecutive pulses were separated by 1 min (Fig. 4D). However, longer interpulse intervals (IPIs) led to a significant release increase in all cells studied. Changes in total release as well as linear and superlinear release were tested by two consecutive pulses using different IPIs (1, 3, and 5 min) in a total of 19 cells. The linear component was quantified by measuring the increase in membrane capacitance from the onset of depolarization until the appearance of the superlinear component. Since we observed variability in the onset of the superlinear component between pulses (see below), linear release was measured in both pulses until the same time point, 0.5 0 -0.5 -1.0 -1.5 ** 1 ** 3 5 IPI (min) that is, until the superlinear onset of the second pulse. For pulses separated by 1 min, release was the same as the initial value (n ⫽ 5 cells) (Fig. 4D). Both components showed nonsignificant differences in the second pulse: superlinear release reduction of 2 ⫾ 17% and linear release reduction of 4 ⫾ 7% (Fig. 4, E and F). Similarly, the superlinear release onset was constant in the second pulse (0.02 ⫾ 0.1 s, n.s.) (Fig. 4G). However, when pulses were separated by 3 min, consecutive pulses unexpectedly led to total release enhancement (38 ⫾ 25% increase, n ⫽ 5 cells, P ⫽ 0.02) (Fig. 4D). Enhancement was also observed for 5-min IPI (52 ⫾ 32%, n ⫽ 9 cells, P ⫽ 0.0001). Release enhancement was not accompanied by an increase in Ca2⫹ load (1 ⫾ 2% increase for 3-min IPI, 5 ⫾ 8% for 5-min IPI). The increase in release resulted from an increase in the superlinear component for IPIs of 3 min (110 ⫾ 42% increase, P ⫽ 0.007) and 5 min (115 ⫾ 39%, P ⫽ 0.0001 J Neurophysiol • doi:10.1152/jn.00559.2015 • www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.5 on June 18, 2017 C 1 232 STORED CALCIUM PROMOTES VESICLE RECRUITMENT TO RIBBON SYNAPSES were no facilitation the baseline for control would be at 0 (as in the linear response) (Fig. 6). The linear release component increased 4%, whereas superlinear release increased by 115% (Fig. 6, B and C). The increases seen in response to a second pulse are used as the comparator for the pharmacological manipulations such that the value for total release in control was 152%, whereas the linear release was 104% and superlinear release 215% of first pulse. The same depolarization protocol was delivered before and after continuous extracellular application of 10 mM caffeine (Fig. 5B) or 10 M ryanodine. Both pulses were separated in time by 5–10 min to ensure drug access through the papilla. In the presence of caffeine, mean total release in the second pulse was 37% of first pulse release (Fig. 6A), where linear release reached 65% and superlinear release 40% of the control pulse. Ryanodine reduced total release to 58% of the initial pulse, where mean linear release reached 85% and superlinear release just 41% of control values. Similarly, when 30 M 8-BrcADPR, a RyR antagonist, was included in the internal solution, total release was 46% of control (Fig. 5C). Addition of 0.5–2 M thapsigargin, a SERCA inhibitor, to the internal solution reduced release to 65% of control, with the linear component maintaining 89% of control and the superlinear component reducing to 56% of control. Inclusion of 1 M xestospongin C, an IP3 receptor blocker, had minimal effects on total release at 85% of control, while 4 M cyclic ADP ribose (cADPR), a RyR agonist, also showed limited efficacy, reaching 95% of control release values. The shift in the superlinear release onset observed in controls was only significantly reduced by ryanodine application (Fig. 6D). Total release was significantly reduced by four of the six compounds tested, with those altering RyRs being more effective (Fig. 6A). Separating effects into release components demonstrates that the largest effects were on the superlinear component. Figure 6B shows that only three compounds significantly affected the linear component of release. Overall linear release in the second pulse was reduced to 84 ⫾ 26% of control, while the superlinear component was reduced to 53 ⫾ 29% of control. Nineteen of twenty-nine pharmacologically treated cells showed a reduction from baseline in superlinear release (Fig. 6, B and C) compared with zero of nine control cells. Thus the magnitude of the pharmacologically driven release reduction was more robust in the superlinear response (compare y-axes in Fig. 6, B and C). Furthermore, the drug effects are likely underestimated when compounds are used in the patch electrode because their effect likely begins prior to the initial measurement. Supporting this contention is the reduction in maximal release rate for the first response in cells where drugs are intracellularly perfused (0.5 ⫾ 0.3 pF/s, n ⫽ 19) compared with control values (0.8 ⫾ 0.6 pF/s, n ⫽ 19). The superlinear component of release could be reduced in several ways: first, the onset of superlinear release could be lengthened; second, the maximal response could be reduced (saturation); and third, the rate of release (slope) could be reduced. Figure 6D demonstrates that the onset of the superlinear response was only affected minimally and only statistically significant for ryanodine. Also, saturation of release did not appear to be the limiting factor, as depicted in the examples of Fig. 5. As also seen in Fig. 5, the maximal release rate (slope) was reduced in the presence of drugs that inhibited CICR. In pharmacologically treated cells where total release J Neurophysiol • doi:10.1152/jn.00559.2015 • www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.5 on June 18, 2017 for 5 min) (Fig. 4F), whereas the linear component did not change significantly (5 ⫾ 11% reduction for 3 min, 4 ⫾ 10% increase for 5 min) (compare y-axes in Fig. 4, E and F). The observed release enhancement was originated by a shortening in the superlinear release onset time for IPIs of 3 min (0.6 ⫾ 0.3 s, P ⫽ 0.01) as well as 5 min (0.5 ⫾ 0.2 s, P ⫽ 0.0001) (Fig. 4, C and G) and not by a change in the rate (slope) of the superlinear release. These data show that 1 min is enough for hair cell buffering mechanisms to reach a steady state in cytoplasmic calcium levels after the long depolarizing pulse. Therefore the observed increase in release after 3–5 min cannot be accounted for by an increase in baseline calcium levels due to buffer saturation. Release enhancement, however, may be consistent with an increase in ER Ca2⫹ reuptake leading to an increase in luminal Ca2⫹ levels for subsequent stimulation pulses. Alternatively, it may underlie the activation of Ca2⫹dependent second messengers that could modulate Ca2⫹ stores or sensitivity to Ca2⫹ influx or activate store-dependent expression of synaptic proteins (Alkon et al. 1998; Benech et al. 1999; Sutton et al. 1999). Although we cannot categorically interpret the physiological relevance of this release shift, the control data are needed for the pharmacological experiments described below. As these data demonstrate that repeated stimulations alter the response, it is critical to characterize the change in order to accurately assess pharmacological manipulations that require repeated measures. To study whether the shift in the superlinear release onset was Ca2⫹ dependent and release specific or could also affect other Ca2⫹-dependent processes taking place distant from the synaptic ribbon, we tested the threshold for SK channel activation, a channel located near efferent terminals (Lioudyno et al. 2004). Hair cells were stimulated and SK was monitored by removing apamin from the external solution (see examples in Fig. 5A). In 120 of 124 cells superlinear release was preceded 0.7 ⫾ 0.9 s by SK activation. Superlinear release onset correlated with SK onset (R2 ⫽ 0.93), demonstrating that Ca2⫹ levels reached intracellular locations not necessarily associated with the vicinity of the ribbon. In mammalian outer hair cells, an ER-like cistern opposing every efferent contact is proposed to act as a Ca2⫹ store to amplify Ca2⫹ levels for SK activation evoked by nicotinic acetylcholine receptor (nAChR) opening (Fuchs 2014; Lioudyno et al. 2004). In those studies, ryanodine and other store modulators alter SK currents, a result that contrasts with our experiments, where such an obvious effect was not observed during continuous application of curare, an inhibitor of nAChRs. The activation of SK channels could be directly triggered by the influx of Ca2⫹ through L-type Ca2⫹ channels, thus circumventing the need for stored Ca2⫹ efflux to activate SK channels during depolarization. Moreover, the sensitivity to Ca2⫹ might be biochemically modulated and thus underlie changes in timing as has been described in neurons (Adelman et al. 2012). Pharmacological manipulation of calcium stores reduces hair cell release. The effect of Ca2⫹ store modulators in hair cell release was tested with the dual-sine capacitance technique (Figs. 5 and 6). Figure 5A shows the release enhancement observed in consecutive control stimulations, mainly due to the early onset of the superlinear component. Total release increased by an average of 52% when comparing two pulses separated by 5 min (Fig. 6A). Data are normalized to account for the facilitation observed in control conditions; thus if there STORED CALCIUM PROMOTES VESICLE RECRUITMENT TO RIBBON SYNAPSES Im (nA) A control 0.4 0.2 0 -0.2 -0.4 Cm (pF) min 10 min 15 min 20 1 0 caffeine (min 17) 0.3 Im (nA) 6 0 2 4 time (s) 6 0 C wash (min 25) 0 -0.3 6 0 -0.1 -0.2 -0.3 8-Br-cADPR (min 10) 2 min 13 min 24 min 32 0.4 Cm (pF) -0.6 Cm (pF) 2 4 time (s) 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) reduction was significant, the mean release rate change (rate 2nd pulse ⫺ rate 1st pulse) was reduced by 40 ⫾ 37% (reduction observed in 18 of 23 cells). In controls, the mean release rate change was reduced by 9 ⫾ 44% (reduction observed in 4 of 9 cells). A reduction in the rate of release can be interpreted as a reduction in vesicle trafficking where trafficking is Ca2⫹ dependent. With the use of dual-sine capacitance recordings, our pharmacological data confirm previous reports of a potential role of intracellular Ca2⫹ stores in hair cell synaptic physiology (Beurg et al. 2005; Evans et al. 2000; Hendricson and Guth 2002; Kennedy and Meech 2002; Lelli et al. 2003; Marcotti et al. 2004). As opposed to the release enhancement observed in controls, hair cell pharmacological treatment led to a reduction in both linear and superlinear release after repeated stimulation, with the superlinear release component most clearly affected. This release reduction was not accompanied by a reduction in the peak Ca2⫹ current and therefore cannot be accounted for by a pharmacological effect of intracellular Ca2⫹ release on Ca2⫹ channel inactivation (Lee et al. 2007). Altogether, these results suggest that the recruitment of vesicles for release during prolonged stimulation might be physiologically linked to a CICR mechanism. Calcium triggers linear and superlinear release more efficiently than barium. Ba2⫹ activates CICR with lower efficacy than Ca2⫹; it slows down exocytosis and reduces the number of vesicles available for release (Neves et al. Fig. 5. Pharmacological disruption of ryanodine receptors (RyRs) reduces hair cell release. A: 3 control 3-s depolarizing pulses were delivered 5 min apart, and current (Im) and Cm were monitored. Consecutive protocols showed release enhancement (dashed line). SK and superlinear 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 3 pulses. B: extracellular 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-BrcADPR (30 M) reduced release, with no effect on the calcium load. Arrowhead indicates superlinear onset. SK current was blocked in this cell by apamin in the external solution. 2000 3000 4000 5000 0 1 2 3 4 5 time (s) 2001; Proks and Ashcroft 1995; Przywara et al. 1993; Seward et al. 1996; von Ruden et al. 1993). Moreover, Ba2⫹ is not reuptaken into the store through SERCA pumps (Kwan and Putney 1990; Przywara et al. 1993). To study whether Ba2⫹ alters linear or superlinear capacitance changes we replaced external Ca2⫹ with equimolar Ba2⫹ (Fig. 7). After superlinear release was evoked with a 3-s depolarization to 50% peak current, extracellular Ca2⫹ was substituted by equimolar Ba2⫹ and release was tested again with the same protocol. As expected, Ba2⫹ abolished SK activation, increased peak currents, and reduced Ca2⫹ channel inactivation (Fig. 7A) (Schnee and Ricci 2003). Ba2⫹ exerted two different effects on release: reduced total release and delayed onset of the initial linear release component (Fig. 7A). Box plots of total release for each stimulus obtained with external Ca2⫹ or Ba2⫹ are shown in Fig. 7B. The response in Ca2⫹ shows release enhancement in the second and third responses, whereas the responses in Ba2⫹ were unchanged or reduced, despite the larger currents observed (487 ⫾ 185 pA in Ba2⫹ vs. 408 ⫾ 173 pA in Ca2⫹, P ⫽ 0.01). Ba2⫹ reduced total release in a second stimulation (72% of control) and even further in a third pulse (46% of control). Normalizing release output more clearly shows the enhancement observed in Ca2⫹ that is absent in the presence of Ba2⫹ (Fig. 7C). The time to initial release was reduced in the presence of Ba2⫹ (Fig. 7D), and the delay was further increased for a third stimulation. The delay may J Neurophysiol • doi:10.1152/jn.00559.2015 • www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.5 on June 18, 2017 B 2 4 time (s) ICa2+ (nA) 0 0.8 233 234 STORED CALCIUM PROMOTES VESICLE RECRUITMENT TO RIBBON SYNAPSES A * ** ** * l C R n e PR g i n ro ne nt ffei DP odi D gar osp o A t i c ca rcA an c s s y ap xe 8B r th (2nd-1st/1st) l C R ne PR gin ro ne nt ffei DP odi D gar osp o A t i c ca rcA an c s s y ap xe 8B r th D superlinear release ** * ** 2nd-1st (2nd-1st/1st) ** ** ** superlinear onset l C R ne PR gin ro ne r nt ffei DP odi sp D a o o c ca rcA an cA sig est y ap x 8B r th be a reflection of Ba2⫹ being less effective at driving release mechanisms (Bhalla et al. 2005). Additionally, Ba2⫹ application promoted a merging of the two components. The linearization of the capacitance response may reflect a single source of divalent ions driving the process as CICR is disabled with Ba2⫹ application. The maximum slope of the superlinear release component for a third stimulation was reduced by 36 ⫾ 30% during Ba2⫹ application, as opposed to controls, which increased by 141 ⫾ 119% (P ⫽ 0.01) of the initial maximum slope in the first pulse (Fig. 7F). Unlike the superlinear decrease in release, the first component showed no change in release following the delay. To make this comparison we measured the first component of release in both Ca2⫹ and Ba2⫹ 500 ms after the onset of the capacitance change, thus bypassing the delay induced by Ba2⫹ (Fig. 7E). Eliminating the onset delays demonstrates that the first release component was not significantly altered in amplitude by the divalent replacement. The delay in the onset of release could be partially explained by a poor sensitivity for Ba2⫹ in the presynaptic Ca2⫹ sensors for vesicle release and recruitment as suggested for SNARE-mediated exocytosis (Bhalla et al. 2005). However, the role of SNARE proteins in vesicle fusion at hair cell ribbon synapses remains controversial (Nouvian et al. 2011). The intensified effect observed with multiple stimulation protocols, together with the marked effect of Ba2⫹ on the superlinear release component (Fig. 7F), are consistent with the existence of a CICR mechanism governing the recruitment of new vesicles for release. l C R ne PR gin ro ne r nt ffei DP odi sp D a o o c ca rcA an cA sig est y ap x 8B r th DISCUSSION The existence of CICR has long been observed in conventional as well as ribbon synapses (Bouchard et al. 2003; Castellano-Munoz and Ricci 2014), yet the functional significance and cellular mechanisms underlying CICR in these cell types are unclear. Here we studied the potential contribution of CICR at hair cell ribbon synapses and obtained the following results. First, spontaneous postsynaptic multiunit activity, along with EPSC frequency, was reduced by pharmacological manipulations that depleted Ca2⫹ stores through RyRs. Second, no presynaptic changes in excitability, like a change in resting potential or sensitivity, could account for the reduction in postsynaptic response. Third, pharmacological presynaptic effects on CICR resulted in a reduction of the superlinear capacitance change that was larger and occurred earlier than changes to the first component of release, consistent with the hypothesis that CICR is important for modulating vesicle trafficking. Fourth, Ca2⫹ substitution by Ba2⫹ delayed the onset of release, likely because of its ability to drive synaptic machinery. It also reduced superlinear release more than the first component of release, and this effect was greater for larger stimulations. Our data suggest a role for RyRs regulating CICR from the ER. Identification of the location of the ER responsible for controlling synaptic vesicle populations remains under investigation. Although mitochondria are highly localized to the synaptic region (Graydon et al. 2011; Schnee et al. 2005), the role of the mitochondria as a Ca2⫹ sink remains suspect, as our pharmacological data show a mild effect but one that cannot J Neurophysiol • doi:10.1152/jn.00559.2015 • www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.5 on June 18, 2017 C linear release (2nd-1st/1st) ** ** ** Fig. 6. Pharmacological disruption of intracellular calcium stores reduces hair cell release. A: caffeine, 8-BrcADPR, ryanodine, and thapsigargin significantly reduced release. See dashed line for comparison with control. B and C: linear (B) and superlinear (C) release were reduced by drugs that interfere with endoplasmic reticulum (ER) RyRs (10 mM caffeine, 30 M 8-BrcADPR, 10 M 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 M thapsigargin and 1 M xestospongin C. Cyclic ADP ribose (cADPR, 4 M), an IP3 receptor agonist, had no obvious effects on release. D: only ryanodine showed a significant effect on superlinear onset. Unpaired ttests show a significant difference: *P ⬍ 0.05, **P ⬍ 0.01. B total release STORED CALCIUM PROMOTES VESICLE RECRUITMENT TO RIBBON SYNAPSES A B Im (nA) 0.2 total release (s) 1 Ca2+ 2.0 -0.2 2 Ba 3 Ba2+ Cm (pF) -0.4 1 2 3 1 total release (pF) 0 2+ 235 control Ba2+ ** 1.5 1.0 0.5 0 0 2 4 6 2nd pulse 3rd pulse D normalized total release 2nd pulse (2nd-1st)/1st (3rd-1st)/1st Ba2+ E control Ba2+ Ba2+ control F initial release ** * 150 50 (3rd-1st)/1st 100 Ba2+ control normalized max release slope (2nd-1st)/1st initial release (pF) ** ** ** control time to release (s) 3rd pulse C 0 1st pulse 2nd pulse 3rd pulse control Ba2+ control Ba2+ Fig. 7. Barium delays release and reduces superlinear release component. A: after substitution of 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 3 pulses. B: release values elicited by pulses delivered 5–10 min apart in control conditions and after substitution of 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. easily be separated from the energy generation role typically ascribed to these organelles. Likely the role of mitochondria at hair cell ribbons is similar to the Ca2⫹ buffering role described at ribbon synapses in the visual pathway, in which mitochondria provide ATP for Ca2⫹ pumps and also sequester Ca2⫹ from cytoplasm (Babai et al. 2010; Cadetti et al. 2006; Krizaj et al. 2003; Suryanarayanan and Slaughter 2006; Wan et al. 2012; Zenisek and Matthews 2000). The clear distinction between linear and superlinear release components can only be experimentally obtained by depolar- izing to potentials that allow less than maximal calcium currents from hyperpolarized holding potentials (Schnee et al. 2011). In turtle hair cells, prepulses to physiological membrane potentials augment release and trigger the convergence of linear and superlinear release components (Schnee et al. 2011). Similarly, in postnatal rat inner hair cells, depolarizing pulses preceded by holding the cell at physiological resting potentials produced an increase in exocytosis and synaptic strength and a shortening of synaptic latency at hair cell ribbon synapses compared with holding the cell at more hyperpolarized poten- J Neurophysiol • doi:10.1152/jn.00559.2015 • www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.5 on June 18, 2017 1st pulse Time (s) 236 STORED CALCIUM PROMOTES VESICLE RECRUITMENT TO RIBBON SYNAPSES lease of Ca2⫹ by CICR has a presynaptic role in neuronal synaptic transmission (Llano et al. 2000; Unni et al. 2004). CICR is observed in hair cells of different animals in both auditory and vestibular organs (Beurg et al. 2005; Evans et al. 2000; Hendricson and Guth 2002; Kennedy and Meech 2002; Lelli et al. 2003; Marcotti et al. 2004; Tucker and Fettiplace 1995). What could the physiological significance of CICR in hair cells be? Hair cells must maintain a tight regulation between Ca2⫹ influx through L-type Ca2⫹ channels and vesicle release at ribbon synapses to allow precise control of release timing. This regulation is achieved in the vicinity of the ribbon by controlling Ca2⫹ levels through buffering and extrusion mechanisms that locally rapidly remove Ca2⫹. Given that the recruitment of vesicles for release during prolonged stimulation is Ca2⫹ dependent, how do hair cells manage to bypass the strong Ca2⫹ regulation near the ribbon to achieve Ca2⫹dependent vesicle recruitment? One possibility is that the Kd of trafficking is much lower than that for release. Alternatively, CICR may serve to amplify and filter the synaptic signal (Fig. 8). Thus, during continuous stimulation, the opening of L-type Ca2⫹ channels could tightly modulate the fusion of vesicles near the ribbon while amplification of this signal away from the synapse through CICR may trigger vesicle recruitment. These calcium-dependent processes may be additionally modulated by the effects of efferent activity on hair cell synaptic calcium levels (Im et al. 2014). We hypothesize that CICR could have a functional role in the recruitment and replenishment of synaptic vesicles to guarantee the availability of vesicles for release during protracted stimulation. Another puzzling finding is the within-cell variability observed with repeated stimulations. This was observed in turtle with the single-sine technique with paired stimulation (Schnee et al. 2011) and often appears as a facilitation of release with repeated measures, particularly when IPIs are short. Here we also identify a slower facilitation that enhances release by shortening the onset time to superlinear release with repeated stimulations that have longer (minute) IPIs. Together these data question measurements where signal averaging is used or where multiple data points need to be obtained via repetitive A Vm 1 B Ca2+ Ca2+ Ca2+ Ca a2 a2 2+ + Ca2 Ca2+ 2+ + 2 EPSCs Cm linear release I Ca 2+ Fig. 8. Ca2⫹-induced Ca2⫹ release (CICR) might sustain the recruitment of synaptic vesicles to the ribbon. Left: a depolarizing pulse triggering an increase in capacitance in response to a calcium current. Right: a hair cell presynaptic terminal and the simultaneous postsynaptic responses (EPSCs). A: at hyperpolarized membrane potential (Vm), 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. 2 1 superlinear release 2 1 linear release 1 2 Ca2+ Ca2+ Ca a a2 2 + Ca2+ 2+ Ca2+ Ca2 C 2 2+ + J Neurophysiol • doi:10.1152/jn.00559.2015 • www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.5 on June 18, 2017 tials (Goutman and Glowatzki 2011). We hypothesize that the superlinear response represents all synaptic release sites being filled and fusion happening at maximal rates because of the enhanced recruitment of synaptic vesicles. We further hypothesize that changes in release rates for the linear component of release may in part represent release from multiple release sites where the number of filled sites increases with Ca2⫹ load. This hypothesis also can explain the variability in obtaining capacitance responses that show depletion (Schnee et al. 2005, 2011), because if the calcium entry is low enough not to trigger recruitment then the pool size will be set by vesicles present at any given moment. It remains a question as to whether superlinear capacitance changes reflect synaptic vesicle fusion, extrasynaptic vesicle fusion, or even fusion of endosomes (Coggins et al. 2007; Zenisek et al. 2000). Our pharmacological approach showed a postsynaptic reduction in spike and EPSC rates that correlates with a presynaptic release reduction. Whereas hair cell linear release was mildly reduced by drugs interfering with CICR, superlinear release was strongly reduced. Similarly, substitution of Ca2⫹ by Ba2⫹ exerted a stronger effect on the superlinear component of release. These results demonstrate that both release components are Ca2⫹ dependent and likely interrelated, suggesting that superlinear release might have a physiological role in synaptic transmission. Whether the release is synaptic or extrasynaptic requires better resolution than what we have at the moment (Chen et al. 2014). However, distributions of glutamate receptors as well as a lack of vesicles at nonsynaptic release sites in hair cells would also support a synaptic role (Lenzi et al. 1999; Liberman et al. 2011; Schnee et al. 2005). Also, given that the postsynaptic response was a dramatic reduction in firing rate and the major presynaptic response was a decrease in the superlinear capacitance response, it is plausible that the superlinear response is a reflection of robust vesicle trafficking to the synapse. In neurons, Ca2⫹ regulates exocytosis of synaptic vesicles as well as the supply of new vesicles to release sites (Dittman and Regehr 1998; Gomis et al. 1999; Stevens and Wesseling 1998; Wang and Kaczmarek 1998). Moreover, the intracellular re- STORED CALCIUM PROMOTES VESICLE RECRUITMENT TO RIBBON SYNAPSES ACKNOWLEDGMENTS We thank the members of our research group for providing helpful comments on the project and the manuscript. GRANTS This work was funded by National Institute on Deafness and Other Communication Disorders Grant DC-009913 to A. J. Ricci and by core grant P30 44992. M. Castellano-Muñoz was supported by a Dean’s Postdoctoral Fellowship from Stanford School of Medicine and a Cajamadrid Foundation Fellowship. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS Author contributions: M.C.-M. and A.J.R. conception and design of research; M.C.-M., M.E.S., and A.J.R. performed experiments; M.C.-M. and A.J.R. analyzed data; M.C.-M., M.E.S., and A.J.R. interpreted results of experiments; M.C.-M. and A.J.R. prepared figures; M.C.-M. and A.J.R. drafted manuscript; M.C.-M., M.E.S., and A.J.R. edited and revised manuscript; M.C.-M. and A.J.R. approved final version of manuscript. REFERENCES Adelman JP, Maylie J, Sah P. Small-conductance Ca2⫹-activated K⫹ channels: form and function. Annu Rev Physiol 74: 245–269, 2012. Albrecht MA, Colegrove SL, Friel DD. Differential regulation of ER Ca2⫹ uptake and release rates accounts for multiple modes of Ca2⫹-induced Ca2⫹ release. J Gen Physiol 119: 211–233, 2002. Alkon DL, Nelson TJ, Zhao W, Cavallaro S. Time domains of neuronal Ca2⫹ signaling and associative memory: steps through a calexcitin, ryanodine receptor, K⫹ channel cascade. Trends Neurosci 21: 529 –537, 1998. 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Ryanodine receptors and BK channels act as a presynaptic depressor of neurotransmission in cochlear inner hair cells. Eur J Neurosci 22: 1109 –1119, 2005. Bhalla A, Tucker WC, Chapman ER. Synaptotagmin isoforms couple distinct ranges of Ca2⫹, Ba2⫹, and Sr2⫹ concentration to SNARE-mediated membrane fusion. Mol Biol Cell 16: 4755– 4764, 2005. Bouchard R, Pattarini R, Geiger JD. Presence and functional significance of presynaptic ryanodine receptors. Prog Neurobiol 69: 391– 418, 2003. Cadetti L, Bryson EJ, Ciccone CA, Rabl K, Thoreson WB. Calciuminduced calcium release in rod photoreceptor terminals boosts synaptic transmission during maintained depolarization. Eur J Neurosci 23: 2983– 2990, 2006. Castellano-Munoz M, Ricci AJ. Role of intracellular calcium stores in hair-cell ribbon synapse. Front Cell Neurosci 8: 162, 2014. Chen M, Krizaj D, Thoreson WB. Intracellular calcium stores drive slow non-ribbon vesicle release from rod photoreceptors. Front Cell Neurosci 8: 20, 2014. Cho S, Li GL, von Gersdorff H. Recovery from short-term depression and facilitation is ultrafast and Ca2⫹ dependent at auditory hair cell synapses. J Neurosci 31: 5682–5692, 2011. Coggins MR, Grabner CP, Almers W, Zenisek D. Stimulated exocytosis of endosomes in goldfish retinal bipolar neurons. J Physiol 584: 853– 865, 2007. Dallos P, He DZ, Lin X, Sziklai I, Mehta S, Evans BN. Acetylcholine, outer hair cell electromotility, and the cochlear amplifier. J Neurosci 17: 2212– 2226, 1997. J Neurophysiol • doi:10.1152/jn.00559.2015 • www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.5 on June 18, 2017 stimuli. More importantly, though, is the question as to the physiological relevance of the variability. The variability appears to be biological, in that no other measured biophysical parameters are changing over this time frame. This enhancement could simply be due to changes in calcium levels or reflect a modulation in the sensitivity triggered by a biochemical modification. In our experiments the onset of SK channel activation paralleled superlinear release onset. Both phenomena are unaffected by CICR pharmacological treatment, thus ruling out the possibility that release enhancement is simply due to calcium baseline modulation. Moreover, the calcium sensitivity of SK2 and SK3 channels depends on the phosphorylation state of SK-bound calmodulin (Adelman et al. 2012), pointing to a phosphorylation modulation as the origin of the release and SK onset shift. One possibility consistent with the present data is that the Ca2⫹ stores are incompletely filled under our recording conditions and initial stimulations serve to fill this pool, which can then be more efficient at driving vesicles to release sites. The stores might be considered in a dynamic equilibrium with cytosolic Ca2⫹ where it can act as both sink and source depending on excitation level. Thus holding a cell at ⫺80 mV and dialyzing with ATP and a Ca2⫹ buffer moves the equilibrium toward store depletion so that Ca2⫹ is driven into stores upon stimulation. Thus this Ca2⫹ is available for release upon further stimulation. Consistent with this idea is the finding that using holding potentials more depolarized leads to more robust release (Schnee et al. 2011). Another confusing component of these data is the variability in responsiveness to the drug applications, even those where drugs were included within the patch pipette. Not all cells responded, and those that did showed a larger than expected variance. Again, the variation appears to be biologically driven and not a function of the biophysical status of the hair cells. One possibility relates to the previous discussion that drug efficacy will be directly determined by the state of the store (how filled it is) at the time of drug administration. Another confounding point is that turtle hair cells have large Ca2⫹ currents and it is likely that large depolarizations that lead to major influx via Ca2⫹ channels can mask the effects of Ca2⫹ stores. Alternatively, there may be additional biochemical modifications that we are not controlling, for example, CaM kinase activity. In summary, the data presented identify a presynaptic role for CICR to modulate hair cell vesicle release. Both pre- and postsynaptic recordings support the argument that CICR may regulate vesicle trafficking. Data demonstrate the involvement of RyRs in this pathway. Capacitance measurements target the major effect to a reduction in the superlinear component of release, previously argued to require vesicle recruitment to the synapses. 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