Download Calcium-induced calcium release supports recruitment of synaptic

Articles in PresS. J Neurophysiol (October 28, 2015). doi:10.1152/jn.00559.2015
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TITLE: Calcium-induced calcium release supports recruitment of synaptic vesicles in
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auditory hair cells.
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ABBREVIATED TITLE: Stored calcium promotes vesicle recruitment to ribbon synapses.
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AUTHOR NAMES AND AFFILIATION: Manuel Castellano-Muñoz1,3, Michael E.
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Schnee1 and Anthony Ricci1,2.
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Department of Otolaryngology1 and Molecular and Cellular Physiology2. Stanford University
School of Medicine. 300 Pasteur Drive. Stanford, CA 94305 (USA).
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Current address: Institute of Bioengineering. Miguel Hernández University. Avenida de la
Universidad, s/n 03202 Elche, Alicante (Spain). 3
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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.
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CONFLICT OF INTEREST: The authors declare that no competing interests exist.
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Copyright © 2015 by the American Physiological Society.
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Abstract
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Hair cells from auditory and vestibular systems transmit continuous sound and balance
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information to the central nervous system through the release of synaptic vesicles at ribbon
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synapses. The high activity experienced by hair cells requires a unique mechanism to sustain
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recruitment and replenishment of synaptic vesicles for continuous release. Using pre and
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postsynaptic electrophysiological recordings, we explored the potential contribution of
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calcium-induced calcium release (CICR) in modulating the recruitment of vesicles to
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auditory hair cell ribbon synapses. Pharmacological manipulation of CICR with agents
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targeting endoplasmic reticulum calcium stores reduced both spontaneous postsynaptic
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multiunit activity and the frequency of excitatory postsynaptic currents (EPSCs).
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Pharmacological treatments had no effect on hair cell resting potential or activation curves
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for calcium and potassium channels. However, these drugs exerted a reduction in vesicle
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release measured by dual-sine capacitance methods. In addition, calcium substitution by
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barium reduced release efficacy by delaying release onset and diminishing vesicle
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recruitment. Together these results demonstrate a role for calcium stores in hair cell ribbon
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synaptic transmission and suggest a novel contribution of CICR in hair cell vesicle
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recruitment. We hypothesize that calcium entry via calcium channels is tightly regulated to
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control timing of vesicle fusion at the synapse whereas CICR is used to maintain a tonic
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calcium signal to modulate vesicle trafficking.
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Keywords
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Hair cell, dual sine capacitance, CICR, intracellular stores, ribbon synapse, synaptic
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transmission
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Introduction
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Hair cells, the sensory receptors in the auditory and vestibular systems, convert mechanical
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information into synaptic activity through the release of neurotransmitter at ribbon synapses.
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Each hair cell contains tens of synaptic ribbons (Schnee et al. 2005; Schnee et al. 2011;
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Sneary 1988), presynaptic specializations surrounded by synaptic vesicles and associated to
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active zones and L-type Ca2+ channels (Issa and Hudspeth 1994; Roberts et al. 1990; Tucker
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and Fettiplace 1995). Similar to other sensory synapses, hair cell ribbon synapses operate in
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a graded fashion, reaching high release rates and exhibiting little fatigue. Both of these
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properties require rapid vesicle replenishment by a mechanism that is not well understood.
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Calcium-induced Ca2+ release (CICR) is a mechanism by which the influx of Ca2+
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through Ca2+ channels in the plasma membrane activates Ca2+ release from intracellular
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stores (Verkhratsky 2005). CICR is implicated in a number of neuronal functions such as
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neuronal excitability, gene expression, synaptic plasticity and release (Bouchard et al. 2003).
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In central synapses, both endoplasmic reticulum (ER) and mitochondria are well-known
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intracellular Ca2+ stores and their Ca2+ homeostatic modulation alter synaptic transmission
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pre- and postsynaptically (Llano et al., 2000; Emptage et al., 2001; Bardo et al., 2006). CICR
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is also suggested to contribute to synaptic transmission at ribbon synapses (Babai et al. 2010;
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Lelli et al. 2003).
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Calcium imaging identified CICR in turtle auditory papilla hair cells (Tucker and
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Fettiplace 1995), frog semicircular canal (Lelli et al. 2003), P6-11 mouse inner hair cells
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(Iosub et al. 2015; Kennedy and Meech 2002) and rat and guinea pig outer hair cells (Evans
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et al. 2000; Mammano et al. 1999). In mammalian outer hair cells, CICR is functionally
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associated to subsynaptic Ca2+ stores in close proximity to efferent terminals (Lioudyno et al.
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2004). In addition, Ca2+ can be released by inositol triphosphate-gated Ca2+ stores at the base
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of the outer hair cell hair bundle (Mammano et al. 1999). Although pharmacological data
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demonstrates the presence of intracellular stores in hair cells, its physiological role is
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debatable. Intracellular Ca2+ stores have been functionally associated to the control of BK
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channel activity in inner hair cells (Beurg et al. 2005; Marcotti et al. 2004) modulation of
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outer hair cell electromotility (Dallos et al. 1997), homeostatic control of presynaptic Ca2+
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levels (Kennedy and Meech 2002; Tucker and Fettiplace 1995), time-dependent segregation
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of afferent and efferent signaling (Im et al. 2014) and regulation of vesicular trafficking,
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exocytosis and synaptic transmission (Hendricson and Guth 2002; Lelli et al. 2003).
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Here, we performed auditory-nerve multiunit and single unit recordings as well as
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hair-cell dual-sine capacitance experiments to study the potential contribution of CICR to
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hair-cell synaptic transmission. Pharmacological and divalent cation substitution results are
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consistent with a role for CICR in the recruitment of vesicles to support maintained release in
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auditory hair cell ribbon synapses.
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Materials & Methods
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Tissue preparation
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The auditory papilla of red-ear sliders (Trachemys scripta elegans) was dissected as
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previously described (Schnee et al. 2005). All animal procedures were approved by the
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Stanford IACUC committee and are in accord with NIH guidelines and standards. Turtle half-
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head preparations were used for multiunit activity measurements from the eighth cranial
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nerve. The turtle head was split in half and pinned in a sylgardTM dissection chamber with
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either an external solution similar to the one used in patch clamp recordings or with
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bicarbonate-buffered perilymph containing (in mM) NaCl 126, KCl 2.5, NaHCO3 13,
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NaH2PO4 1.7, CaCl2 1.8, MgCl2 1 and glucose 5 (continuously bubbled with 95% O2 and 5%
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CO2). The brain was removed and the auditory nerve exposed (Figure 1A), cutting the
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connections to posterior ampulla and saccule. The ventral otic membrane was trimmed to
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allow access to perfusion prior to mounting the preparation in the recording dish. A gravity-
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controlled perfusion pipet was located approximately 5 mm above the otic capsule and was
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connected to a perfusion system with a flow rate of roughly 1 ml/min.
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For intracellular hair-cell recordings, the inner ear was dissected from the otic capsule
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in external solution containing 128 mM NaCl, 0.5 mM KCl, 2.8 CaCl2, 2.2 mM MgCl2, 10
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mM HEPES, 6 mM glucose, 2mM creatine monohydrate, 2mM ascorbate, 2 mM pyruvate
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and the pH was adjusted to 7.6 and osmolality adjusted to 275 mosml/kg. The external
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solution was supplemented with 20 µM curare to eliminate efferent activity and 100 nM
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apamin was included in some of the experiments to block SK activity. We found no evidence
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of remaining efferent activity when incubated with curare in both pre and postsynaptic
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recordings. To disrupt mechanotransduction channels, after trimming extraneous tissue and
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removing the otoconia, the papilla was incubated for 15 min in external solution and perfused
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with 200 µl of 5 mM BAPTA before and after removing the tectorial membrane with a fine
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insect pin. In some experiments where the tectorial membrane was left intact, cell
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visualization was impaired but no obvious electrophysiological effects were observed. The
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basilar papilla was transferred to the recording chamber and secured with single strands of
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dental floss. Cells were imaged with an Axioskop 2 FS plus (Zeiss, Thornwood, NY) with
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bright field optics using a 60x 0.9 NA water objective (LUMPlan Fl/IR, Olympus). Perfusion
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of bath and drugs was delivered using a Minipuls 3 pump (Gilson, Midleton, WI).
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Electrophysiology
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For multiunit activity we used the turtle half-head preparation, where the auditory nerve was
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inserted in an hourglass-shaped suction electrode with a micromanipulator (Narishige, East
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Meadow, NY) and compound action potentials were recorded using a differential AC
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preamplifier (Grass, P55 Asto-Med, Westwarwick, RI). One electrode was inserted into the
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borosilicate suction pipet and the neutral electrode was in contact with the bath. The signal
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was band-passed filtered (1Hz-1kHz) and amplified 1,000 times.
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potentials were collected through a data acquisition interface (CED Micro 1401 mkII,
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Cambridge Electronic Design, UK) and analyzed with Spike 2 software (Cambridge
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Electronic Design, UK). Noise levels were identified by blocking afferent activity with 1 μM
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tetrodoxin (TTX) or prolonged high potassium concentration and spike threshold was set to
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3xSD of baseline noise. Drugs were applied via the local perfusion system after a baseline
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firing rate was established. Control perfusion with the external solution (sans drug) was used
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to confirm no mechanical artifacts and firing stability throughout recording (96 ± 2 % after
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20 min, n = 3).
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For hair-cell patch clamp experiments, thick-wall borosilicate electrodes of resistance 2.5-3.5
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MΩ were used with internal solution containing 110 mM CsCl, 1 mM EGTA, 5 mM creatine
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phosphate, 3 mM Na2ATP, 10 mM HEPES, 3 mM MgCl2, 2 mM ascorbate and pH adjusted
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to 7.2 and osmolality at 255 mosml/kg. Stimulus protocols were performed starting 10 min
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after whole-cell configuration to allow solution equilibration and run up stabilization of the
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Ca2+ current (Schnee 2003). Hair cells were voltage clamped with an Axopatch 200B (Axon
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Instruments-Molecular Devices, Sunnyvale, CA) or a VE-2 amplifier (Alembic Instruments,
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Montreal, Canada). Data were collected at 100-200 kHz with an IOTech Daq/3000
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acquisition board (MC Measurement Computing, Norton, MA) driven by jClamp software
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(Scisoft). Voltage was intentionally not corrected for junction potential or series resistance to
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match the values used in two-sine capacitance protocols (Schnee et al. 2011). Dual sinusoidal
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stimulation was performed to compensate in-cell stray capacitance at different frequencies
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before running capacitance protocols. A dual sinusoid with amplitudes and frequencies of 20-
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30 mV at 3.1-6.2 kHz and 6.2-9.4 kHz was delivered superimposed to the desired voltage
Compound action
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step. Capacitance measurements were low-pass filtered at 40 Hz and the onset-offset gating
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capacitative transients were removed offline.
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Afferent fiber patch recordings were done using similar solutions as described for hair cell
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recordings (Schnee et al. 2013). Patch electrodes were smaller tipped and had resistances of
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9-11 MΩ. Series resistance was compensated to 70% resulting in uncompensated SR of 11 +
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3 MΩ (n = 5).
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Data analysis
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In hair-cell patch-clamp experiments, the initial capacitance of cells located at the center of
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the papilla was 12.4 ± 1.4 pF (n=47) 1 min after establishment of whole cell configuration.
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Cells were discarded when the uncompensated series resistance was larger than 12 MΩ due
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to the difficulty in the neutralization of in-cell stray capacitance. Cells were also discarded
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when the leak current was larger than 50 pA after 9 min of recording to avoid calcium
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channel inactivation due to slow calcium loading of the cells. Data and graphics show mean ±
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SD and number of experiments (n) unless noted. Statistical analyses were performed using a
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two-tailed Student’s t-test assuming normal distribution. Box plots are presented with raw
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data [symbols], mean [star] and standard deviation of the mean [box]. In the capacitance
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experiments where multiple release protocols were evoked, normalized percentage values are
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related to the value evoked in the first pulse. In figures, the y-axis represents the amount of
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release in a second stimulation protocol normalized by a first stimulation protocol (2nd-1st
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pulse/ 1st pulse); therefore values below 0 represent a reduction in the 2nd pulse, whereas
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values near 1 represent twice as much release in the 2nd pulse.
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Results
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Pharmacological manipulation of calcium stores reduces auditory nerve activity
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The ability of neuronal endoplasmic reticulum (ER) and mitochondria to store and release
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Ca2+ has been extensively characterized (Nicholls 2009; Tang and Zucker 1997; Verkhratsky
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2005; Wan et al. 2012). To study the potential contribution of these Ca2+ stores in hair-cell
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synaptic release, we first tested the pharmacological effect of Ca2+ store modulators on the
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auditory nerve firing rate using an extracellular multiunit preparation (Figure 1A). Spike
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activity in control experiments was abolished by TTX (1 μM), kynurenic acid (2 mM) or
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DNQX (1 μM) (Figure 1C), confirming the glutamatergic nature of the hair-cell ribbon
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synapse and that neural activity was driven by synaptic activity. Caffeine (10-20 mM), a non-
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specific ER modulator that keeps ryanodine receptors (RyRs) in a semi-opened state (Zucchi
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and Ronca-Testoni 1997), reduced the spike rate to 28 ± 21% of its initial rate (n = 8, p=
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0.0001) (Figure 1B-C). BHQ, a blocker of the SERCA pump in the ER, reduced the spike
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activity to 44 ± 31% of its initial rate (n = 6, p = 0.001). Similarly, ryanodine, which blocks
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RyRs at high concentrations (60 μM), reduced multiunit activity to 73 ± 26% of control (n =
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6, p= n.s.). Ruthenium red (40 μM), a non specific inhibitor of RyR, also reduced spike
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activity (n=1, data not shown). Incubation with drugs known to reduce mitochondrial Ca2+
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buffering
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tetraphenylphosphonium (TPP+) and antimycin A, reduced spike activity to a lesser extent.
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Spiking was reduced by TPP+ (100 μM) to 74 ± 4% of control (n = 5, p = 0.002) and by
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antimycin A (10 μM) to 77 ± 22% of control (n = 4, p = n.s.). Although pharmacological
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manipulation suggested a potential contribution of both mitochondria and CICR Ca2+ stores
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to synaptic activity, we concentrated our attention in the ER, which provided more robust
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effects.
by
interfering
with
mitochondrial
membrane
potential,
such
as
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The contribution of CICR to hair-cell synaptic transmission was first observed using
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vestibular nerve recordings (Hendricson and Guth 2002; Lelli et al. 2003; Rossi et al. 2006).
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In our experiments, application of caffeine reduced the spontaneous spiking rate, consistent
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with the ER depletion effect reported in other systems (Albrecht et al. 2002; Alonso et al.
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1999; Hongpaisan et al. 2001; Pozzo-Miller et al. 1997). Additional application of 100 nM
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apamin, a blocker of the Ca2+-dependent SK channel, did not vary the spontaneous activity
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reduction obtained with caffeine (data not shown), thus discarding a potential contribution of
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SK-evoked hyperpolarization due to Ca2+ release from ER. Reduction during prolonged
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caffeine application could alternatively be explained by eventual Ca2+ depletion in the ER
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and a consequent impairment of a potential CICR mechanism.
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Caffeine reduces postsynaptic activity in auditory fibers
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The pharmacological effects observed in our multiunit preparation cannot, however,
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distinguish between a pre or postsynaptic contribution of stores to auditory synaptic
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transmission (Fitzjohn and Collingridge 2002). To obtain further evidence of a potential role
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for CICR in synaptic activity, we tested the effect of caffeine on excitatory postsynaptic
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currents (EPSCs) measured from individual afferent fibers from the auditory nerve (Schnee et
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al. 2013). Postsynaptic afferent patch clamp recordings were made and spontaneous activity
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recorded from the neurons (Figure 2). Application of caffeine (10 mM) resulted in a net
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decrease in EPSC frequency that could be recovered upon washout (Figure 2A). Change in
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frequency for 9 fibers is presented in Figure 2C. Of these, 5 are whole cell recordings and 4
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are cell attached recordings where spike rate could be monitored. In all cases the frequency of
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release was reduced. Frequency histograms for EPSC amplitudes were also generated; an
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example is presented in Figure 2B. The mean EPSC amplitude tended to be reduced, maybe
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due to a loss of synchrony (Schnee et al. 2013); however, this reduction was not statistically
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significant (Figure 2D). In three of four fiber recordings there was a transient increase in
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EPSC frequency followed by a decrease. Additionally, upon washout in three of four cells
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there was an initial overshoot in EPSC frequency (data no shown), indicative of an increased
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permeability to calcium, maybe due to the activation of store-operated calcium channels
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(Lukyanenko et al. 2001). Together these data support the conclusion that there is a
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presynaptic role for CICR in regulating synaptic vesicle release.
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Both
multiunit
and
single
postsynaptic
bouton
recordings
suggest
that
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pharmacological manipulations of CICR modulates action potential rate by reducing EPSC
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frequency. The frequency of EPSCs is driven presynaptically as the rate of vesicle fusion. In
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turn, fusion can be modulated at multiple levels that are direct or indirect. For example,
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hyperpolarizing the hair cell will reduce release, inactivating Ca2+ channels might reduce
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release directly, reduction in the Ca2+ current might also alter release properties or vesicle
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trafficking and recycling (Grant and Fuchs 2008; Johnson et al. 2008; Lee et al. 2007;
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Magistretti et al. 2015). The following experiments systematically evaluate each potential
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mechanism for reducing synaptic vesicle fusion.
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Caffeine has no effect on presynaptic electrical properties
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A presynaptic effect is postulated to underlie the reduction in postsynaptic activity
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after pharmacological manipulation of Ca2+ stores (Bouchard et al. 2003). The observed
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reduction
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hyperpolarization. Caffeine could trigger the release of Ca2+ from intracellular stores and
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activate SK potassium channels, thus hyperpolarizing the hair cell and reducing release
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probability. To examine this possibility, we tested the effect of caffeine perfusion on the
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electrical properties of hair cells (Figure 3). The responses of hair cells recorded in current-
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clamp to 10 pA current injects are presented in Figure 3A in the absence and presence of
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caffeine. We found no change in resting potential and a variable effect on the electrical
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resonance response. A summary of the resting potential data is presented in the box plots of
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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
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resonance was reduced, but this was not statistically different, nor did it typically recover
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upon washing.
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It is also possible that the reduced frequency of release obtained with caffeine is due
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to the efflux of calcium from calcium stores, leading to Ca2+ channel inactivation at the
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synapse (Schnee and Ricci, 2003). We compared hair cell Ca2+ current amplitudes in
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response to a depolarizing pulse before and during drug application (caffeine or ryanodine) in
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the external solution (Figure 3C). The same protocol (two 3-s depolarizing pulses separated
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by 5 min) was performed using drugs in the internal solution (8Br-cAMP or thapsigargin)
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(Figure 3D). Data summarized in Figure 3C,D suggest that the Ca2+ current amplitude was
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not significantly altered by drug application. Thus, the most parsimonious explanation for the
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reduction in vesicle release is a synaptically-driven mechanism.
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We also investigated the macroscopic currents elicited in voltage-clamp (Figure 3E-
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H). Macroscopic currents elicited from both hyperpolarizing and depolarizing voltage steps
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did not vary significantly in the presence of caffeine. Current-voltage plots elicited from tail
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currents at the time point indicated by the blue line and plotted in Figure 3G were also not
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significantly different between control and caffeine treated. A summary of the voltages of
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half activation shown in Figure 3H were also not significantly different. Therefore, these data
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indicate that the changes recorded postsynaptically during caffeine perfusion are likely to be
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a direct effect on synaptic properties within the hair cell.
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Time dependence of superlinear release
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The two sine technique (Schnee et al., 2011) was first used to monitor vesicle release during
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cell depolarization to avoid the intercellular variability found in hair cells, particularly with
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repeated measures (Levic et al. 2011; Patel et al. 2012; Quinones et al. 2012; Rutherford and
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Roberts 2006). Two components of release were observed, an initial linear component,
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previously demonstrated to be accounted for by vesicles within 0.7 µms of the synapses, and
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a larger superlinear component that requires recruitment of additional vesicles (Figure 4A)
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(Schnee et al. 2005; Schnee et al. 2011). The first linear release component is proportional to
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the Ca2+ load (integral of calcium current) and correlates with the vesicle population near to
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the synapses (Schnee et.al., 2005, 2011). In our hands the traditional pool descriptions of
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rapidly and readily released are less valuable in that pool size as measured by depletion varies
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within a given cell quite substantially and is often difficult to observe, suggesting a very
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dynamic population of vesicles with robust recruitment (Schnee et.al., 2005, 2011). The first
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component includes these pools but likely additional vesicles from immediately near the
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synapse and potentially some vesicles that are recruited during depolarization. The second
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superlinear component presents a higher release rate and is independent of Ca2+ load. Similar
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superlinear release components are observed in other secretory cells (Andersson et al. 2011;
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Seward et al. 1996) as well as photoreceptors and hair cells by applying trains of short
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stimulation pulses using single sine capacitance techniques (Duncker et al. 2013; Innocenti
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and Heidelberger 2008; Moser and Beutner 2000). At present, we consider the superlinear
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release to represent the hair cell´s exocytotic activity when release sites are maximally filled
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and releasing. This release component requires significant recruitment of vesicles (Schnee et.
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al., 2005, 2011). This interpretation remains a hypothesis requiring further investigation.
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Calcium imaging experiments also showed a non-linear rise in the intracellular Ca2+ levels
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accompanying the superlinear capacitance change (Schnee et al. 2011). One possible
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interpretation of this nonlinear signaling is a potential second internal source of Ca2+, maybe
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supporting CICR.
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Our postsynaptic results point to a potential role of hair cell ER Ca2+ homeostasis in
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the ability to sustain ribbon synaptic transmission. To test whether ER Ca2+ homeostasis
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modulates vesicle release, we used the dual sine capacitance technique to monitor release
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properties under control conditions (Figure 4) and during pharmacological treatment (Figure
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5-7). Previous experiments using single sine stimuli have demonstrated a presynaptic
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facilitation effect that results in variation in responses to repeated stimuli which could make
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interpretation of pharmacological manipulations requiring repeated measures difficult (Cho et
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al. 2011; Schnee et al. 2011). Further, since repeated stimulation using long depolarizing
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pulses has not been described, we first characterized release properties of control hair cells in
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order to establish a comparator for subsequent pharmacological experiments. Control cells
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were voltage-clamped to -85 mV and consecutive 3-s stimulation pulses to 50% of peak Ca2+
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current (as estimated from IV plots generated for each cell) were tested. These relatively long
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protocols were selected to ensure observation and separation of the two release components,
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while at the same time not overtaxing the cell so that it could replenish for multiple
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stimulations (Schnee et al. 2005). Also, these stimulations are closer to physiological than
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traditional depolarizations where potentials are stepped to elicit peak calcium currents.
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Figure 4B shows an example in which hair cell calcium current and capacitance were
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measured sequentially in response to a stimulation that elicited a linear and superlinear
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component. In this example, multiple depolarizing pulses separated by 1 min lead to a slight
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release reduction, consistent with vesicle depletion. Conversely, release was unexpectedly
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enhanced when pulses were separated by 3 min (Fig. 4B). The amount of release was not
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significantly modified when two consecutive pulses were separated by 1 min (Figure 4D).
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However, longer interpulse intervals (IPI) lead to a significant release increase in all cells
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studied. Changes in total release as well as linear and superlinear release were tested by two
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consecutive pulses using different IPIs (1, 3 and 5 min) in a total of 19 cells. The linear
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component was quantified by measuring the increase in membrane capacitance from the
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onset of depolarization until the appearance of the superlinear component. Since we observed
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variability in the onset of the superlinear component between pulses (see below), linear
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release was measured in both pulses until the same time point, that is, until the superlinear
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onset of the second pulse. For pulses separated by 1 min, release was the same as the initial
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value (n= 5 cells) (Fig. 4D). Both components showed non-significant differences in the
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second pulse: superlinear release reduction of 2 ± 17% and linear release reduction of 4 ± 7%
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(Figure 4E-F). Similarly, the superlinear release onset was constant in the second pulse (0.02
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± 0.1s, n.s.) (Figure 4G). However, when pulses were separated by 3 min, consecutive pulses
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unexpectedly lead to total release enhancement (38 ± 25% increase, n = 5 cells, p= 0.02) (Fig.
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4D). Enhancement was also observed for 5-min interpulse interval (IPI) (52 ± 32%, n = 9
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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
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increase in the superlinear component for IPIs of 3-min (110 ± 42% increase, p=0.007) and 5-
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min (115 ± 39%, p= 0.0001, for 5 min)(Fig. 4F), whereas the linear component did not
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change significantly (5 ± 11% reduction for 3 min, 4 ± 10% increase for 5 min)(compare y-
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axis in Figure 4E and 4F). The observed release enhancement was originated by a shortening
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in the superlinear release onset time for IPIs of 3-min (0.6 ± 0.3s, p= 0.01) as well as 5-min
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(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.
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Therefore the observed increase in release after 3-5 minutes cannot be accounted for by an
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increase in baseline calcium levels due to buffer saturation. Release enhancement, however,
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may be consistent with an increase in ER Ca2+ reuptake leading to an increase in luminal Ca2+
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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
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activate store-dependent expression of synaptic proteins (Alkon et al. 1998; Benech et al.
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1999; Sutton et al. 1999). Although we cannot categorically interpret the physiological
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relevance of this release shift, the control data are needed for the pharmacological
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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
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manipulations that require repeated measures.
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To study whether the shift in the superlinear release onset was Ca2+-dependent and
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release-specific or could also affect other Ca2+-dependent processes taking place distant to the
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synaptic ribbon, we tested the threshold for SK channel activation, a channel located near
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efferent terminals (Lioudyno et al. 2004). Hair cells were stimulated and SK was monitored
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by removing apamin from the external solution (see examples in Figure 5A). In 120 of 124
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cells superlinear release was preceded 0.7 ± 0.9 s by SK activation. Superlinear release onset
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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
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cells, an ER-like cistern opposing every efferent contact is proposed to act as a Ca2+ store to
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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
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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
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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.
Albrecht MA, Colegrove SL, and 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, and 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.
Alonso MT, Barrero MJ, Michelena P, Carnicero E, Cuchillo I, Garcia AG, Garcia-Sancho J, Montero
M, and Alvarez J. Ca2+-induced Ca2+ release in chromaffin cells seen from inside the ER with
targeted aequorin. J Cell Biol 144: 241-254, 1999.
Andersson SA, Pedersen MG, Vikman J, and Eliasson L. Glucose-dependent docking and SNARE
protein-mediated exocytosis in mouse pancreatic alpha-cell. Pflugers Arch 462: 443-454, 2011.
Babai N, Morgans CW, and Thoreson WB. Calcium-induced calcium release contributes to synaptic
release from mouse rod photoreceptors. Neuroscience 165: 1447-1456, 2010.
Benech JC, Crispino M, Kaplan BB, and Giuditta A. Protein synthesis in presynaptic endings from
squid brain: modulation by calcium ions. J Neurosci Res 55: 776-781, 1999.
Beurg M, Hafidi A, Skinner LJ, Ruel J, Nouvian R, Henaff M, Puel JL, Aran JM, and Dulon D.
Ryanodine receptors and BK channels act as a presynaptic depressor of neurotransmission in
cochlear inner hair cells. Eur J Neurosci 22: 1109-1119, 2005.
609
610
611
612
613
614
615
616
617
618
619
620
621
622
623
624
625
626
627
628
629
630
631
632
633
634
635
636
637
638
639
640
641
642
643
644
645
646
647
648
649
650
651
652
653
654
655
656
657
Bhalla A, Tucker WC, and 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, and Geiger JD. Presence and functional significance of presynaptic
ryanodine receptors. Prog Neurobiol 69: 391-418, 2003.
Cadetti L, Bryson EJ, Ciccone CA, Rabl K, and Thoreson WB. Calcium-induced calcium release in rod
photoreceptor terminals boosts synaptic transmission during maintained depolarization. Eur J
Neurosci 23: 2983-2990, 2006.
Castellano-Munoz M, and Ricci AJ. Role of intracellular calcium stores in hair-cell ribbon synapse.
Front Cell Neurosci 8: 162, 2014.
Coggins MR, Grabner CP, Almers W, and Zenisek D. Stimulated exocytosis of endosomes in
goldfish retinal bipolar neurons. J Physiol 584: 853-865, 2007.
Chen M, Krizaj D, and Thoreson WB. Intracellular calcium stores drive slow non-ribbon vesicle
release from rod photoreceptors. Front Cell Neurosci 8: 20, 2014.
Cho S, Li GL, and 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.
Dallos P, He DZ, Lin X, Sziklai I, Mehta S, and Evans BN. Acetylcholine, outer hair cell
electromotility, and the cochlear amplifier. J Neurosci 17: 2212-2226, 1997.
Dittman JS, and Regehr WG. Calcium dependence and recovery kinetics of presynaptic depression
at the climbing fiber to Purkinje cell synapse. J Neurosci 18: 6147-6162, 1998.
Duncker SV, Franz C, Kuhn S, Schulte U, Campanelli D, Brandt N, Hirt B, Fakler B, Blin N, Ruth P,
Engel J, Marcotti W, Zimmermann U, and Knipper M. Otoferlin couples to clathrin-mediated
endocytosis in mature cochlear inner hair cells. J Neurosci 33: 9508-9519, 2013.
Evans MG, Lagostena L, Darbon P, and Mammano F. Cholinergic control of membrane conductance
and intracellular free Ca2+ in outer hair cells of the guinea pig cochlea. Cell Calcium 28: 195-203,
2000.
Fitzjohn SM, and Collingridge GL. Calcium stores and synaptic plasticity. Cell Calcium 32: 405-411,
2002.
Fuchs PA. A 'calcium capacitor' shapes cholinergic inhibition of cochlear hair cells. J Physiol 592:
3393-3401, 2014.
Gomis A, Burrone J, and Lagnado L. Two actions of calcium regulate the supply of releasable
vesicles at the ribbon synapse of retinal bipolar cells. J Neurosci 19: 6309-6317, 1999.
Goutman JD, and Glowatzki E. Short-term facilitation modulates size and timing of the synaptic
response at the inner hair cell ribbon synapse. J Neurosci 31: 7974-7981, 2011.
Grant L, and Fuchs P. Calcium- and calmodulin-dependent inactivation of calcium channels in inner
hair cells of the rat cochlea. J Neurophysiol 99: 2183-2193, 2008.
Graydon CW, Cho S, Li GL, Kachar B, and von Gersdorff H. Sharp Ca(2)(+) nanodomains beneath the
ribbon promote highly synchronous multivesicular release at hair cell synapses. J Neurosci 31:
16637-16650, 2011.
Hendricson AW, and Guth PS. Transmitter release from Rana pipiens vestibular hair cells via
mGluRs: a role for intracellular Ca(++) release. Hear Res 172: 99-109, 2002.
Hongpaisan J, Pivovarova NB, Colegrove SL, Leapman RD, Friel DD, and Andrews SB. Multiple
modes of calcium-induced calcium release in sympathetic neurons II: a [Ca2+](i)- and locationdependent transition from endoplasmic reticulum Ca accumulation to net Ca release. J Gen Physiol
118: 101-112, 2001.
Im GJ, Moskowitz HS, Lehar M, Hiel H, and Fuchs PA. Synaptic calcium regulation in hair cells of the
chicken basilar papilla. J Neurosci 34: 16688-16697, 2014.
Innocenti B, and Heidelberger R. Mechanisms contributing to tonic release at the cone
photoreceptor ribbon synapse. J Neurophysiol 99: 25-36, 2008.
658
659
660
661
662
663
664
665
666
667
668
669
670
671
672
673
674
675
676
677
678
679
680
681
682
683
684
685
686
687
688
689
690
691
692
693
694
695
696
697
698
699
700
701
702
703
704
705
706
707
708
Iosub R, Avitabile D, Grant L, Tsaneva-Atanasova K, and Kennedy HJ. Calcium-Induced Calcium
Release during Action Potential Firing in Developing Inner Hair Cells. Biophys J 108: 1003-1012,
2015.
Issa NP, and Hudspeth AJ. Clustering of Ca2+ channels and Ca(2+)-activated K+ channels at
fluorescently labeled presynaptic active zones of hair cells. Proc Natl Acad Sci U S A 91: 7578-7582,
1994.
Johnson SL, Forge A, Knipper M, Munkner S, and Marcotti W. Tonotopic variation in the calcium
dependence of neurotransmitter release and vesicle pool replenishment at mammalian auditory
ribbon synapses. J Neurosci 28: 7670-7678, 2008.
Kennedy HJ, and Meech RW. Fast Ca2+ signals at mouse inner hair cell synapse: a role for Ca2+induced Ca2+ release. J Physiol 539: 15-23, 2002.
Krizaj D, Lai FA, and Copenhagen DR. Ryanodine stores and calcium regulation in the inner
segments of salamander rods and cones. J Physiol 547: 761-774, 2003.
Kwan CY, and Putney JW, Jr. Uptake and intracellular sequestration of divalent cations in resting
and methacholine-stimulated mouse lacrimal acinar cells. Dissociation by Sr2+ and Ba2+ of
agonist-stimulated divalent cation entry from the refilling of the agonist-sensitive intracellular
pool. J Biol Chem 265: 678-684, 1990.
Lee S, Briklin O, Hiel H, and Fuchs P. Calcium-dependent inactivation of calcium channels in
cochlear hair cells of the chicken. J Physiol 583: 909-922, 2007.
Lelli A, Perin P, Martini M, Ciubotaru CD, Prigioni I, Valli P, Rossi ML, and Mammano F. Presynaptic
calcium stores modulate afferent release in vestibular hair cells. J Neurosci 23: 6894-6903, 2003.
Lenzi D, Runyeon JW, Crum J, Ellisman MH, and Roberts WM. Synaptic vesicle populations in
saccular hair cells reconstructed by electron tomography. J Neurosci 19: 119-132, 1999.
Levic S, Bouleau Y, and Dulon D. Developmental acquisition of a rapid calcium-regulated vesicle
supply allows sustained high rates of exocytosis in auditory hair cells. PLoS One 6: e25714, 2011.
Liberman LD, Wang H, and Liberman MC. Opposing gradients of ribbon size and AMPA receptor
expression underlie sensitivity differences among cochlear-nerve/hair-cell synapses. J Neurosci 31:
801-808, 2011.
Lioudyno M, Hiel H, Kong JH, Katz E, Waldman E, Parameshwaran-Iyer S, Glowatzki E, and Fuchs
PA. A "synaptoplasmic cistern" mediates rapid inhibition of cochlear hair cells. J Neurosci 24:
11160-11164, 2004.
Lukyanenko V, Viatchenko-Karpinski S, Smirnov A, Wiesner TF, and Gyorke S. Dynamic regulation
of sarcoplasmic reticulum Ca(2+) content and release by luminal Ca(2+)-sensitive leak in rat
ventricular myocytes. Biophys J 81: 785-798, 2001.
Llano I, Gonzalez J, Caputo C, Lai FA, Blayney LM, Tan YP, and Marty A. Presynaptic calcium stores
underlie large-amplitude miniature IPSCs and spontaneous calcium transients. Nat Neurosci 3:
1256-1265, 2000.
Magistretti J, Spaiardi P, Johnson SL, and Masetto S. Elementary properties of Ca(2+) channels and
their influence on multivesicular release and phase-locking at auditory hair cell ribbon synapses.
Front Cell Neurosci 9: 123, 2015.
Mammano F, Frolenkov GI, Lagostena L, Belyantseva IA, Kurc M, Dodane V, Colavita A, and Kachar
B. ATP-Induced Ca(2+) release in cochlear outer hair cells: localization of an inositol triphosphategated Ca(2+) store to the base of the sensory hair bundle. J Neurosci 19: 6918-6929, 1999.
Marcotti W, Johnson SL, and Kros CJ. Effects of intracellular stores and extracellular Ca(2+) on
Ca(2+)-activated K(+) currents in mature mouse inner hair cells. J Physiol 557: 613-633, 2004.
Moser T, and Beutner D. Kinetics of exocytosis and endocytosis at the cochlear inner hair cell
afferent synapse of the mouse. Proc Natl Acad Sci U S A 97: 883-888, 2000.
Neves G, Neef A, and Lagnado L. The actions of barium and strontium on exocytosis and
endocytosis in the synaptic terminal of goldfish bipolar cells. J Physiol 535: 809-824, 2001.
Nicholls DG. Mitochondrial calcium function and dysfunction in the central nervous system.
Biochim Biophys Acta 1787: 1416-1424, 2009.
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Nouvian R, Neef J, Bulankina AV, Reisinger E, Pangrsic T, Frank T, Sikorra S, Brose N, Binz T, and
Moser T. Exocytosis at the hair cell ribbon synapse apparently operates without neuronal SNARE
proteins. Nat Neurosci 14: 411-413, 2011.
Patel SH, Salvi JD, D OM, and Hudspeth AJ. Frequency-selective exocytosis by ribbon synapses of
hair cells in the bullfrog's amphibian papilla. J Neurosci 32: 13433-13438, 2012.
Pozzo-Miller LD, Pivovarova NB, Leapman RD, Buchanan RA, Reese TS, and Andrews SB. Activitydependent calcium sequestration in dendrites of hippocampal neurons in brain slices. J Neurosci
17: 8729-8738, 1997.
Proks P, and Ashcroft FM. Effects of divalent cations on exocytosis and endocytosis from single
mouse pancreatic beta-cells. J Physiol 487 ( Pt 2): 465-477, 1995.
Przywara DA, Chowdhury PS, Bhave SV, Wakade TD, and Wakade AR. Barium-induced exocytosis is
due to internal calcium release and block of calcium efflux. Proc Natl Acad Sci U S A 90: 557-561,
1993.
Quinones PM, Luu C, Schweizer FE, and Narins PM. Exocytosis in the frog amphibian papilla. J
Assoc Res Otolaryngol 13: 39-54, 2012.
Roberts WM, Jacobs RA, and Hudspeth AJ. Colocalization of ion channels involved in frequency
selectivity and synaptic transmission at presynaptic active zones of hair cells. J Neurosci 10: 36643684, 1990.
Rossi ML, Prigioni I, Gioglio L, Rubbini G, Russo G, Martini M, Farinelli F, Rispoli G, and Fesce R. IP3
receptor in the hair cells of frog semicircular canal and its possible functional role. Eur J Neurosci
23: 1775-1783, 2006.
Rutherford MA, and Roberts WM. Frequency selectivity of synaptic exocytosis in frog saccular hair
cells. Proc Natl Acad Sci U S A 103: 2898-2903, 2006.
Schnee ME, Castellano-Munoz M, and Ricci AJ. Response properties from turtle auditory hair cell
afferent fibers suggest spike generation is driven by synchronized release both between and
within synapses. J Neurophysiol 110: 204-220, 2013.
Schnee ME, Lawton DM, Furness DN, Benke TA, and Ricci AJ. Auditory hair cell-afferent fiber
synapses are specialized to operate at their best frequencies. Neuron 47: 243-254, 2005.
Schnee ME, and Ricci AJ. Biophysical and pharmacological characterization of voltage-gated
calcium currents in turtle auditory hair cells. J Physiol 549: 697-717, 2003.
Schnee ME, Santos-Sacchi J, Castellano-Munoz M, Kong JH, and Ricci AJ. Calcium-dependent
synaptic vesicle trafficking underlies indefatigable release at the hair cell afferent fiber synapse.
Neuron 70: 326-338, 2011.
Seward EP, Chernevskaya NI, and Nowycky MC. Ba2+ ions evoke two kinetically distinct patterns
of exocytosis in chromaffin cells, but not in neurohypophysial nerve terminals. J Neurosci 16: 13701379, 1996.
Sneary MG. Auditory receptor of the red-eared turtle: II. Afferent and efferent synapses and
innervation patterns. J Comp Neurol 276: 588-606, 1988.
Stevens CF, and Wesseling JF. Activity-dependent modulation of the rate at which synaptic vesicles
become available to undergo exocytosis. Neuron 21: 415-424, 1998.
Suryanarayanan A, and Slaughter MM. Synaptic transmission mediated by internal calcium stores
in rod photoreceptors. J Neurosci 26: 1759-1766, 2006.
Sutton KG, McRory JE, Guthrie H, Murphy TH, and Snutch TP. P/Q-type calcium channels mediate
the activity-dependent feedback of syntaxin-1A. Nature 401: 800-804, 1999.
Tang Y, and Zucker RS. Mitochondrial involvement in post-tetanic potentiation of synaptic
transmission. Neuron 18: 483-491, 1997.
Tucker T, and Fettiplace R. Confocal imaging of calcium microdomains and calcium extrusion in
turtle hair cells. Neuron 15: 1323-1335, 1995.
Unni VK, Zakharenko SS, Zablow L, DeCostanzo AJ, and Siegelbaum SA. Calcium release from
presynaptic ryanodine-sensitive stores is required for long-term depression at hippocampal CA3CA3 pyramidal neuron synapses. J Neurosci 24: 9612-9622, 2004.
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761
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765
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770
771
772
773
774
775
Verkhratsky A. Physiology and pathophysiology of the calcium store in the endoplasmic reticulum
of neurons. Physiol Rev 85: 201-279, 2005.
von Ruden L, Garcia AG, and Lopez MG. The mechanism of Ba(2+)-induced exocytosis from single
chromaffin cells. FEBS Lett 336: 48-52, 1993.
Wan QF, Nixon E, and Heidelberger R. Regulation of presynaptic calcium in a mammalian synaptic
terminal. J Neurophysiol 108: 3059-3067, 2012.
Wang LY, and Kaczmarek LK. High-frequency firing helps replenish the readily releasable pool of
synaptic vesicles. Nature 394: 384-388, 1998.
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.
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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 (**).
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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.
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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
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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.
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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 (**).
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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.
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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 (**).
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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 (**).
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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
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co caf rcA ano cA sig sto
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8B r
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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
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ap xe
8B r
th
l
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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++