Download Calcium-induced calcium release supports recruitment of synaptic

J Neurophysiol 115: 226 –239, 2016.
First published October 28, 2015; doi:10.1152/jn.00559.2015.
Calcium-induced calcium release supports recruitment of synaptic vesicles in
auditory hair cells
X Manuel Castellano-Muñoz,1 Michael E. Schnee,1 and Anthony J. Ricci1,2
1
Department of Otolaryngology, Stanford University School of Medicine, Stanford, California; and 2Department of Molecular
and Cellular Physiology, Stanford University School of Medicine, Stanford, California
Submitted 8 June 2015; accepted in final form 23 October 2015
hair cell; dual-sine capacitance; Ca2⫹-induced Ca2⫹ release; intracellular stores; ribbon synapse; synaptic transmission
HAIR CELLS, the sensory receptors in the auditory and vestibular
systems, convert mechanical information into synaptic activity
through the release of neurotransmitter at ribbon synapses.
Each hair cell contains tens of synaptic ribbons (Schnee et al.
2005, 2011; Sneary 1988), presynaptic specializations surrounded by synaptic vesicles and associated to active zones and
L-type Ca2⫹ channels (Issa and Hudspeth 1994; Roberts et al.
1990; Tucker and Fettiplace 1995). Similar to other sensory
synapses, hair cell ribbon synapses operate in a graded fashion,
reaching high release rates and exhibiting little fatigue. Both of
these properties require rapid vesicle replenishment by a mechanism that is not well understood.
Ca2⫹-induced Ca2⫹ release (CICR) is a mechanism by
which the influx of Ca2⫹ through Ca2⫹ channels in the plasma
membrane activates Ca2⫹ release from intracellular stores
(Verkhratsky 2005). CICR is implicated in a number of neu-
Address for reprint requests and other correspondence: M. CastellanoMuñoz, Inst. of Bioengineering, Miguel Hernández Univ., Avenida de la
Universidad, s/n 03202 Elche, Alicante, Spain (e-mail: [email protected]).
226
ronal functions such as neuronal excitability, gene expression,
and synaptic plasticity and release (Bouchard et al. 2003). In
central synapses both endoplasmic reticulum (ER) and mitochondria are well-known intracellular Ca2⫹ stores, and their
Ca2⫹ homeostatic modulation alters synaptic transmission preand postsynaptically (Bardo et al. 2006; Emptage et al. 2001;
Llano et al. 2000). CICR is also suggested to contribute to
synaptic transmission at ribbon synapses (Babai et al. 2010;
Lelli et al. 2003).
Calcium imaging identified CICR in turtle auditory papilla
hair cells (Tucker and Fettiplace 1995), frog semicircular canal
(Lelli et al. 2003), P6 –P11 mouse inner hair cells (Iosub et al.
2015; Kennedy and Meech 2002), and rat and guinea pig outer
hair cells (Evans et al. 2000; Mammano et al. 1999). In
mammalian outer hair cells, CICR is functionally associated to
subsynaptic Ca2⫹ stores in close proximity to efferent terminals (Lioudyno et al. 2004). In addition, Ca2⫹ can be released
by inositol triphosphate-gated Ca2⫹ stores at the base of the
outer hair cell hair bundle (Mammano et al. 1999). Although
pharmacological data demonstrate the presence of intracellular
stores in hair cells, their physiological role is debatable. Intracellular Ca2⫹ stores have been functionally associated with the
control of BK channel activity in inner hair cells (Beurg et al.
2005; Marcotti et al. 2004), modulation of outer hair cell
electromotility (Dallos et al. 1997), homeostatic control of
presynaptic Ca2⫹ levels (Kennedy and Meech 2002; Tucker
and Fettiplace 1995), time-dependent segregation of afferent
and efferent signaling (Im et al. 2014), and regulation of
vesicular trafficking, exocytosis, and synaptic transmission
(Hendricson and Guth 2002; Lelli et al. 2003).
Here we performed auditory nerve multiunit and single-unit
recordings as well as hair cell dual-sine capacitance experiments to study the potential contribution of CICR to hair cell
synaptic transmission. Pharmacological and divalent cation
substitution results are consistent with a role for CICR in the
recruitment of vesicles to support maintained release in auditory hair cell ribbon synapses.
MATERIALS AND METHODS
Tissue preparation. The auditory papilla of red-eared sliders (Trachemys scripta elegans) was dissected as previously described (Schnee et al. 2005). All animal procedures were approved by the Stanford
Institutional Animal Care and Use Committee (IACUC) and are in
accord with National Institutes of Health guidelines and standards.
Turtle half-head preparations were used for multiunit activity measurements from the eighth cranial nerve. The turtle head was split in
half and pinned in a Sylgard dissection chamber either with an
external solution similar to that used in patch-clamp recordings or
with bicarbonate-buffered perilymph containing (in mM) 126 NaCl,
0022-3077/16 Copyright © 2016 the American Physiological Society
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Castellano-Muñoz M, Schnee ME, Ricci AJ. Calcium-induced
calcium release supports recruitment of synaptic vesicles in auditory
hair cells. J Neurophysiol 115: 226 –239, 2016. First published October 28, 2015; doi:10.1152/jn.00559.2015.—Hair cells from auditory
and vestibular systems transmit continuous sound and balance information to the central nervous system through the release of synaptic
vesicles at ribbon synapses. The high activity experienced by hair
cells requires a unique mechanism to sustain recruitment and replenishment of synaptic vesicles for continuous release. Using pre- and
postsynaptic electrophysiological recordings, we explored the potential contribution of calcium-induced calcium release (CICR) in modulating the recruitment of vesicles to auditory hair cell ribbon synapses. Pharmacological manipulation of CICR with agents targeting
endoplasmic reticulum calcium stores reduced both spontaneous postsynaptic multiunit activity and the frequency of excitatory postsynaptic currents (EPSCs). Pharmacological treatments had no effect on
hair cell resting potential or activation curves for calcium and potassium channels. However, these drugs exerted a reduction in vesicle
release measured by dual-sine capacitance methods. In addition,
calcium substitution by barium reduced release efficacy by delaying
release onset and diminishing vesicle recruitment. Together these
results demonstrate a role for calcium stores in hair cell ribbon
synaptic transmission and suggest a novel contribution of CICR in
hair cell vesicle recruitment. We hypothesize that calcium entry via
calcium channels is tightly regulated to control timing of vesicle
fusion at the synapse, whereas CICR is used to maintain a tonic
calcium signal to modulate vesicle trafficking.
STORED CALCIUM PROMOTES VESICLE RECRUITMENT TO RIBBON SYNAPSES
2.5 KCl, 13 NaHCO3, 1.7 NaH2PO4, 1.8 CaCl2, 1 MgCl2, and 5
glucose (continuously bubbled with 95% O2-5% CO2). The brain was
removed and the auditory nerve exposed (Fig. 1A), cutting the connections to posterior ampulla and saccule. The ventral otic membrane
was trimmed to allow access to perfusion prior to mounting of the
A
Drug Delivery
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Fig. 1. Pharmacological disruption of intracellular calcium stores reduces
extracellular spike activity in the 8th cranial nerve. A: low-power view of
half-head preparation with the recording suction electrode and drug application
pipette labeled. B, top: effect of caffeine application on spontaneous nerve
activity. Bottom: spontaneous spike rate. Asterisk shows the time point of
maximum effect after drug delivery selected for rate quantification. C: box
plots illustrating spike rate reduction by drugs that interfere with endoplasmic
reticulum (ER) calcium homeostasis (10 mM caffeine, 50 –100 ␮M BHQ, and
60 ␮M ryanodine). In this and subsequent figures, box plots present raw data
(symbols), mean (star), and SD (box). Pharmacological modulation of mitochondrial calcium homeostasis [100 ␮M tetraphenylphosphonium (TPP⫹) and
10 ␮M antimycin A] reduced spontaneous rate to a lesser extent. Kynurenic
acid (KynA) and DNQX, 2 glutamatergic receptor antagonists, were used as
controls. **Paired t-tests show a significant difference at P ⬍ 0.01 level.
preparation in the recording dish. A gravity-controlled perfusion
pipette was located ⬃5 mm above the otic capsule and was connected
to a perfusion system with a flow rate of roughly 1 ml/min.
For intracellular hair cell recordings, the inner ear was dissected
from the otic capsule in external solution containing (in mM) 128
NaCl, 0.5 KCl, 2.8 CaCl2, 2.2 MgCl2, 10 HEPES, 6 glucose, 2
creatine monohydrate, 2 ascorbate, and 2 pyruvate and pH was
adjusted to 7.6 and osmolality to 275 mosmol/kg. The external
solution was supplemented with 20 ␮M curare to eliminate efferent
activity, and 100 nM apamin was included in some of the experiments
to block SK activity. We found no evidence of remaining efferent
activity with incubation with curare in both pre- and postsynaptic
recordings. To disrupt mechanotransduction channels, after extraneous tissue was trimmed and the otoconia removed the papilla was
incubated for 15 min in external solution and perfused with 200 ␮l of
5 mM BAPTA before and after removal of the tectorial membrane
with a fine insect pin. In some experiments where the tectorial
membrane was left intact, cell visualization was impaired but no
obvious electrophysiological effects were observed. The basilar papilla was transferred to the recording chamber and secured with single
strands of dental floss. Cells were imaged with an Axioskop 2 FS plus
(Zeiss, Thornwood, NY) with bright field optics using a ⫻60 0.9 NA
water objective (LUMPlan Fl/IR, Olympus). Perfusion of bath and
drugs was delivered with a Minipuls 3 pump (Gilson, Middleton, WI).
Electrophysiology. For multiunit activity we used the turtle halfhead preparation, in which the auditory nerve was inserted into an
hourglass-shaped suction electrode with a micromanipulator (Narishige, East Meadow, NY) and compound action potentials were
recorded with a differential AC preamplifier (Grass, P55 Astro-Med,
West Warwick, RI). One electrode was inserted into the borosilicate
suction pipette, and the neutral electrode was in contact with the bath.
The signal was band-pass filtered (1 Hz–1 kHz) and amplified 1,000
times. Compound action potentials were collected through a data
acquisition interface (CED Micro 1401 mkII, Cambridge Electronic
Design) and analyzed with Spike2 software (Cambridge Electronic
Design). Noise levels were identified by blocking afferent activity
with 1 ␮M tetrodotoxin (TTX) or prolonged high potassium concentration, and spike threshold was set to 3 ⫻ SD of baseline noise. Drugs
were applied via the local perfusion system after a baseline firing rate
was established. Control perfusion with the external solution (sans
drug) was used to confirm absence of mechanical artifacts and firing
stability throughout recording (96 ⫾ 2% after 20 min, n ⫽ 3).
For hair cell patch-clamp experiments, thick-walled borosilicate
electrodes of resistance 2.5–3.5 M⍀ were used with internal solution
containing (in mM) 110 CsCl, 1 EGTA, 5 creatine phosphate, 3
Na2ATP, 10 HEPES, 3 MgCl2, and 2 ascorbate, pH adjusted to 7.2
and osmolality at 255 mosmol/kg. Stimulus protocols were performed
starting 10 min after whole cell configuration to allow solution
equilibration and run up stabilization of the Ca2⫹ current (Schnee and
Ricci 2003). Hair cells were voltage clamped with an Axopatch 200B
(Axon Instruments-Molecular Devices, Sunnyvale, CA) or a VE-2
amplifier (Alembic Instruments, Montreal, ON, Canada). Data were
collected at 100 –200 kHz with an IOTech Daq/3000 acquisition board
(MC Measurement Computing, Norton, MA) driven by jClamp software (SciSoft). Voltage was intentionally not corrected for junction
potential or series resistance to match the values used in two-sine
capacitance protocols (Schnee et al. 2011). Dual sinusoidal stimulation was performed to compensate in-cell stray capacitance at different frequencies before capacitance protocols were run. A dual sinusoid with amplitudes and frequencies of 20 –30 mV at 3.1– 6.2 kHz
and 6.2–9.4 kHz was delivered superimposed to the desired voltage
step. Capacitance measurements were low-pass filtered at 40 Hz, and
the onset-offset gating capacitative transients were removed off-line.
Afferent fiber patch recordings were done with solutions similar to
those described for hair cell recordings (Schnee et al. 2013). Patch
electrodes were smaller tipped and had resistances of 9 –11 M⍀.
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B
Nose
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STORED CALCIUM PROMOTES VESICLE RECRUITMENT TO RIBBON SYNAPSES
RESULTS
Pharmacological manipulation of calcium stores reduces
auditory nerve activity. The ability of neuronal ER and mitochondria to store and release Ca2⫹ has been extensively characterized (Nicholls 2009; Tang and Zucker 1997; Verkhratsky
2005; Wan et al. 2012). To study the potential contribution of
these Ca2⫹ stores in hair cell synaptic release, we first tested
the pharmacological effect of Ca2⫹ store modulators on the
auditory nerve firing rate, using an extracellular multiunit
preparation (Fig. 1A). Spike activity in control experiments
was abolished by TTX (1 ␮M), kynurenic acid (2 mM), or
DNQX (1 ␮M) (Fig. 1C), confirming the glutamatergic nature
of the hair cell ribbon synapse and that neural activity was
driven by synaptic activity. Caffeine (10 –20 mM), a nonspecific ER modulator that keeps ryanodine receptors (RyRs) in a
semiopened state (Zucchi and Ronca-Testoni 1997), reduced
the spike rate to 28 ⫾ 21% of its initial rate (n ⫽ 8, P ⫽
0.0001) (Fig. 1, B and C). BHQ, a blocker of the SERCA pump
in the ER, reduced the spike activity to 44 ⫾ 31% of its initial
rate (n ⫽ 6, P ⫽ 0.001). Similarly, ryanodine, which blocks
RyRs at high concentrations (60 ␮M), reduced multiunit activity to 73 ⫾ 26% of control [n ⫽ 6, P ⫽ not significant
(n.s.)]. Ruthenium red (40 ␮M), a nonspecific inhibitor of RyR,
also reduced spike activity (n ⫽ 1, data not shown). Incubation
with drugs known to reduce mitochondrial Ca2⫹ buffering by
interfering with mitochondrial membrane potential, such as
tetraphenylphosphonium (TPP⫹) and antimycin A, reduced
spike activity to a lesser extent. Spiking was reduced by TPP⫹
(100 ␮M) to 74 ⫾ 4% of control (n ⫽ 5, P ⫽ 0.002) and by
antimycin A (10 ␮M) to 77 ⫾ 22% of control (n ⫽ 4, P ⫽ n.s.).
Although pharmacological manipulation suggested a potential
contribution of both mitochondrial and CICR Ca2⫹ stores to
synaptic activity, we concentrated our attention on the ER,
which provided more robust effects.
The contribution of CICR to hair cell synaptic transmission
was first observed with vestibular nerve recordings (Hendricson and Guth 2002; Lelli et al. 2003; Rossi et al. 2006). In our
experiments, application of caffeine reduced the spontaneous
spiking rate, consistent with the ER depletion effect reported in
other systems (Albrecht et al. 2002; Alonso et al. 1999;
Hongpaisan et al. 2001; Pozzo-Miller et al. 1997). Additional
application of 100 nM apamin, a blocker of the Ca2⫹-depen-
dent SK channel, did not vary the spontaneous activity reduction obtained with caffeine (data not shown), thus ruling out a
potential contribution of SK-evoked hyperpolarization due to
Ca2⫹ release from ER. Reduction during prolonged caffeine
application could alternatively be explained by eventual Ca2⫹
depletion in the ER and a consequent impairment of a potential
CICR mechanism.
Caffeine reduces postsynaptic activity in auditory fibers. The
pharmacological effects observed in our multiunit preparation
cannot, however, distinguish between a presynaptic and a
postsynaptic contribution of stores to auditory synaptic transmission (Fitzjohn and Collingridge 2002). To obtain further
evidence of a potential role for CICR in synaptic activity, we
tested the effect of caffeine on excitatory postsynaptic currents
(EPSCs) measured from individual afferent fibers from the
auditory nerve (Schnee et al. 2013). Postsynaptic afferent
patch-clamp recordings were made and spontaneous activity
recorded from the neurons (Fig. 2). Application of caffeine (10
mM) resulted in a net decrease in EPSC frequency that could
be recovered upon washout (Fig. 2A). Change in frequency for
nine fibers is presented in Fig. 2C. Of these, five are whole cell
recordings and four are cell-attached recordings where spike
rate could be monitored. In all cases the frequency of release
was reduced. Frequency histograms for EPSC amplitudes were
also generated; an example is presented in Fig. 2B. The mean
EPSC amplitude tended to be reduced, perhaps because of a
loss of synchrony (Schnee et al. 2013); however, this reduction
was not statistically significant (Fig. 2D). In three of four fiber
recordings there was a transient increase in EPSC frequency
followed by a decrease. Additionally, upon washout in three of
four cells there was an initial overshoot in EPSC frequency
(data not shown), indicative of an increased permeability to
calcium, perhaps due to the activation of store-operated calcium channels (Lukyanenko et al. 2001). Together these data
support the conclusion that there is a presynaptic role for CICR
in regulating synaptic vesicle release.
Both multiunit and single postsynaptic bouton recordings
suggest that pharmacological manipulations of CICR modulate
action potential rate by reducing EPSC frequency. The frequency of EPSCs is driven presynaptically as the rate of vesicle
fusion. In turn, fusion can be modulated at multiple levels that
are direct or indirect. For example, hyperpolarizing the hair cell
will reduce release, inactivating Ca2⫹ channels might reduce
release directly, and reduction in the Ca2⫹ current might also
alter release properties or vesicle trafficking and recycling
(Grant and Fuchs 2008; Johnson et al. 2008; Lee et al. 2007;
Magistretti et al. 2015). The following experiments systematically evaluate each potential mechanism for reducing synaptic
vesicle fusion.
Caffeine has no effect on presynaptic electrical properties.
A presynaptic effect is postulated to underlie the reduction in
postsynaptic activity after pharmacological manipulation of
Ca2⫹ stores (Bouchard et al. 2003). The observed reduction in
EPSC frequency could be explained by caffeine-driven hair
cell hyperpolarization. Caffeine could trigger the release of
Ca2⫹ from intracellular stores and activate SK potassium
channels, thus hyperpolarizing the hair cell and reducing release probability. To examine this possibility, we tested the
effect of caffeine perfusion on the electrical properties of hair
cells (Fig. 3). The responses of hair cells recorded in current
clamp to 10-pA current injections are presented in Fig. 3A in
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Series resistance was compensated to 70%, resulting in uncompensated series resistance of 11 ⫾ 3 M⍀ (n ⫽ 5).
Data analysis. In hair cell patch-clamp experiments, the initial
capacitance of cells located at the center of the papilla was 12.4 ⫾ 1.4
pF (n ⫽ 47) 1 min after establishment of whole cell configuration.
Cells were discarded when the uncompensated series resistance was
⬎12 M⍀ because of the difficulty in the neutralization of in-cell stray
capacitance. Cells were also discarded when the leak current was ⬎50
pA after 9 min of recording to avoid calcium channel inactivation due
to slow calcium loading of the cells. Data and graphics show means ⫾
SD and number of experiments (n) unless noted. Statistical analyses
were performed with a two-tailed Student’s t-test assuming normal
distribution. In the capacitance experiments where multiple release
protocols were evoked, normalized percentage values are related to
the value evoked in the first pulse. In figures, the y-axis represents the
amount of release in a second stimulation protocol normalized by a
first stimulation protocol (2nd ⫺ 1st pulse/1st pulse); therefore values
below 0 represent a reduction in the second pulse, whereas values near
1 represent twice as much release in the second pulse.
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Fig. 2. Postsynaptic patch recordings from afferent terminals show a reduction in excitatory postsynaptic current (EPSC) frequency and firing rate with caffeine
application. A: spontaneous EPSCs from a whole cell recording of an afferent terminal. Red traces are in the presence of 10 mM caffeine, and blue traces are
activity during washout of the drug. B: frequency histograms for EPSC amplitudes in the absence (black) and presence (red) of caffeine, where solid lines are
Gaussian fits to the histograms. C: EPSC frequency (or extracellular spike frequency) before and after caffeine administration. Paired t-tests show a significant
difference at P ⬍ 0.001 level. D: mean EPSC amplitude as measured from the fits to the Gaussian curves in B. Although a trend toward smaller mean amplitudes
was found, no statistical difference was observed.
the absence and presence of caffeine. We found no change in
resting potential and a variable effect on the electrical resonance response. A summary of the resting potential data is
presented in the box plots of Fig. 3B, where no consistent
change was observed. In several cells the quality of the resonance was reduced, but this was not statistically different, nor
did it typically recover upon washout.
It is also possible that the reduced frequency of release
obtained with caffeine is due to the efflux of calcium from
calcium stores, leading to Ca2⫹ channel inactivation at the
synapse (Schnee and Ricci 2003). We compared hair cell Ca2⫹
current amplitudes in response to a depolarizing pulse before
and during drug application (caffeine or ryanodine) in the
external solution (Fig. 3C). The same protocol (two 3-s depolarizing pulses separated by 5 min) was performed with drugs
in the internal solution (8-BrcAMP or thapsigargin) (Fig. 3D).
Data summarized in Fig. 3, C and D, suggest that Ca2⫹ current
amplitude was not significantly altered by drug application.
Thus the most parsimonious explanation for the reduction in
vesicle release is a synaptically driven mechanism.
We also investigated the macroscopic currents elicited in
voltage clamp (Fig. 3, E–H). Macroscopic currents elicited
from both hyperpolarizing and depolarizing voltage steps did
not vary significantly in the presence of caffeine. Currentvoltage plots elicited from tail currents at the time point
indicated in Fig. 3E and plotted in Fig. 3G were also not
significantly different between control and caffeine treated.
The voltages of half-activation summarized in Fig. 3H were
also not significantly different. Therefore, these data indicate
that the changes recorded postsynaptically during caffeine
perfusion are likely to be a direct effect on synaptic properties
within the hair cell.
Time dependence of superlinear release. The two-sine technique (Schnee et al. 2011) was first used to monitor vesicle
release during cell depolarization to avoid the intercellular
variability found in hair cells, particularly with repeated measures (Levic et al. 2011; Patel et al. 2012; Quinones et al. 2012;
Rutherford and Roberts 2006). Two components of release
were observed, an initial linear component, previously demonstrated to be accounted for by vesicles within 0.7 ␮m of the
synapses, and a larger superlinear component that requires
recruitment of additional vesicles (Fig. 4A) (Schnee et al. 2005,
2011). The first linear release component is proportional to the
Ca2⫹ load (integral of calcium current) and correlates with the
vesicle population near the synapses (Schnee et al. 2005,
2011). In our hands the traditional pool descriptions of rapidly
and readily released are less valuable in that pool size as
measured by depletion varies within a given cell quite substantially and is often difficult to observe, suggesting a very
dynamic population of vesicles with robust recruitment (Schnee et al. 2005, 2011). The first component includes these
pools but likely additional vesicles from immediately near the
synapse and potentially some vesicles that are recruited during
depolarization. The second superlinear component presents a
higher release rate and is independent of Ca2⫹ load. Similar
superlinear release components are observed in other secretory
cells (Andersson et al. 2011; Seward et al. 1996) as well as
photoreceptors and hair cells by application of trains of short
stimulation pulses with single-sine capacitance techniques
(Duncker et al. 2013; Innocenti and Heidelberger 2008; Moser
and Beutner 2000). At present, we consider the superlinear
release to represent the hair cell’s exocytotic activity when
release sites are maximally filled and releasing. This release
component requires significant recruitment of vesicles (Schnee
et al. 2005, 2011). This interpretation remains a hypothesis
requiring further investigation. Calcium imaging experiments
also showed a nonlinear rise in the intracellular Ca2⫹ levels
accompanying the superlinear capacitance change (Schnee et
al. 2011). One possible interpretation of this nonlinear signal-
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EPSC Peak (nA)
wash
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ing is a potential second internal source of Ca2⫹, perhaps
supporting CICR.
Our postsynaptic results point to a potential role of hair cell
ER Ca2⫹ homeostasis in the ability to sustain ribbon synaptic
transmission. To test whether ER Ca2⫹ homeostasis modulates
vesicle release, we used the dual-sine capacitance technique to
monitor release properties under control conditions (Fig. 4) and
during pharmacological treatment (Figs. 5–7). Previous experiments using single-sine stimuli have demonstrated a presynaptic facilitation effect that results in variation in responses to
repeated stimuli, which could make interpretation of pharmacological manipulations requiring repeated measures difficult
(Cho et al. 2011; Schnee et al. 2011). Furthermore, since
repeated stimulation using long depolarizing pulses has not
been described, we first characterized release properties of
0
20
Control
caffeine
control hair cells in order to establish a comparator for subsequent pharmacological experiments. Control cells were voltage-clamped to ⫺85 mV, and consecutive 3-s stimulation
pulses to 50% of peak Ca2⫹ current (as estimated from currentvoltage plots generated for each cell) were tested. These
relatively long protocols were selected to ensure observation
and separation of the two release components, while at the
same time not overtaxing the cell so that it could replenish for
multiple stimulations (Schnee et al. 2005). Also, these stimulations are closer to physiological than traditional depolarizations where potentials are stepped to elicit peak calcium
currents.
Figure 4B shows an example in which hair cell calcium
current and capacitance were measured sequentially in response to a stimulation that elicited a linear and a superlinear
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Fig. 3. Presynaptic excitability is not responsible for the reduction in vesicle release. A:
membrane potential responses from a hair
cell elicited by a 10-pA current injection
about the cell’s resting potential: control
(top) and in the presence of 10 mM caffeine
(bottom). The slight reduction in quality of
resonance is not significantly different. B:
zero-current potential (Vz) in the absence
(black) and presence (red) of caffeine. No
statistical difference is noted. C: calcium current (ICa) elicited in paired stimulus for cells
in the absence and presence of drug (red,
caffeine; blue, ryanodine; black, control). No
change is identified. D: summary of calcium
currents elicited from 1st (darker colors) and
2nd (lighter shades) stimuli for drugs that
were applied internally. No significant differences were found. E and F: voltage-clamp
responses for step voltage changes between
⫺104 and ⫹60 in 20-mV increments from a
holding potential of ⫺84 mV for control (E)
and caffeine (F). G: tail current voltage plots
generated at the time point indicated by
dashed line in E. Black, control; red, caffeine. No difference was found between
plots. H: half-activating voltages (V1/2) obtained from data in G. No significant difference was observed.
STORED CALCIUM PROMOTES VESICLE RECRUITMENT TO RIBBON SYNAPSES
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0.2
0
-0.2
F
superlinear release
**
2
G
1
3
5
IPI (min)
1
0
1
Δ superlinear onset
**
-1
-0.4
1.5
1
pulse 1 (pF)
2nd-1st (s)
linear release
(2nd-1st)/1st
(2nd-1st)/1st
E
Fig. 4. Long interpulse intervals (IPIs) result in
an increase in hair cell superlinear release. A:
representative example showing linear and superlinear components of release using 2-sine
capacitance method. Cm, membrane capacitance. B: consecutive 3-s depolarizing pulses to
50% of peak current were delivered in whole
cell. Top: release. Bottom: calcium currents.
Note the increase in release for 3-min IPI
(scale: 100 fF/10 pA, 1 s). C: traces from A
superimposed. Note that superlinear release onset (arrowheads) started earlier after longer
IPIs. D: normalized data showing that release
was enhanced only for 3- to 5-min IPIs (n ⫽ 19
cells). E and F: box plots showing that superlinear release increased only for 3- to 5-min
intervals whereas linear release was unchanged. G: superlinear onset shortened only
for 3- to 5-min intervals. **Paired t-tests show
a significant difference at P ⬍ 0.01 level.
3
5
IPI (min)
component. In this example, multiple depolarizing pulses separated by 1 min led to a slight release reduction, consistent with
vesicle depletion. Conversely, release was unexpectedly enhanced when pulses were separated by 3 min (Fig. 4B). The
amount of release was not significantly modified when two
consecutive pulses were separated by 1 min (Fig. 4D). However, longer interpulse intervals (IPIs) led to a significant
release increase in all cells studied. Changes in total release as
well as linear and superlinear release were tested by two
consecutive pulses using different IPIs (1, 3, and 5 min) in a
total of 19 cells. The linear component was quantified by
measuring the increase in membrane capacitance from the
onset of depolarization until the appearance of the superlinear
component. Since we observed variability in the onset of the
superlinear component between pulses (see below), linear
release was measured in both pulses until the same time point,
0.5
0
-0.5
-1.0
-1.5
**
1
**
3
5
IPI (min)
that is, until the superlinear onset of the second pulse. For
pulses separated by 1 min, release was the same as the initial
value (n ⫽ 5 cells) (Fig. 4D). Both components showed
nonsignificant differences in the second pulse: superlinear
release reduction of 2 ⫾ 17% and linear release reduction of
4 ⫾ 7% (Fig. 4, E and F). Similarly, the superlinear release
onset was constant in the second pulse (0.02 ⫾ 0.1 s, n.s.) (Fig.
4G). However, when pulses were separated by 3 min, consecutive pulses unexpectedly led to total release enhancement (38
⫾ 25% increase, n ⫽ 5 cells, P ⫽ 0.02) (Fig. 4D). Enhancement was also observed for 5-min IPI (52 ⫾ 32%, n ⫽ 9 cells,
P ⫽ 0.0001). Release enhancement was not accompanied by an
increase in Ca2⫹ load (1 ⫾ 2% increase for 3-min IPI, 5 ⫾ 8%
for 5-min IPI). The increase in release resulted from an increase in the superlinear component for IPIs of 3 min (110 ⫾
42% increase, P ⫽ 0.007) and 5 min (115 ⫾ 39%, P ⫽ 0.0001
J Neurophysiol • doi:10.1152/jn.00559.2015 • www.jn.org
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C
1
232
STORED CALCIUM PROMOTES VESICLE RECRUITMENT TO RIBBON SYNAPSES
were no facilitation the baseline for control would be at 0 (as
in the linear response) (Fig. 6). The linear release component
increased 4%, whereas superlinear release increased by 115%
(Fig. 6, B and C). The increases seen in response to a second
pulse are used as the comparator for the pharmacological
manipulations such that the value for total release in control
was 152%, whereas the linear release was 104% and superlinear release 215% of first pulse. The same depolarization
protocol was delivered before and after continuous extracellular application of 10 mM caffeine (Fig. 5B) or 10 ␮M ryanodine. Both pulses were separated in time by 5–10 min to ensure
drug access through the papilla. In the presence of caffeine,
mean total release in the second pulse was 37% of first pulse
release (Fig. 6A), where linear release reached 65% and superlinear release 40% of the control pulse. Ryanodine reduced
total release to 58% of the initial pulse, where mean linear
release reached 85% and superlinear release just 41% of
control values. Similarly, when 30 ␮M 8-BrcADPR, a RyR
antagonist, was included in the internal solution, total release
was 46% of control (Fig. 5C). Addition of 0.5–2 ␮M thapsigargin, a SERCA inhibitor, to the internal solution reduced
release to 65% of control, with the linear component maintaining 89% of control and the superlinear component reducing to
56% of control. Inclusion of 1 ␮M xestospongin C, an IP3
receptor blocker, had minimal effects on total release at 85% of
control, while 4 ␮M cyclic ADP ribose (cADPR), a RyR
agonist, also showed limited efficacy, reaching 95% of control
release values. The shift in the superlinear release onset observed in controls was only significantly reduced by ryanodine
application (Fig. 6D).
Total release was significantly reduced by four of the six
compounds tested, with those altering RyRs being more effective (Fig. 6A). Separating effects into release components
demonstrates that the largest effects were on the superlinear
component. Figure 6B shows that only three compounds significantly affected the linear component of release. Overall
linear release in the second pulse was reduced to 84 ⫾ 26% of
control, while the superlinear component was reduced to 53 ⫾
29% of control. Nineteen of twenty-nine pharmacologically
treated cells showed a reduction from baseline in superlinear
release (Fig. 6, B and C) compared with zero of nine control
cells. Thus the magnitude of the pharmacologically driven
release reduction was more robust in the superlinear response
(compare y-axes in Fig. 6, B and C). Furthermore, the drug
effects are likely underestimated when compounds are used in
the patch electrode because their effect likely begins prior to
the initial measurement. Supporting this contention is the
reduction in maximal release rate for the first response in cells
where drugs are intracellularly perfused (0.5 ⫾ 0.3 pF/s, n ⫽
19) compared with control values (0.8 ⫾ 0.6 pF/s, n ⫽ 19).
The superlinear component of release could be reduced in
several ways: first, the onset of superlinear release could be
lengthened; second, the maximal response could be reduced
(saturation); and third, the rate of release (slope) could be
reduced. Figure 6D demonstrates that the onset of the superlinear response was only affected minimally and only statistically significant for ryanodine. Also, saturation of release did
not appear to be the limiting factor, as depicted in the examples
of Fig. 5. As also seen in Fig. 5, the maximal release rate
(slope) was reduced in the presence of drugs that inhibited
CICR. In pharmacologically treated cells where total release
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for 5 min) (Fig. 4F), whereas the linear component did not
change significantly (5 ⫾ 11% reduction for 3 min, 4 ⫾ 10%
increase for 5 min) (compare y-axes in Fig. 4, E and F). The
observed release enhancement was originated by a shortening
in the superlinear release onset time for IPIs of 3 min (0.6 ⫾
0.3 s, P ⫽ 0.01) as well as 5 min (0.5 ⫾ 0.2 s, P ⫽ 0.0001)
(Fig. 4, C and G) and not by a change in the rate (slope) of the
superlinear release. These data show that 1 min is enough for
hair cell buffering mechanisms to reach a steady state in
cytoplasmic calcium levels after the long depolarizing pulse.
Therefore the observed increase in release after 3–5 min cannot
be accounted for by an increase in baseline calcium levels due
to buffer saturation. Release enhancement, however, may be
consistent with an increase in ER Ca2⫹ reuptake leading to an
increase in luminal Ca2⫹ levels for subsequent stimulation
pulses. Alternatively, it may underlie the activation of Ca2⫹dependent second messengers that could modulate Ca2⫹ stores
or sensitivity to Ca2⫹ influx or activate store-dependent expression of synaptic proteins (Alkon et al. 1998; Benech et al.
1999; Sutton et al. 1999). Although we cannot categorically
interpret the physiological relevance of this release shift, the
control data are needed for the pharmacological experiments
described below. As these data demonstrate that repeated
stimulations alter the response, it is critical to characterize the
change in order to accurately assess pharmacological manipulations that require repeated measures.
To study whether the shift in the superlinear release onset
was Ca2⫹ dependent and release specific or could also affect
other Ca2⫹-dependent processes taking place distant from the
synaptic ribbon, we tested the threshold for SK channel activation, a channel located near efferent terminals (Lioudyno et
al. 2004). Hair cells were stimulated and SK was monitored by
removing apamin from the external solution (see examples in
Fig. 5A). In 120 of 124 cells superlinear release was preceded
0.7 ⫾ 0.9 s by SK activation. Superlinear release onset correlated with SK onset (R2 ⫽ 0.93), demonstrating that Ca2⫹
levels reached intracellular locations not necessarily associated
with the vicinity of the ribbon. In mammalian outer hair cells,
an ER-like cistern opposing every efferent contact is proposed
to act as a Ca2⫹ store to amplify Ca2⫹ levels for SK activation
evoked by nicotinic acetylcholine receptor (nAChR) opening
(Fuchs 2014; Lioudyno et al. 2004). In those studies, ryanodine
and other store modulators alter SK currents, a result that
contrasts with our experiments, where such an obvious effect
was not observed during continuous application of curare, an
inhibitor of nAChRs. The activation of SK channels could be
directly triggered by the influx of Ca2⫹ through L-type Ca2⫹
channels, thus circumventing the need for stored Ca2⫹ efflux to
activate SK channels during depolarization. Moreover, the
sensitivity to Ca2⫹ might be biochemically modulated and thus
underlie changes in timing as has been described in neurons
(Adelman et al. 2012).
Pharmacological manipulation of calcium stores reduces
hair cell release. The effect of Ca2⫹ store modulators in hair
cell release was tested with the dual-sine capacitance technique
(Figs. 5 and 6). Figure 5A shows the release enhancement
observed in consecutive control stimulations, mainly due to the
early onset of the superlinear component. Total release increased by an average of 52% when comparing two pulses
separated by 5 min (Fig. 6A). Data are normalized to account
for the facilitation observed in control conditions; thus if there
STORED CALCIUM PROMOTES VESICLE RECRUITMENT TO RIBBON SYNAPSES
Im (nA)
A
control
0.4
0.2
0
-0.2
-0.4
Cm (pF)
min 10
min 15
min 20
1
0
caffeine
(min 17)
0.3
Im (nA)
6
0
2
4
time (s)
6
0
C
wash
(min 25)
0
-0.3
6
0
-0.1
-0.2
-0.3
8-Br-cADPR
(min 10)
2
min 13
min 24
min 32
0.4
Cm (pF)
-0.6
Cm (pF)
2
4
time (s)
1
0
0
8-Br-cADPR
(min 15)
1000
0
2 4
time (s)
6
0
2 4 6 0
time (s)
2 4 6
time (s)
reduction was significant, the mean release rate change (rate
2nd pulse ⫺ rate 1st pulse) was reduced by 40 ⫾ 37%
(reduction observed in 18 of 23 cells). In controls, the mean
release rate change was reduced by 9 ⫾ 44% (reduction
observed in 4 of 9 cells). A reduction in the rate of release can
be interpreted as a reduction in vesicle trafficking where
trafficking is Ca2⫹ dependent.
With the use of dual-sine capacitance recordings, our pharmacological data confirm previous reports of a potential role of
intracellular Ca2⫹ stores in hair cell synaptic physiology
(Beurg et al. 2005; Evans et al. 2000; Hendricson and Guth
2002; Kennedy and Meech 2002; Lelli et al. 2003; Marcotti et
al. 2004). As opposed to the release enhancement observed in
controls, hair cell pharmacological treatment led to a reduction
in both linear and superlinear release after repeated stimulation, with the superlinear release component most clearly
affected. This release reduction was not accompanied by a
reduction in the peak Ca2⫹ current and therefore cannot be
accounted for by a pharmacological effect of intracellular Ca2⫹
release on Ca2⫹ channel inactivation (Lee et al. 2007). Altogether, these results suggest that the recruitment of vesicles for
release during prolonged stimulation might be physiologically
linked to a CICR mechanism.
Calcium triggers linear and superlinear release more
efficiently than barium. Ba2⫹ activates CICR with lower
efficacy than Ca2⫹; it slows down exocytosis and reduces
the number of vesicles available for release (Neves et al.
Fig. 5. Pharmacological disruption of ryanodine receptors (RyRs) reduces hair cell release. A: 3 control 3-s depolarizing pulses
were delivered 5 min apart, and current (Im)
and Cm were monitored. Consecutive protocols showed release enhancement (dashed
line). SK and superlinear release onsets were
triggered earlier in successive stimulations.
Arrowheads show SK activation, and arrows
show initial linear release onset. Baseline was
subtracted in order to compare the 3 pulses.
B: extracellular caffeine application (10 mM)
reduced release, whereas SK current behaved
as in control experiments. Caffeine additionally reduced peak current in 4 of 6 cells. C:
intracellular application of 8-BrcADPR (30
␮M) reduced release, with no effect on the
calcium load. Arrowhead indicates superlinear onset. SK current was blocked in this cell
by apamin in the external solution.
2000
3000
4000
5000
0 1 2 3 4 5
time (s)
2001; Proks and Ashcroft 1995; Przywara et al. 1993;
Seward et al. 1996; von Ruden et al. 1993). Moreover, Ba2⫹
is not reuptaken into the store through SERCA pumps
(Kwan and Putney 1990; Przywara et al. 1993). To study
whether Ba2⫹ alters linear or superlinear capacitance
changes we replaced external Ca2⫹ with equimolar Ba2⫹
(Fig. 7). After superlinear release was evoked with a 3-s
depolarization to 50% peak current, extracellular Ca2⫹ was
substituted by equimolar Ba2⫹ and release was tested again
with the same protocol. As expected, Ba2⫹ abolished SK
activation, increased peak currents, and reduced Ca2⫹ channel inactivation (Fig. 7A) (Schnee and Ricci 2003). Ba2⫹
exerted two different effects on release: reduced total release and delayed onset of the initial linear release component (Fig. 7A). Box plots of total release for each stimulus
obtained with external Ca2⫹ or Ba2⫹ are shown in Fig. 7B.
The response in Ca2⫹ shows release enhancement in the
second and third responses, whereas the responses in Ba2⫹
were unchanged or reduced, despite the larger currents
observed (487 ⫾ 185 pA in Ba2⫹ vs. 408 ⫾ 173 pA in Ca2⫹,
P ⫽ 0.01). Ba2⫹ reduced total release in a second stimulation (72% of control) and even further in a third pulse (46%
of control). Normalizing release output more clearly shows
the enhancement observed in Ca2⫹ that is absent in the
presence of Ba2⫹ (Fig. 7C). The time to initial release was
reduced in the presence of Ba2⫹ (Fig. 7D), and the delay
was further increased for a third stimulation. The delay may
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B
2
4
time (s)
ICa2+ (nA)
0
0.8
233
234
STORED CALCIUM PROMOTES VESICLE RECRUITMENT TO RIBBON SYNAPSES
A
*
**
**
*
l
C
R n e PR g i n
ro ne
nt ffei DP odi
D gar osp
o
A
t
i
c ca rcA an
c
s
s
y
ap xe
8B r
th
(2nd-1st/1st)
l
C
R ne PR gin
ro ne
nt ffei DP odi
D gar osp
o
A
t
i
c ca rcA an
c
s
s
y
ap xe
8B r
th
D
superlinear release
**
*
**
2nd-1st
(2nd-1st/1st)
** ** **
superlinear onset
l
C
R ne PR gin
ro ne
r
nt ffei DP odi
sp
D
a
o
o
c ca rcA an
cA sig est
y
ap x
8B r
th
be a reflection of Ba2⫹ being less effective at driving release
mechanisms (Bhalla et al. 2005). Additionally, Ba2⫹ application promoted a merging of the two components. The
linearization of the capacitance response may reflect a single
source of divalent ions driving the process as CICR is
disabled with Ba2⫹ application.
The maximum slope of the superlinear release component
for a third stimulation was reduced by 36 ⫾ 30% during
Ba2⫹ application, as opposed to controls, which increased
by 141 ⫾ 119% (P ⫽ 0.01) of the initial maximum slope in
the first pulse (Fig. 7F). Unlike the superlinear decrease in
release, the first component showed no change in release
following the delay. To make this comparison we measured
the first component of release in both Ca2⫹ and Ba2⫹ 500 ms
after the onset of the capacitance change, thus bypassing the
delay induced by Ba2⫹ (Fig. 7E). Eliminating the onset
delays demonstrates that the first release component was not
significantly altered in amplitude by the divalent replacement. The delay in the onset of release could be partially
explained by a poor sensitivity for Ba2⫹ in the presynaptic
Ca2⫹ sensors for vesicle release and recruitment as suggested for SNARE-mediated exocytosis (Bhalla et al. 2005).
However, the role of SNARE proteins in vesicle fusion at
hair cell ribbon synapses remains controversial (Nouvian et
al. 2011). The intensified effect observed with multiple
stimulation protocols, together with the marked effect of
Ba2⫹ on the superlinear release component (Fig. 7F), are
consistent with the existence of a CICR mechanism governing the recruitment of new vesicles for release.
l
C
R ne PR gin
ro ne
r
nt ffei DP odi
sp
D
a
o
o
c ca rcA an
cA sig est
y
ap x
8B r
th
DISCUSSION
The existence of CICR has long been observed in conventional as well as ribbon synapses (Bouchard et al. 2003;
Castellano-Munoz and Ricci 2014), yet the functional significance and cellular mechanisms underlying CICR in these cell
types are unclear. Here we studied the potential contribution of
CICR at hair cell ribbon synapses and obtained the following
results. First, spontaneous postsynaptic multiunit activity,
along with EPSC frequency, was reduced by pharmacological
manipulations that depleted Ca2⫹ stores through RyRs. Second, no presynaptic changes in excitability, like a change in
resting potential or sensitivity, could account for the reduction
in postsynaptic response. Third, pharmacological presynaptic
effects on CICR resulted in a reduction of the superlinear
capacitance change that was larger and occurred earlier than
changes to the first component of release, consistent with the
hypothesis that CICR is important for modulating vesicle
trafficking. Fourth, Ca2⫹ substitution by Ba2⫹ delayed the
onset of release, likely because of its ability to drive synaptic
machinery. It also reduced superlinear release more than the
first component of release, and this effect was greater for larger
stimulations.
Our data suggest a role for RyRs regulating CICR from the
ER. Identification of the location of the ER responsible for
controlling synaptic vesicle populations remains under investigation. Although mitochondria are highly localized to the
synaptic region (Graydon et al. 2011; Schnee et al. 2005), the
role of the mitochondria as a Ca2⫹ sink remains suspect, as our
pharmacological data show a mild effect but one that cannot
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C
linear release
(2nd-1st/1st)
** ** **
Fig. 6. Pharmacological disruption of intracellular calcium stores reduces hair cell release. A:
caffeine, 8-BrcADPR, ryanodine, and thapsigargin significantly reduced release. See dashed
line for comparison with control. B and C: linear
(B) and superlinear (C) release were reduced by
drugs that interfere with endoplasmic reticulum
(ER) RyRs (10 mM caffeine, 30 ␮M
8-BrcADPR, 10 ␮M ryanodine). Reduction was
more pronounced in the superlinear component
of release (note y-axis values in B vs. C). Superlinear release was also reduced significantly
by 0.5–2 ␮M thapsigargin and 1 ␮M xestospongin C. Cyclic ADP ribose (cADPR, 4 ␮M),
an IP3 receptor agonist, had no obvious effects
on release. D: only ryanodine showed a significant effect on superlinear onset. Unpaired ttests show a significant difference: *P ⬍ 0.05,
**P ⬍ 0.01.
B
total release
STORED CALCIUM PROMOTES VESICLE RECRUITMENT TO RIBBON SYNAPSES
A
B
Im (nA)
0.2
total release (s)
1 Ca2+
2.0
-0.2
2 Ba
3 Ba2+
Cm (pF)
-0.4
1
2
3
1
total release (pF)
0
2+
235
control
Ba2+
**
1.5
1.0
0.5
0
0
2
4
6
2nd pulse
3rd pulse
D
normalized total release
2nd pulse
(2nd-1st)/1st
(3rd-1st)/1st
Ba2+
E
control
Ba2+
Ba2+
control
F
initial release
**
*
150
50
(3rd-1st)/1st
100
Ba2+
control
normalized max release slope
(2nd-1st)/1st
initial release (pF)
**
**
**
control
time to release (s)
3rd pulse
C
0
1st pulse
2nd pulse
3rd pulse
control
Ba2+
control
Ba2+
Fig. 7. Barium delays release and reduces superlinear release component. A: after substitution of calcium by barium in the extracellular solution, linear release
component onset was delayed and maximal release slopes reduced. Distinction between linear and superlinear components was blurred with subsequent
depolarization protocols. Baseline was subtracted in order to compare the 3 pulses. B: release values elicited by pulses delivered 5–10 min apart in control
conditions and after substitution of calcium by barium in the external solution. C: in controls, release enhancement was magnified by subsequent stimulation
(black circles). Conversely, barium substitution reduced release (triangles). D: barium substitution delayed initial release. E: initial release measured 500 ms after
release onset was not significantly altered by barium. F: maximal release slope increased with successive stimulations in controls but was reduced by barium
substitution. Unpaired t-tests show a significant difference: *P ⬍ 0.05,**P ⬍ 0.01.
easily be separated from the energy generation role typically
ascribed to these organelles. Likely the role of mitochondria at
hair cell ribbons is similar to the Ca2⫹ buffering role described
at ribbon synapses in the visual pathway, in which mitochondria provide ATP for Ca2⫹ pumps and also sequester Ca2⫹
from cytoplasm (Babai et al. 2010; Cadetti et al. 2006; Krizaj
et al. 2003; Suryanarayanan and Slaughter 2006; Wan et al.
2012; Zenisek and Matthews 2000).
The clear distinction between linear and superlinear release
components can only be experimentally obtained by depolar-
izing to potentials that allow less than maximal calcium currents from hyperpolarized holding potentials (Schnee et al.
2011). In turtle hair cells, prepulses to physiological membrane
potentials augment release and trigger the convergence of
linear and superlinear release components (Schnee et al. 2011).
Similarly, in postnatal rat inner hair cells, depolarizing pulses
preceded by holding the cell at physiological resting potentials
produced an increase in exocytosis and synaptic strength and a
shortening of synaptic latency at hair cell ribbon synapses
compared with holding the cell at more hyperpolarized poten-
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1st pulse
Time (s)
236
STORED CALCIUM PROMOTES VESICLE RECRUITMENT TO RIBBON SYNAPSES
lease of Ca2⫹ by CICR has a presynaptic role in neuronal
synaptic transmission (Llano et al. 2000; Unni et al. 2004).
CICR is observed in hair cells of different animals in both
auditory and vestibular organs (Beurg et al. 2005; Evans et al.
2000; Hendricson and Guth 2002; Kennedy and Meech 2002;
Lelli et al. 2003; Marcotti et al. 2004; Tucker and Fettiplace
1995). What could the physiological significance of CICR in
hair cells be? Hair cells must maintain a tight regulation
between Ca2⫹ influx through L-type Ca2⫹ channels and vesicle
release at ribbon synapses to allow precise control of release
timing. This regulation is achieved in the vicinity of the ribbon
by controlling Ca2⫹ levels through buffering and extrusion
mechanisms that locally rapidly remove Ca2⫹. Given that the
recruitment of vesicles for release during prolonged stimulation is Ca2⫹ dependent, how do hair cells manage to bypass the
strong Ca2⫹ regulation near the ribbon to achieve Ca2⫹dependent vesicle recruitment? One possibility is that the Kd of
trafficking is much lower than that for release. Alternatively,
CICR may serve to amplify and filter the synaptic signal (Fig.
8). Thus, during continuous stimulation, the opening of L-type
Ca2⫹ channels could tightly modulate the fusion of vesicles
near the ribbon while amplification of this signal away from the
synapse through CICR may trigger vesicle recruitment. These
calcium-dependent processes may be additionally modulated
by the effects of efferent activity on hair cell synaptic calcium
levels (Im et al. 2014). We hypothesize that CICR could have
a functional role in the recruitment and replenishment of
synaptic vesicles to guarantee the availability of vesicles for
release during protracted stimulation.
Another puzzling finding is the within-cell variability observed with repeated stimulations. This was observed in turtle
with the single-sine technique with paired stimulation (Schnee
et al. 2011) and often appears as a facilitation of release with
repeated measures, particularly when IPIs are short. Here we
also identify a slower facilitation that enhances release by
shortening the onset time to superlinear release with repeated
stimulations that have longer (minute) IPIs. Together these
data question measurements where signal averaging is used or
where multiple data points need to be obtained via repetitive
A
Vm
1
B
Ca2+
Ca2+
Ca2+
Ca
a2
a2
2+
+
Ca2
Ca2+
2+
+
2
EPSCs
Cm
linear release
I Ca 2+
Fig. 8. Ca2⫹-induced Ca2⫹ release (CICR) might sustain the
recruitment of synaptic vesicles to the ribbon. Left: a depolarizing pulse triggering an increase in capacitance in response
to a calcium current. Right: a hair cell presynaptic terminal
and the simultaneous postsynaptic responses (EPSCs). A: at
hyperpolarized membrane potential (Vm), a small calcium
influx triggers the fusion of vesicles near the synaptic ribbon.
Prolonged depolarization leads to vesicle depletion and a
reduction in EPSC frequency. B: larger calcium influx triggers
the fusion of vesicles near the synaptic ribbon as well as CICR
from intracellular stores, allowing vesicle recruitment and an
increase in EPSC frequency. The fusion of new recruited
vesicles due to CICR is experimentally observed as a superlinear component of release. Note that under physiological
conditions hair cells are maintained at depolarized membrane
potentials, a situation in which both linear and superlinear
components of release might merge.
2
1
superlinear release
2
1
linear release
1
2
Ca2+
Ca2+
Ca
a
a2
2
+
Ca2+
2+
Ca2+
Ca2
C 2
2+
+
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tials (Goutman and Glowatzki 2011). We hypothesize that the
superlinear response represents all synaptic release sites being
filled and fusion happening at maximal rates because of the
enhanced recruitment of synaptic vesicles. We further hypothesize that changes in release rates for the linear component of
release may in part represent release from multiple release sites
where the number of filled sites increases with Ca2⫹ load. This
hypothesis also can explain the variability in obtaining capacitance responses that show depletion (Schnee et al. 2005,
2011), because if the calcium entry is low enough not to trigger
recruitment then the pool size will be set by vesicles present at
any given moment.
It remains a question as to whether superlinear capacitance
changes reflect synaptic vesicle fusion, extrasynaptic vesicle
fusion, or even fusion of endosomes (Coggins et al. 2007;
Zenisek et al. 2000). Our pharmacological approach showed a
postsynaptic reduction in spike and EPSC rates that correlates
with a presynaptic release reduction. Whereas hair cell linear
release was mildly reduced by drugs interfering with CICR,
superlinear release was strongly reduced. Similarly, substitution of Ca2⫹ by Ba2⫹ exerted a stronger effect on the superlinear component of release. These results demonstrate that
both release components are Ca2⫹ dependent and likely interrelated, suggesting that superlinear release might have a physiological role in synaptic transmission. Whether the release is
synaptic or extrasynaptic requires better resolution than what
we have at the moment (Chen et al. 2014). However, distributions of glutamate receptors as well as a lack of vesicles at
nonsynaptic release sites in hair cells would also support a
synaptic role (Lenzi et al. 1999; Liberman et al. 2011; Schnee
et al. 2005). Also, given that the postsynaptic response was a
dramatic reduction in firing rate and the major presynaptic
response was a decrease in the superlinear capacitance response, it is plausible that the superlinear response is a reflection of robust vesicle trafficking to the synapse.
In neurons, Ca2⫹ regulates exocytosis of synaptic vesicles as
well as the supply of new vesicles to release sites (Dittman and
Regehr 1998; Gomis et al. 1999; Stevens and Wesseling 1998;
Wang and Kaczmarek 1998). Moreover, the intracellular re-
STORED CALCIUM PROMOTES VESICLE RECRUITMENT TO RIBBON SYNAPSES
ACKNOWLEDGMENTS
We thank the members of our research group for providing helpful comments on the project and the manuscript.
GRANTS
This work was funded by National Institute on Deafness and Other
Communication Disorders Grant DC-009913 to A. J. Ricci and by core grant
P30 44992. M. Castellano-Muñoz was supported by a Dean’s Postdoctoral
Fellowship from Stanford School of Medicine and a Cajamadrid Foundation
Fellowship.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: M.C.-M. and A.J.R. conception and design of research; M.C.-M., M.E.S., and A.J.R. performed experiments; M.C.-M. and
A.J.R. analyzed data; M.C.-M., M.E.S., and A.J.R. interpreted results of
experiments; M.C.-M. and A.J.R. prepared figures; M.C.-M. and A.J.R. drafted
manuscript; M.C.-M., M.E.S., and A.J.R. edited and revised manuscript;
M.C.-M. and A.J.R. approved final version of manuscript.
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