Response Properties from Turtle Auditory Hair Cell Afferent Fibers Suggest Spike Generation Is Driven by Synchronized Release Bo

J Neurophysiol 110: 204–220, 2013. First published April 17, 2013; doi:10.1152/jn.00121.2013.

Response properties from turtle auditory hair cell afferent ﬁbers suggest spike generation is driven by synchronized release both between and within synapses

M. E. Schnee,1 M. Castellano-Muñoz,1 and A. J. Ricci2 1Department of Otolaryngology, Stanford University School of Medicine, Stanford, California; and 2Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California

Submitted 15 February 2013; accepted in ﬁnal form 10 April 2013

Schnee ME, Castellano-Muñoz M, Ricci AJ. Response properties making 20–50 synaptic contacts (Martinez-Dunst et al. 1997; Downloaded from from turtle auditory hair cell afferent fibers suggest spike generation Schnee et al. 2011; Sneary 1988). In mammalian inner hair is driven by synchronized release both between and within synapses. cells, 15–20 afferent fibers innervate each hair cell, but each J Neurophysiol 110: 204–220, 2013. First published April 17, 2013; afferent fiber has a single synapse (Meyer et al. 2009). The doi:10.1152/jn.00121.2013.—Inner ear hair cell afferent fiber syn- functional significance of the different innervation patterns is a apses are capable of transferring information at high rates for long periods of time with extraordinary fidelity. As at other sensory focus of the present work. synapses, hair cells rely on graded receptor potentials and unique Postsynaptic recordings of auditory fibers present a remark- http://jn.physiology.org/ vesicle trafficking and release properties of ribbon synapses to relay ably broad range of excitatory postsynaptic current (EPSC) intensity information. Postsynaptic recordings from afferent fibers of amplitudes, varying by as much as 20-fold (Glowatzki and the turtle auditory papilla identified excitatory postsynaptic currents Fuchs 2002). Rise and decay times are generally very similar (EPSCs) that were fast AMPA receptor-based responses with rapid between the smallest and largest EPSCs measured in a given onset and decay times. EPSCs varied in amplitude by ϳ15ϫ per fiber, fiber. Together these data support the hypothesis of multive- with kinetics that showed a tendency to slow at larger amplitudes. sicular release (Glowatzki and Fuchs 2002). Multivesicular Complex EPSCs were produced by temporal summation of single release may be due to synchronized fusion of multiple single events, likely across synapses. Complex EPSCs were more efficient at vesicles, to release of large prefused vesicles, or perhaps to a generating action potentials than single EPSCs. Potassium-evoked by 10.220.33.4 on September 23, 2016 release increased the frequency of EPSCs, in particular complex combination of the two (Fuchs et al. 2003; Matthews and events, but did not increase EPSC amplitudes. Temporal summation Fuchs 2010). The data presented here support both possibili- of EPSCs across synapses may underlie action potential generation at ties. these synapses. Broad amplitude histograms were probed for mecha- In turtle auditory papilla, afferent fibers are tuned between 2ϩ nisms of multivesicular release with reduced external Ca or the 30 and 700 Hz, with Q10 ranging from 0.5 to 7.5, and show introduction of Cd2ϩ or Sr2ϩ to uncouple release. The results are phase-locking throughout the frequency range (Crawford and consistent with broad amplitude histograms being generated by a Fettiplace 1980). Hair cells contain calcium channels clustered combination of the variability in synaptic vesicle size and coordinated at synapses responsible for both tuning and synaptic release release of these vesicles. It is posited that multivesicular release plays (Crawford and Fettiplace 1981; Ricci et al. 2000; Tucker and less of a role in multisynaptic ribbon synapses than in single synaptic Fettiplace 1995). Release, as measured via capacitance afferent fibers. changes, varies with calcium entry and is comprised of multi- auditory hair cell afferent fibers; multivesicular release; excitatory ple components that correlate with morphologically identified postsynaptic currents vesicle pools (Schnee et al. 2011). No tonotopic differences have been observed at turtle hair cell synapses, other than in the prevalence of synapses in high-frequency hair cells, and PRIMARY AUDITORY afferent fibers transfer information regarding thus far no evidence exists to suggest that synapses within a frequency, intensity, and timing to the central nervous system given hair cell vary morphologically or in calcium channel (CNS). The high temporal precision and indefatigable nature of density (Schnee et al. 2005, 2011). This is in contrast to the response is a result of pre- and postsynaptic specializations. mammalian hair cells, in which variability in ribbon sizes, Fibers typically fire spontaneously at rates up to 100 Hz and ϩ Ca2 channel number, and postsynaptic glutamate receptor translate stimulus intensity in terms of firing rate (Keen and density shows heterogeneity in both pre- and postsynaptic Hudspeth 2006). Presynaptically, the sensory hair cell has specializations (Frank et al. 2009; Liberman et al. 2011). specialized ribbon-type synapses that are found at a variety of The data presented here describe afferent response proper- sensory receptors responsible for translating graded inputs into ties of turtle auditory papilla fibers. The results support the tonic outputs (Matthews and Fuchs 2010). The specific func- hypothesis that synchronized release of vesicles between syn- tion of these presynaptic specializations remains unknown. apses is critical for action potential (AP) generation. The data Innervation patterns vary between auditory end organs. In also support a role for multivesicular release. lower vertebrates, like frog, turtle, and bird, one or at most two afferent fibers innervate a single hair cell, with each fiber MATERIALS AND METHODS Address for reprint requests and other correspondence: A. J. Ricci, Dept. of Otolaryngology, Stanford Univ., 300 Pasteur Dr., Edwards Bldg. R145, Stan- Tissue preparation. Turtles (Trachemys scripta elegans, carapace ford, CA 94304 (e-mail: [email protected]). length 3–6 in.) were euthanized as previously described, and inner ear

204 0022-3077/13 Copyright © 2013 the American Physiological Society www.jn.org SYNCHRONOUS RELEASE AT AUDITORY HAIR CELL-AFFERENT FIBER SYNAPSE 205 tissue was removed (Farris et al. 2006; Schnee et al. 2011). All animal with the external solution (sans drug) was used to confirm that there procedures were approved by the Administrative Panel on Laboratory were no mechanical artifacts. Animal Care of Stanford University and are in accordance with the Data analysis. Data were analyzed with Mini Analysis (Synap- American Physiological Society’s “Guiding Principles for the Care tosoft) or Clampfit software packages. Origin (Microcal) was also and Use of Vertebrate Animals in Research and Training” and all used for statistical analysis, curve fitting, and figure generation. Final federal regulations. The auditory papilla was separated from other figures were assembled with Adobe Illustrator. Box plots are pre- tissue and pinned in a Sylgard-bottomed dish, and the tectorial sented with individual data points; boxes represent SD and the open membrane was removed. No enzyme treatments were used. The star symbol represents the mean of the data. Histograms were fit with auditory papilla was placed in a coverslipped bottom recording the sum of Gaussian distributions of either one, two, or three peaks. chamber and held in place with single strands of dental floss. The Best fit was selected both by the correlation coefficient (r2) and by eye RESULTS external solution contained (in mM) 125 NaCl, 2.8 KCl, 2.8 CaCl2, when discriminating multiple peaks (see for more details). 2.2 MgCl2, 10 HEPES, 6 glucose, 2 ascorbate, 2 creatine, and 2 Paired Student’s t-tests were used for comparisons of data sets; P pyruvate. pH was buffered to 7.6 with NaOH, and the osmolality was values are given with data. All data are presented as means Ϯ SD. maintained at 275 mosmol/kgH2O. To ensure that the hair cells were not overly hyperpolarized, 100 nM apamin was added to the bath RESULTS

solution to block hair cell small-conductance potassium channels Downloaded from ϳ ␮ A total of 58 recordings were made from afferent fibers of (Tucker and Fettiplace 1996). An apical perfusion pipette ( 50 min the turtle auditory papilla. Membrane capacitance, as estimated diameter) was placed near the recording site. In addition, a larger bath from a single-decay model fit to a Ϫ5-mV step in voltage pipette was used to maintain circulation at 1–2 ml/min. Recording Ϯ ϭ chambers were placed onto a specialized stage coupled to a BX51 clamp, averaged 4.2 1.4 pF (n 58). Fiber membrane upright Olympus microscope and viewed with a Q-imaging camera potentials were held to Ϫ89 mV, with a mean holding current (Q-imaging). of Ϫ107 Ϯ 71 pA (n ϭ 58). Series resistances were corrected Afferent fiber recordings. Thick-walled borosilicate patch elec- up to 70%, resulting in values of 13 Ϯ 7M⍀ (n ϭ 58). Series trodes fire polished to resistances between 5 and 10 M⍀ were used for resistance compensation was critical for obtaining accurate http://jn.physiology.org/ all recordings. Internal solutions contained (in mM) 115 KCl, 10 information regarding peak current distributions as well as rise HEPES, 3 Na2ATP, 5 creatine phosphate, and 4 MgCl2; EGTA or in and decay kinetics. Figure 1A presents a representative exam- some cases CsCl replaced the KCl. pH was buffered to 7.2 with either ple of spontaneous fiber activity, where a range of simple and KOH or CsOH, and osmolality was maintained at 255 mosmol/ complex EPSCs were consistently observed. Spontaneous ac- kgH2O. A larger electrode, filled with external solution, was used to make a hole from the apical surface of the papilla to clear a path for tivity is used to define postsynaptic recordings under standard the patch electrode. This electrode was used to clean the afferent fiber external conditions in which the hair cell is not being stimu- as well. Patch electrodes were brought up to the nerve ending either lated. Previous experiments have demonstrated that in vitro near the base of the hair cell or along one of the fingerlike projections. hair cells are typically more hyperpolarized than under physi- by 10.220.33.4 on September 23, 2016 Alexa 488 (50 ␮M) was often included in the patch electrode to label ological conditions with resting potentials closer to Ϫ60 mV the ending being recorded. No fiber was found to innervate more than (Farris et al. 2006). At these potentials there is a small calcium one hair cell. Data were collected with the Multiclamp amplifier current that can drive release (Schnee and Ricci 2003), but, as (Molecular Probes) connected to a Daq3000 (IOtech) A/D, D/A can be seen, release is at very low rates. Thus spontaneous interface with a Dell personal computer. Data were collected at 10 activity is driven by calcium entry via calcium channels. We kHz. Voltage protocols and all recordings were obtained with the define evoked activity as release observed under elevated jClamp software package. Multiunit recording. Turtle half-head preparations were used for potassium conditions in which the hair cell will be depolarized multiunit activity measurements from the eighth cranial nerve. The to potentials activating a larger proportion of the calcium turtle head was split in half and pinned in a Sylgard dissection current. Single EPSCs were defined as those that started and chamber with either an external solution similar to that used in ended at the baseline current and had single rise times as well patch-clamp recordings or bicarbonate-buffered perilymph containing as exponential decays (Fig. 1B). All other events were consid-

(in mM) 126 NaCl, 2.5 KCl, 13 NaHCO3, 1.7 NaH2PO4, 1.8 CaCl2, ered complex (or bursts) and could present in different forms 1 MgCl2, and 5 glucose (continuously bubbled with 95% O2-5% (Fig. 1, C–E). Steplike increments in the rising phase were CO2). The brain was removed and the auditory nerve exposed, cutting observed (Fig. 1C) and assumed to indicate summation of the connections to posterior ampulla and saccule. The ventral otic unique events. Often burstlike behavior was observed, where membrane was trimmed to allow access to perfusion prior to mount- the responses did not return to baseline before the next event ing the preparation in the recording chamber. A perfusion pipette was located ϳ5 mm above the otic capsule and connected to a gravity occurred; these events could be separated in time, revealing perfusion system with a flow rate of roughly 1 ml/min. The auditory more clearly the individual events (Fig. 1D) or more overlap- nerve was inserted in an hourglass-shaped suction electrode with a ping (Fig. 1E), where rise and decays could not be assessed. In micromanipulator, and compound APs were recorded with a differ- both cases peaks could be counted to determine the minimum ential AC preamplifier (Grass P55, Astro-Med, West Warwick, RI). number of EPSCs (complex events) forming the burst. Ampli- One electrode was inserted into the borosilicate suction pipette, and tudes for the single events were measured as the difference the neutral electrode was in contact with the bath. The signal was between the peak current reached and the baseline prior to the band-pass filtered (1 Hz–1 kHz) and amplified 1,000 times. Com- event occurring. Similarly for complex events, the peak am- pound APs were collected through a data acquisition interface (CED plitudes were measured as the difference in current between Micro 1401 mkII, Cambridge Electronic Design) and analyzed with each peak value and the start of each event in the burst Spike 2 software (Cambridge Electronic Design). Noise levels were confirmed by blocking afferent activity with 1 ␮M tetrodoxin (TTX) (typically not a constant baseline value; see Fig. 1E, dotted or the glutamate antagonists 6,7-dinitroquinoxaline-2,3-dione (DNQX; lines). For spontaneous activity, complex events represented 1 ␮M) or kynurenic acid (2 mM), and spike threshold was set to 3 ϫ Ͻ20% of the total events measured. One representation of this SD of baseline noise. Drugs were applied via the local perfusion distribution is the use of a recurrence plot, often used to system after a baseline firing rate was established. Control perfusion identify complex phase relationships, where sequential inter-

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Fig. 1. A: spontaneous postsynaptic measurements from an afferent fiber innervating an auditory hair cell from a low-frequency position of the turtle auditory papilla. The fiber was voltage-clamped at Ϫ84 mV. B–E: a variety of response types were measured: singles, defined as excitatory postsynaptic currents (EPSCs) with a single rise and decay time starting and ending at the same baseline current (B), and complex responses, defined as having multiple inflections on the rise or decay component (C; see arrow for inflection), as being shifted from baseline for the rise or decay time (D; see dashed line as indicating baseline), and/or as having multiple peaks prior to decaying back to the original baseline (E). The dotted lines in E depict how amplitude was measured for overlapping EPSCs in complex events. A burst is equivalent to D or E. F: measurements made including threshold as 3ϫ the baseline rms noise (red), rise time measurements (green) (10–90% measurements), and decay time constants (blue line), measured as single exponential decays from the peak. G and H: amplitude histograms for the single (G) and complex (H) events measured. Plots were fit with single, double, or triple Gaussian functions as shown. Often single or double Gaussians could be fit; as indicated by arrow in G, in each case the best fit with the fewest peaks was selected (here a single). Fits to individual Gaussians are shown in red and yellow in H and the summed Gaussian in blue. Numbers above each peak represent how peaks are defined in subsequent figures. I: an expanded view of spontaneous EPSCs with the interpulse intervals times 1 and 2 noted. J: time 1 vs. time 2 in a recurrence plot to identify patterns that might suggest differences between individual synapses onto this fiber. Blue shading demarks the time range that would require the interval to be reflecting complex events, as a single EPSC would take longer to complete. pulse intervals (IPIs) are plotted against each other. Fig. 1, I For single events, rise times were measured as the 10–90% and J, illustrate this approach, where Fig. 1I is an expanded time to peak; as will be seen, these can often be quite fast and view of spontaneous EPSCs where IPIs are indicated. Time 1 is likely limited by voltage clamp speed. Decay times were fit first plotted against time 2, and then time 2 is plotted (as time with an exponential, and it is the time constant of this fit that 1) against the next interval. The recurrence plot in Fig. 1J is reported (Fig. 1F). Histograms for EPSC amplitudes were shows that the vast majority of events have IPIs Ͼ 3 ms, generated for each fiber with 10-pA bins and sampled for corresponding to single events (a single EPSC takes ϳ3ms periods of 1–30 s, depending on response frequency. Most from start to finish). The relatively broad scatter in this plot often, histograms had a single major peak based on the relative suggests stochastic behavior of the synapses contributing to the area contributed, with minor peaks of smaller area contribution responses. of either greater or lesser mean amplitude. In cases as depicted

J Neurophysiol • doi:10.1152/jn.00121.2013 • www.jn.org SYNCHRONOUS RELEASE AT AUDITORY HAIR CELL-AFFERENT FIBER SYNAPSE 207 in Fig. 1G, single Gaussians were used, as they represented the (Fig. 2, A and B). A reversal potential of Ϫ3 Ϯ 3mVwas data as well as a double function would. For other ﬁbers, obtained, showing the mixed cation-conducting behavior typ- however, the best ﬁt was a double or triple Gaussian with ical of glutamatergic receptors. A comparison of amplitude minor peaks of both greater and lesser value than the main peak histograms was made in the absence and presence of TTX in as in Fig. 1H. order to ensure that unclamped sodium currents were not Pharmacology. The reversal potential of the EPSCs was altering the shapes of the EPSCs measured. The peak ampli- determined in three cells by clamping the membrane potential tudes are plotted in Fig. 2C, where a tendency toward smaller at different values long enough to obtain an amplitude distri- peaks was observed with TTX. For this reason all measure- bution for the events. The major peak current amplitude of ments reported are in the presence of 1 ␮M TTX. In addition, each histogram was then plotted against holding potential to ensure that all measured values were of AMPA receptor Downloaded from

Fig. 2. Synaptic potentials are carried by AMPA receptors. Current-voltage plots for spontaneous EPSCs were generated by creating amplitude histograms at different po- http://jn.physiology.org/ tentials. Current examples are presented in A, and then the peak Gaussian ﬁt to the amplitude histogram is plotted against potential in B. The reversal potential was Ϫ3 Ϯ 3 mV for 3 cells with a slope of 13 Ϯ 6 mVϪ1. C: contamination by non-voltage- clamped sodium currents was investigated by comparing frequency histograms in the absence and presence of tetrodotoxin (TTX;

1 ␮M); peaks of these histograms are plotted by 10.220.33.4 on September 23, 2016 for individual cells, showing no significant change. D: block by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) demonstrates that the postsynaptic potential measurements were carried by glutamate AMPA receptor channels. Complete block was observed with 10 ␮M CNQX. E: cyclothiazide (CTZ) application (red trace, below the control example) suggests that desensitization plays a role in shaping EPSCs. F: single EPSCs of similar magnitude in the absence and presence of CTZ to illustrate the slower rise and decay times. Exponential fits to the decay times are also shown. G and H: summaries of mean rise time (G) and decay time constant (␶; H) are presented in box plots where the box outlines the SD, the star the mean for the group, and the diamonds the individual fiber responses. I: bar graph breaking down the number of individual peaks in each complex event. CTZ increases the number of EPSCs per complex event. J: multiunit recordings from the 8th cranial nerve were measured from the half-head preparation, and the frequency response was plotted. K: application of CTZ consistently reduced firing rates as shown in the box plot, intimating a presynaptic effect on release.

J Neurophysiol • doi:10.1152/jn.00121.2013 • www.jn.org 208 SYNCHRONOUS RELEASE AT AUDITORY HAIR CELL-AFFERENT FIBER SYNAPSE origin and to assess baseline levels for setting detection thresh- (Fig. 3F). The similarity in properties between the complex and olds, 10 ␮M 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) single events is consistent with the hypothesis that the complex was used to completely abolish EPSCs (Fig. 2D). Curare was events simply represent increased activity resulting in temporal also tested under both normal and high potassium levels to test overlap, likely due to release from multiple synapses or per- potential efferent effects altering the responses. No effects of haps multiple release sites at a given synapse. In evaluating the either 10 or 100 ␮M curare were observed (data not shown). afferent fibers with multiple peaked amplitude histograms, a Cyclothiazide. To investigate the importance of EPSC kinet- question arises as to whether the underlying mechanisms re- ics, particularly in determining the proportion of single com- sponsible for achieving each peak were similar. Comparing the pared with complex events, the effects of cyclothiazide (CTZ) relative variance (measured from the width of the individual were investigated. CTZ is most commonly used to alter desen- histogram) to the mean of the histogram provides a measure sitization, but it has also been shown to alter glutamate affinity where a constant ratio among peaks is predicted for similar for receptors (Dzubay and Jahr 1999; Partin et al. 1993, 1994, processes, whereas greater or lower ratio values would suggest 1996). Regardless of the specific mechanism of action, CTZ is more or less complexity. We unexpectedly found less variance useful because it can alter EPSC kinetics. Both rise and decay for the histograms with the largest mean value (Fig. 3G), times were slowed by 100 ␮M CTZ (P Ͻ 0.05 for rise times perhaps suggesting a more simple release process for these Downloaded from and P Ͻ 0.001 for decay time constants; Fig. 2, E–H). Decay large-amplitude events. times were slowed by a factor of almost 3 (Fig. 2H), a more In general, EPSC amplitudes varied by more than an order of dramatic effect compared with the rise times. As a result of the magnitude for individual fibers. The mean maximum ampli- slowing of EPSCs, the fraction of complex events increased to tude was 555 Ϯ 202 pA, while the minimum was 37 Ϯ 14 pA 86 Ϯ 5% (n ϭ 3) after CTZ treatment. The number of unitary (n ϭ 42). The range of minimum and maximum EPSC ampli- EPSCs per complex event also increased in the presence of tudes covaried with a slope of 15 Ϯ 1 (Fig. 3H)(r2 ϭ 0.94). CTZ. Fig. 2I presents a frequency histogram of events per burst This relationship is interesting in that it suggests a constant http://jn.physiology.org/ showing that in the presence of CTZ there are many more range of amplitudes within a given fiber that scales between bursts with multiple peaks. This result is not surprising and fibers. Hair cells from the turtle papilla have Ͼ20 synapses per supports the argument that bursts (complex events) are simply fiber, and so likely the EPSCs measured are in part the sum the temporal summation of single events. In addition to the across these synapses (Schnee et al. 2005; Sneary 1988). change in release kinetics and the increase in complex events, However, the broad range in amplitude histograms is quite the frequency of spontaneous release was reduced in four of six typical among ribbon synapses, even those with only single cells after CTZ treatment. Changes in frequency of release are synapses per fiber (Glowatzki and Fuchs 2002; Grant et al. typically indicative of a presynaptic action, and thus a decrease 2010). Therefore, the breadth of amplitude histograms is not by 10.220.33.4 on September 23, 2016 in frequency might suggest a presynaptic effect of CTZ. simply due to summation across synapses but likely a common Further support of this decrease in release frequency comes feature of individual ribbon synapses. from multiunit recordings from the turtle half-head preparation Given the pseudocalcyceal nature of the turtle postsynaptic (Fig. 2J). Application of CTZ (50 ␮M) reduced afferent firing fiber, it is possible that the space clamp is a potential error rates to 74 Ϯ 10% of initial rate (n ϭ 7, P ϭ 0.02), an effect source for both kinetic and amplitude values. Plotting the rise that disappeared after washout of the drug (Fig. 2K). This time or decay time constant against the peak amplitude of reduction occurs despite the increase in complex events that are EPSC histograms provides some insight into this issue (Fig. 4, expected to increase firing rates (see below). A and B). Rise times varied two- to threefold within individual Spontaneous activity. Spontaneous activity was observed in fibers, as did the decay time constant. The envelope of the plots 26 of 33 cells, with EPSC frequencies up to 50 Hz; the mean showed a relationship in which the rise time slowed as current value was 22 Ϯ 18 Hz (n ϭ 27), with 6 fibers having rates Ͻ amplitude grew. This is opposite that expected for a space 5 Hz. A total of 83 Ϯ 17% of the spontaneous EPSCs measured clamp or clamp speed error, where current amplitudes should in each fiber were single events. EPSC amplitude histograms decrease and kinetics slow as the clamp speed as well as the were fit to each of the responding fibers, where 13 were best fit space clamp is compromised. Additionally, it is clear from the by single Gaussian functions, 7 by double Gaussian functions, scattergrams that EPSCs may be selected across amplitudes to and 6 by triples. The peak currents, as measured from the find very similar kinetic properties, simply by moving horizon- Gaussian fits, are presented in box plots (Fig. 3A), with the tally across the scatter (Fig. 4D). Conversely, quite different major peak (based on area of the histogram) plotted as peak 1, kinetics can be observed simply by moving vertically along the the smaller mean amplitude peak as peak 2, and the larger scatter (Fig. 4C). Figure 4C provides examples for small- and mean amplitude peak as peak 3 (see Fig. 1H). In 20 of 26 cells, large-amplitude responses where the kinetics are quite different the major peak represented at least 80% of the total distribution between the EPSCs, while Fig. 4D provides an example of a (Fig. 3B). Individual fiber peak amplitudes are presented in large-amplitude EPSC with a small (but scaled) EPSC of Fig. 3C, showing the broad range of amplitudes. The relative comparable kinetics. change between peak amplitudes was similar between units (as Summaries of the envelope fits for the plots of both the rise seen by the similar slopes), indicative of a simple scaling and decay times against peak amplitude (Fig. 4, E and F) process. illustrate that every fiber consistently showed a slowing of The complex EPSCs represent on average 17% of the total kinetics with larger EPSC amplitudes. Scattergrams were also spontaneous activity. The distribution of peak amplitudes as created for rise times against decay times to determine whether well as the relative area of each peak are presented in Fig. 3, D this kinetics covaried. For all fibers tested no consistent rela- and E. These values are comparable to those of the single tionship was observed. The scattergram shown in Fig. 4G EPSCs, as is the relationship between peaks within given units represents the closest relationship observed, and it is clear from

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Fig. 3. Gaussian fits to frequency histograms for spontaneous EPSC amplitudes were generated. A: the peaks to these fits. B: each Gaussian had a major peak based on the relative area, typically ϳ80% of total area of histogram. Peak 1 is defined as the peak with the largest area (see Fig. 1H), peak 2 represents amplitudes smaller than the major peak, and peak 3 represents those with peak currents greater than the major peak. C: peak amplitudes for individual cells, where red boxes are those cells with single Gaussian peaks, green are those with 2 peaks, and blue are those with 3 peaks; open symbols indicate a minor peak of smaller peak current than the primary peak, and filled symbols represent those with a minor peak of greater mean amplitude than the primary peak. D–F: complex EPSCs presented in a manner similar to the singles presented in A–C. G: SD of each peak divided by the mean value for each peak to illustrate the relative variance between peaks. Surprisingly, the largest amplitude peaks showed the smallest relative variance and were statistically different from either of the other peaks (t-test, 2-tailed, P Ͻ 0.05) H: a relationship was observed between minimum and maximum amplitude EPSC where the red line represents a linear fit with a slope of 15 Ϯ 1 and intercept of 0 (R2 ϭ 0.94). this plot that no pattern is present; a linear fit produced an r2 to previous work in turtle (Crawford and Fettiplace 1980). Just value of 0.02. A lack of relationship argues against space as in voltage clamp, both single and complex events were clamp dictating these results and rather supports the idea that observed. A higher percentage of complex events was ob- there is considerable physiological variability in EPSC kinet- served in current clamp (39 Ϯ 19%) compared with voltage ics, perhaps in part due to the timing of vesicle release or to the clamp (17 Ϯ 16, n ϭ 5), most likely as a consequence of size of vesicles fusing. EPSPs being significantly slower than EPSCs, thus allowing Current clamp. To explore the efficacy of EPSCs in gener- for more temporal overlap (much like the CTZ experiments). ating postsynaptic APs, current-clamp recordings were ob- Spikes were defined by identifying the inflection point in tained using Kϩ as the major intracellular cation. An example voltage change indicative of Naϩ channel activation (see of these measurements is presented in Fig. 5, where it is arrows in Fig. 5B). The mean voltage change for a spike was apparent that not all excitatory postsynaptic potentials (EPSPs) 56 Ϯ 22 mV (n ϭ 15), with a range of 22–97 mV. The spike were capable of generating APs. This differs from the recent amplitude variance suggests that spike generation is at a report in mammalian afferent fibers (Rutherford et al. 2012) variable distance from the recording site. Similarly, the occur- but is similar to that observed in young rats (Yi et al. 2010) and rence of Naϩ currents that were not well voltage-clamped,

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Fig. 4. Kinetic analysis of the rise and decay time course for spontaneous EPSCs (singles). A and B: rise time (A) and decay ␶ (B) plotted against peak current for individual

EPSCs showing a great deal of scatter, with http://jn.physiology.org/ both currents and rise times varying 4-fold. A line could be fit to the envelope of the response indicating a trend for larger EPSCs to be slower than smaller (see red and blue lines, respectively). C: examples of EPSCs of similar sizes but different kinetic behavior for both large and small EPSCs. Blue traces show differences in the decay times, while red shows differences in rise time. D:a similar plot where small and large EPSCs by 10.220.33.4 on September 23, 2016 were scaled to show that the kinetics were similar. E and F: envelope fits for individual cells for both rise (E) and decay (F) times. Each cell similarly showed the trends for slowing with larger current amplitudes. G: the rise time for a given cell plotted against its decay time to demonstrate that no clear relationship exists (fast rise times are observed with slow decay and vice versa). Red line indicates a fit with r2 ϭ 0.02.

likely due to space clamp issues, supports this contention (data evoked from single and complex modes are presented in Fig. not shown). To ensure that afferent properties were not af- 5C (there was no difference between recording modes). EPSPs fected by obtaining the whole cell recording conﬁguration, were relatively inefﬁcient at generating APs, with 35% of cell-attached patch data were also investigated. Both current- EPSPs resulting in spikes (Fig. 5D, AP/event). Of the total APs and voltage-clamp modes were used, and examples of APs generated, more than half were generated from complex events

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Fig. 5. A: current-clamp responses from afferent fibers showing excitatory postsynaptic potentials (EPSPs) and EPSP-evoked spikes. B: spikes elicited from single (left) and complex (right) EPSPs. Arrows indicate the inflection that is indicative of regenerative action potentials (APs). C: cell-attached recordings in current clamp of single event-driven (left) and complex event-driven (right) APs. D: the fraction of APs generated from all EPSPs (red), the proportion of APs generated from complex EPSPs (green), the proportion of complex events (bursts) that result in APs (cAP) (dark blue), and the proportion of single EPSPs that result in APs (light blue). Open symbols are from whole cell recordings and filled symbols from cell-attached recordings. E: a fiber’s membrane potential response to current step injections. Box plots in F–H present summary data for resting potential (F), spike threshold (G) as measured from the inflection point (see arrow in B), and the current injection required to reach threshold (H). [Fig. 5D, cAP/AP (complex AP/all AP)], despite complex ing the argument that temporal summation was important for events representing only 39% of all events. More than 80% of spike generation. The ability of synaptic events to generate complex events generated APs (Fig. 5D, cAP/burst) compared APs varied considerably between fibers, from as low as 3% to with only 17% for single events (Fig. 5D, AP/single), support- as high as 90% (Fig. 5D). The reason for this variation is

J Neurophysiol • doi:10.1152/jn.00121.2013 • www.jn.org 212 SYNCHRONOUS RELEASE AT AUDITORY HAIR CELL-AFFERENT FIBER SYNAPSE unclear but did seem to correlate with the fraction of complex tions, prior to Kϩ treatment, 5 showed no activity in control, 6 events for a given unit. These data suggest that spike timing were single Gaussian distributions, 2 showed three peaks, and and initiation may be controlled by the ability of synapses to 1 showed a single secondary peak of higher amplitude than the release synchronously, thus creating suprathreshold complex major peak. After Kϩ exposure all fibers showed activity. The events. five originally silent fibers produced double Gaussian distribu- To determine whether the lack of AP efficiency was in part tions, where the secondary peak was larger than the major due to postsynaptic properties, fibers were injected with current peak. The complex Gaussian amplitude distributions for spon- at different levels until APs were elicited (Fig. 5E). AP thresh- taneous activity remained the same with the Kϩ-evoked re- old response was measured as Ϫ48 Ϯ 8mV(n ϭ 14; Fig. 5G) Ϫ Ϯ ϭ sponses. Of the six fibers with single Gaussian peaks, two compared with a resting potential of 66 3mV(n 19; remained singles, two had a smaller minor peak, and two had F Fig. 5 ), indicating that EPSPs needed to depolarize the fiber a minor larger peak. These additional larger peaks were within 18 mV on average to evoke a spike. The average current the range of peaks measured across fibers for spontaneous injection required to elicit a spike was 167 Ϯ 70 pA (n ϭ 16; activity as well. Thus the increased frequency of EPSCs with Fig. 5H). No distinct populations of fibers were identified to ϩ suggest that fibers had different thresholds. K application did not result in larger EPSCs. Downloaded from Potassium-evoked activity. Spontaneous activity from the To further explore the idea that the increase in complex afferent fibers was low in our recording conditions. This most events was simply a function of the increase in release rate, the likely stems from a lack of active mechanotransduction and an average release frequency in each fiber was plotted against the improper endolymph-like environment serving to hyperpolar- proportion of complex events for both spontaneous and evoked ize hair cells away from their normal resting potential (Farris et responses (Fig. 6K). An exponential relationship was observed al. 2006). To mimic more physiological hair cell potentials and with a correlation coefficient of 0.91, again supporting the idea to provide more events for analysis, hair cells were exposed to that the change in proportion of complex events was simply a http://jn.physiology.org/ 50 mM Kϩ while the afferent fiber was voltage-clamped to function of the increase in probability of release, leading to Ϫ89 mV. Figure 6, A and B, present an example of a fiber in more temporal overlap in EPSCs. 2.8 mM and 50 mM Kϩ. Release rates increased from 12 Ϯ 14 The recurrence plot for a fiber in the absence and presence Hz (n ϭ 13) to 211 Ϯ 144 Hz (n ϭ 13) under these conditions. of 50 mM Kϩ in Fig. 6J supports the conclusion that higher Fatigue was not observed, and release rates were maintained frequencies result in more complex events; the shaded region indefinitely throughout the Kϩ application, which on average again demarcates where complex events would be occurring lasted 4 Ϯ 1 min (n ϭ 14), attesting to the robustness of these and Kϩ shifts the responses from single events to more com- synapses and their ability to maintain vesicle populations for plex events. In this example and in many of the generated plots, by 10.220.33.4 on September 23, 2016 release. Concomitant with the increase in release rate was an there are several hot spots suggesting some coordination in the increase in the proportion of complex events from 19 Ϯ 6% to release properties. It is possible that depolarization with Kϩ 54 Ϯ 25% (n ϭ 13). The number of EPSC peaks within each brings the hair cell closer to its resonant frequency and it is the complex event was 2–16 for evoked release but only up to 6 for intrinsic presynaptic resonance that generates coordinated re- spontaneous release, with the vast majority being of the dou- sponses (Andor-Ardo et al. 2010). blet variety. Along these lines there are several outliers in the frequency Amplitude histograms were created for both single and vs. percent complex plot in Fig. 6K, where the percentage of complex EPSCs under elevated-Kϩ conditions (Fig. 6, C, D, F, complex events was much higher than predicted by the release and G). For both single (Fig. 6, C and D) and complex (Fig. 6, frequency. Figure 7 plots three examples of these outliers, with F and G) events, there was an increase in the number of fibers one example of a more traditional response (Fig. 7, top), with multi-Gaussian distributions in elevated Kϩ compared showing both 1-s examples of the measurements (Fig. 7A)as with spontaneous activity. Peak current amplitudes for the well as the recurrence plot (Fig. 7B). Each example shows the major peaks and both secondary peaks were not different prevalence of complex events but also the clustering of re- between single and complex events (Fig. 6, E and H; see sponses suggesting presynaptic coordination of release. Likely yellow symbol as mean value from spontaneous recordings). these outliers represent hair cells whose resting potential is Together these data also support the argument that evoked more depolarized (likely having some remaining mechano- EPSCs are not larger than spontaneous EPSCs. To further transduction current) allowing for electrical resonance to co- explore this idea, the amplitude peaks measured from the ordinate release properties. This being the case, it would Gaussian fits from spontaneous firing fibers were plotted support the argument that temporal summation between syn- against the same peaks in elevated Kϩ (Fig. 6I); the error bars apses to generate complex events that have a high probability are the width of the Gaussian fits. This relationship has a slope of generating APs is an important mechanism used by fibers of 1 and intercept of 0 and so also supports the argument that with multiple synaptic endings. the amplitude of EPSCs does not increase with stimulation and The kinetics of EPSCs was also investigated under stimu- that the observed changes are simply a function of the in- lated conditions. Plots of rise times and decay time constants creased probability of release. Interestingly, the shape of the against peak current for spontaneous and evoked EPSCs are EPSC amplitude distributions varied in some of the fibers presented in Fig. 8, A and B. These plots basically overlapped. during the application of Kϩ. The data in Fig. 6 demonstrate Fits to the envelope under both conditions were also not that the amplitude histograms do not change when comparing different. The rise time slopes were 0.23 Ϯ 0.06 ms/nA and spontaneous and Kϩ-evoked measurements; however, they do 0.25 Ϯ 0.13 ms/nA (n ϭ 8) for 2.8 and 50 mM Kϩ, respec- not directly assess those peaks that were not present in spon- tively. Slopes for the decay plots were 0.8 Ϯ 0.4 ms/nA and taneous activity. In 14 cells investigated under control condi- 0.8 Ϯ 0.5 ms/nA (n ϭ 12) for 2.8 and 50 mM Kϩ, respectively.

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Fig. 6. Summary data for Kϩ-evoked EPSC activity where A presents spontaneous EPSC activity while B presents activity in the presence of 50 mM Kϩ from a single fiber. C and F plot the amplitude histograms for single and complex events in 2.8 mM Kϩ, while D and G present similar plots from the same fiber in 50 mM Kϩ. E and H: peak amplitudes from the Gaussian fits are plotted for both single (E) and complex (H) EPSCs. Filled symbols represent the individual cells, stars represent the mean value, and the box presents the SD. Yellow circles represent the mean values from the spontaneously occurring events in Fig. 3. I: peak currents in 2.8 mM Kϩ plotted against the corresponding peak current in 50 mM Kϩ. Filled squares indicate peak 1 singles, red circles peak 2 singles, blue triangles peak 3 singles, and open squares complex peak 1 values. The line has an intercept of 0 and a slope of 1, showing no change in peak values between control and stimulated conditions. J: an example of a recurrence plot where black symbols represent data from 2.8 mM Kϩ and red symbols represent data from 50 mM Kϩ. The blue shaded area represents the region for complex events. K: frequency of total events (single and bursts) plotted against the proportion of complex events for spontaneous (black) and Kϩ-induced (red) release. Line is an exponential fit to the data having an r2 of 0.91.

Effects of divalents. Strontium (Sr2ϩ) is often used to replace triggering multiple vesicle fusions in a synchronous (coordi- Ca2ϩ in order to desynchronize release (Xu-Friedman and nated) manner. Sr2ϩ, therefore might interfere with these re- Regehr 1999, 2000). Typically these experiments are per- lease processes and result in reduced frequency and, more formed in response to presynaptic stimulation where calcium importantly, reduced-amplitude EPSCs. We would also predict entry is synchronized and becomes desynchronized with Sr2ϩ a reduction in rise and decay times because temporal precision as Sr2ϩ replaces calcium less efﬁciently in triggering release. of these coordinated vesicles might be compromised such that In addition, Sr2ϩ is not well buffered, resulting in continued mistimed summation will lengthen the activation and deacti- release after stimulus. Here, there is no directed presynaptic vation times. Thus the effect of Sr2ϩ on EPSCs was tested to stimulation; however, as activity is driven by Ca2ϩ entry, the determine whether the broad range in EPSC amplitude might in hypothesis is that clustered calcium channels summate Ca2ϩ part be due to synchronous release of vesicles. In the presence

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Fig. 7. Investigating the relationship between release frequency and complex events revealed several outliers where complex events were at a higher proportion than predicted by frequency. A: examples of spontaneous activity from 4 different cells; the example at top was http://jn.physiology.org/ the most typically observed, and the following 3 examples are outliers from Fig. 6K. B: recurrence plots for each. The outlier plots show a larger percentage of complex events largely due to the patterned responses, suggesting coordinated release between synapses. by 10.220.33.4 on September 23, 2016

of Sr2ϩ, release rates, the proportion of complex events, and complex events was also reduced, likely as a result of the the peak amplitudes for each major peak in each afferent fiber overall decrease in release frequency (Fig. 6K). These data sit were reduced. Examples of the EPSCs obtained in the presence exactly on the control data, thus suggesting that the change in of Sr2ϩ are presented in Fig. 9, A and B. Amplitude reductions complex events is solely due to the frequency change. The rise ranged from 30% to 60% of control peak values (Fig. 9, C and and decay times still showed a broad spread (Fig. 8, C and D). D). Amplitude histograms for one fiber are presented in Fig. 9C However, the slopes of the envelopes were steeper, indicating to illustrate shift in peak amplitudes. A summary plot of peak that the rise and decay times were slowed in the presence of amplitudes (Fig. 9D) uses a color scheme similar to previous Sr2ϩ (Fig. 9, F and G), as predicted if coordinated release were figures (where green is minor smaller peak, red is major being compromised. Plotting the absolute mean values for rise intermediate amplitude peak, and blue is smaller peak of higher and decay times (Fig. 8, E and F) results in no difference for amplitude) and shows a consistent decrease in each peak Sr2ϩ compared with Kϩ for the rise time; however, the decay amplitude after Sr2ϩ treatment. However, these amplitudes do time was significantly slower (P Ͻ 0.05, 2-tailed paired t-test). not converge onto a unitary event size; rather, it appears as if In conjunction with the reduced activity and the shift to smaller each peak of the Gaussian represents a unique unitary distri- values for the peaks in the amplitude histograms, there was a bution, perhaps based on vesicle size. Thus these data would major increase in the number of events with peaks below 100 suggest that synchronous vesicle fusion may provide an im- pA (Fig. 9E). The increase in small-amplitude events was portant component of multivesicular release that is superim- broad and did not lend itself to fitting but suggests that posed onto a broad range of vesicle sizes. The fraction of desynchronizing release results in smaller-amplitude EPSCs.

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Fig. 8. Changing release rates does not alter the kinetics of EPSCs. A and B: plots of rise (A) and decay (B) times Downloaded from against EPSC amplitude in 2.8 mM or 50 mM Kϩ are presented to demonstrate that the envelope of these plots is invariant. Envelopes are indicated by the lines. C and D: similar plots of rise (C) and decay (D) rates in the absence (control) and presence of 10 mM Sr2ϩ. Here the envelopes of the plots differ, being steeper in the presence of Sr2ϩ. Figure 9 summarizes these data. E: average rise times for all EPSCs in a given ﬁber for http://jn.physiology.org/ control (red), in the presence of Sr2ϩ (green), or in the low divalent (or 0.1 mM Cd2ϩ; blue). *Signiﬁcantly different from control data at the P Ͻ 0.05 level. F: similar plots of decay time constants under these conditions, demonstrating that Sr2ϩ slows the average EPSC decay rate (P Ͻ 0.05). by 10.220.33.4 on September 23, 2016

Together these data support the argument that the broad range Further attempts at interfering with synchronous release of EPSC amplitudes is in part dictated by synchronous release were made by lowering external calcium or using cadmium of vesicles but do not suggest that this is the exclusive (Cd2ϩ), a known blocker of hair cell calcium channels, to block mechanism. calcium entry (Fig. 1) (Schnee and Ricci 2003). Both lowering Sr2ϩ might directly alter AMPA receptor conductance, external calcium (Fig. 10, A–C) and introducing Cd2ϩ extra- thereby generating smaller EPSCs simply because the single- cellularly (Fig. 10, D and E) produced a dramatic reduction in channel conductances were reduced. Although many investi- the frequency of EPSC release and also reduced the EPSC gations using Sr2ϩ have not identified this as a potential amplitude. The time course for frequency reduction is plotted mechanism, it remains plausible (Bender et al. 2006; Clements for the EGTA solution applied externally in Fig. 10B. Ampli- and Silver 2000). To test this possibility, we recorded EPSCs tude reduction closely followed the same time course (Fig. in the absence and presence of Sr2ϩ at positive and negative 10C). The EGTA solution ultimately resulted in no or ex- potentials. At positive potentials a divalent block of the pore tremely few EPSCs that did not allow for generation of would be removed, thus increasing the mean EPSC amplitude frequency histograms; however, the Cd2ϩ experiments main- compared with Ϫ80 mV. In four cells tested the mean EPSC tained release at rates where frequency histograms could be amplitude changed from 253 Ϯ 72 pA to 223 Ϯ 143 pA in generated (Fig. 10F). The example of a control histogram in normal Ca2ϩ at Ϫ84 and ϩ76 mV, respectively, while in the Fig. 10F was best fit with the equation for a triple Gaussian presence of Sr2ϩ the change was from 150 Ϯ 15 pA to 129 Ϯ curve, having peaks and half-widths of 0.08 Ϯ 0.06, 0.15 Ϯ 80 pA for Ϫ84 and ϩ76 mV, respectively. These data dem- 0.1, and 0.28 Ϯ 0.1 nA (r2 ϭ 0.97). The histogram generated onstrate that the divalents were not altering the AMPA in the presence of Cd2ϩ was best fit with a double Gaussian conductance. equation with peaks and half-widths of 0.04 Ϯ 0.008 and

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Fig. 9. Sr2ϩ reduces frequency and amplitude of EPSC responses. A and B: afferent fiber responses in the presence of 50 mM Kϩ (A) and the same fiber in the presence of 50 mM Kϩ and 10 mM Sr2ϩ (B). C: amplitude histograms in the absence (gray histogram) and presence (red histogram) of Sr2ϩ. D: peak amplitudes decreased in the presence of Sr2ϩ. Color code is as in previous figures, with red being the primary peak based on histogram area, green the smaller amplitude peak, and blue the larger. E: there is a large increase in the number of small events, those Ͻ100 pA. F and G: the envelopes of the rise (F) and decay (G) plots (Fig. 8) are steeper in the presence than in the absence of Sr2ϩ.

0.07 Ϯ 0.04 nA (r2 ϭ 0.88). Thus in this case Cd2ϩ unmasked fact, discrete events were observed down to the noise ﬂoor and a smaller population of EPSCs and shifted larger peaks to multiple larger peaks were also observed. smaller values very similar to the Sr2ϩ data. Figure 10G plots the relative change in EPSC amplitudes under control and DISCUSSION altered divalent conditions to assess amplitude changes under Recordings from afferent ﬁbers innervating hair cells of the conditions in which the frequency of responses was too low to ϩ turtle auditory papilla provide insight into the functioning of generate histograms. Under each condition, Sr2 or lowered this synapse. A broad range of EPSC amplitudes and frequen- ϩ ϩ Ca2 or Cd2 , the EPSC amplitude was reduced. The reduc- cies were observed for both single and complex events. Depo- tion in EPSC amplitude did not reach a quantal value, as is also larization increased both the frequency and the proportion of illustrated by the example in Fig. 10A and from these mean complex events but did not appear to directly increase the values. If all release were a function of a single vesicle size and amplitude of the EPSCs. Fatigue was not observed, attesting to the divalent exchange experiments were capable of interfering the ability of these synapses to rapidly replenish their supply of with coordinated release so as to unveil the individual events, vesicles. Complex events appear to be bursts of single events then the EPSC amplitudes would have reduced to a single that overlap in time and are simply a function of the release rate distribution about a minimal mean amplitude. This was not of the individual synapses. Bursts as described in immature observed, in that a minimal mean value was not reached; in mammalian cochlea hair cells were observed in only 1 of 55

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Fig. 10. Reducing external calcium or blocking calcium channels with cadmium reduced EPSC amplitude and frequency. A: EPSCs at the start (top) and stop (bottom) of a perfusion of an EGTA-buffered external solution. The time course of reduction in frequency is shown in B. The reduction in frequency of release correlated with the reduction of EPSC amplitude. C: individual EPSC amplitudes plotted over perfusion time, demonstrating that although EPSC amplitudes are reduced, large events remain. Similar results Downloaded from were obtained by perfusing 0.1 mM Cd2ϩ onto the preparation. D: examples of EPSCs prior to (top) and at the end of (bottom) perfusion. E: time course of amplitude reduction during Cd2ϩ application where individual EPSC amplitudes are plotted at vari- ous time points. F: amplitude histograms for the cell in D for the control and in the http://jn.physiology.org/ presence of Cd2ϩ. The control data were ﬁt with the equation for a triple Gaussian, resulting in peaks and half-widths of 0.084 Ϯ 0.06 nA, 0.148 Ϯ 0.1 nA, and 0.28 Ϯ 0.1 nA (r2 ϭ 0.97), while the data in the presence of Cd2ϩ were ﬁt with the equation for a double Gaussian, with peaks and half-widths of 0.04 Ϯ 0.008 nA and 0.07 Ϯ 0.04 nA (r2 ϭ 0.88). G: relative change in peak amplitude

of EPSCs under different conditions includ- by 10.220.33.4 on September 23, 2016 ing Kϩ (red) Sr2ϩ (green), or low divalent 0 mM Ca2ϩ or Cd2ϩ (blue). *Signiﬁcant difference at P Ͻ 0.05.

recordings from the mature turtle papilla (Glowatzki and Fuchs broad histograms were observed, often with multiple peaks. 2002). Bursts in mammalian immature afferent fibers differ The number of peaks increased with release frequency. At least from what was found here in that the complex events under- two scenarios could explain the broad amplitude distribution of lying the burst were all small-amplitude responses that when EPSCs: vesicle size variability and/or coordinated release of summed were the equivalent of a single EPSC. Complex vesicles. Previous measurements of synaptic vesicle diameters events in turtle auditory papilla had peaks that were compara- followed a Gaussian distribution with a mean of 42 nm and a ble to those of the single EPSC events, suggesting that in the half-width of 11 nm (Schnee et al. 2005). The range of vesicle turtle bursts are due to temporal overlap of EPSCs arising from size was 25–60 nm, providing an expected volume difference different synapses while in mammalian fibers bursts are con- of 14ϫ between the smallest and largest vesicle. This ratio is sidered desynchronized EPSCs at a single synapse (Grant et al. similar to the range of EPSC amplitudes measured, and so 2011). vesicle size could account for the amplitude range of EPSCs The wide range of EPSC amplitudes measured at ribbon measured. Whether the vesicle size distribution reflects prefu- synapses is the basis for arguments invoking a multivesicular sion of single vesicles and thus a form of multivesicular release release process whose underlying mechanism could be coor- remains to be determined. dinated release of single vesicles, release of prefused large Reduction of calcium entry reduced the EPSC range to 7ϫ vesicles, or a combination of both. Direct evidence for either while desynchronizing release with Sr2ϩ (Xu-Friedman and mechanism is limited but compelling (Graydon et al. 2011; Regehr 2000) reduced it to 9ϫ, in both cases shifting ampli- Matthews and Sterling 2008), and the basic tenet that multi- tudes to lower values (Schnee et al. 2005). The changes in peak vesicular release is a property of ribbon synapses required to response with external Sr2ϩ did not reduce the amplitudes to a explain amplitude histogram distributions has been validated at unitary size, as was reported in frog when hair cells were several ribbon synapses (Glowatzki and Fuchs 2002; Grant et hyperpolarized (Li et al. 2009). This difference in part may be al. 2011; Graydon et al. 2011; Li et al. 2009). In the turtle, accounted for by Sr2ϩ being more effective at replacing Ca2ϩ

J Neurophysiol • doi:10.1152/jn.00121.2013 • www.jn.org 218 SYNCHRONOUS RELEASE AT AUDITORY HAIR CELL-AFFERENT FIBER SYNAPSE at hair cell synapses than at more conventional synapses. Given prevalent type of complex event). The sum of complex events that the molecules involved in release are presumed to be creates a burst. The proportion of complex events varied with different and also that Ca2ϩ channels are clustered so tightly the frequency of release for a given fiber, as did the number of (Beurg et al. 2010; Dulon et al. 2009; Reisinger et al. 2011; individual events comprising a single burst. No differences Ricci et al. 2000; Roux et al. 2006; Safieddine and Wenthold were found between peak amplitudes of single and complex 1999; Tucker and Fettiplace 1995), this conclusion is plausible. events for a given fiber, in contrast to those of mammalian That incomplete desynchronization by Sr2ϩ may be occurring afferent fibers (Grant et al. 2011). The simplest conclusion is supported by the slowing of both the rise and decay times of from these results is that complex events result from over- the remaining EPSCs recorded. Recent data from rod bipolar lapping release events that might arise from individual cells similarly found a decrease in EPSC amplitude with synapses. Most importantly, it does not appear that any calcium channel blocker application (Mehta et al. 2013). These separate mechanism is needed to account for these events. data may support a general property of ribbon synapses and These results are in good agreement with work in frog clustered calcium channels, i.e., to coordinate release such that amphibian papilla (Andor-Ardo et al. 2012), in which a larger EPSCs are generated for a higher probability of reaching deconvolution algorithm was used to investigate complex threshold. events under spontaneous and stimulated conditions and Downloaded from Together the morphological data and the pharmacological came to very similar conclusions. results are consistent with both synchronous vesicle fusion and Complex events were more efficient at generating APs than perhaps prerelease vesicle fusion (to account for vesicle size single events, implicating summation between synapses as a variability) largely dictating the range of EPSC amplitudes. mechanism by which these pseudocalyceal endings coordinate Data from frog hair cells have similarly argued for synchro- output to ensure reaching threshold. This would be a particu- nous release (Graydon et al. 2011), where amplitude histo- larly useful mechanism for hair cells that are intrinsically grams have widths comparable to those in turtle. However, tuned, as it would directly translate both frequency and timing http://jn.physiology.org/ frog data show a much narrower distribution of vesicle sizes, information synaptically. The recurrence plots also support this with vesicles ranging in diameter from 24 to 50 nm, which conclusion, showing clusters of responses corresponding to means a volume difference of only 9ϫ, thus requiring synchro- complex events. The limitation, however, is the upper fre- nous release to account for at least some of the variation in quency level because the requirement of temporal summation amplitude histogram (Graydon et al. 2011). One problem in to achieve threshold would ostensibly limit the repetition rate each of these studies is the clear identification of synaptic of these complex events to the width of the complex event. The vesicles compared with bulk-endocytosed membrane, sorting reduction in synapses per afferent fiber in higher-frequency end and recycling endosomes, or endoplasmic reticulum. Location organs (as in birds and mammals) might be a manifestation of by 10.220.33.4 on September 23, 2016 near the dense body was typically the selected criterion, but this limitation (Martinez-Dunst et al. 1997). Reducing the this may not be rigorous enough. number of synapses per fiber and increasing the role of mul- The vesicle size variability might reflect a form of multive- tivesicular release could also represent the underlying evolu- sicular release where prerelease fusion of vesicles creates a tionary driving force that culminated at mammalian auditory broader distribution of potential EPSC measurements. Support- synapses. For example, the largest EPSCs reported in mam- ing this potential mechanism are the data from divalent ions as mals are 800–1,000 pA, values rarely if ever observed in turtle well as Sr2ϩ treatment. In each case, lowering external cal- hair cells (Glowatzki and Fuchs 2002). Multivesicular release cium, blocking calcium channels with Cdϩ, or desynchroniz- may guarantee APs in higher-frequency end organs because the ing release with Sr2ϩ resulted in reduced EPSC amplitudes, EPSCs remain temporally brief. although this reduction did not reach a single quantal value. The ability of ribbon synapses to sustain release indefinitely The reduction supports an argument for synchronization; how- has been the focus of work from many laboratories. The turtle ever, the lack of a quantal distribution in EPSCs supports a auditory papilla is no exception in this ability, with rates over broad distribution of vesicle sizes prior to release. 400 Hz measured with an event simply defined as an EPSC On average, the width of the amplitude histograms was regardless of amplitude (so clearly underestimating vesicle ϳ15ϫ. Interestingly, although the width of the EPSC fre- numbers). Recordings were obtained largely from low-frequency histograms was relatively constant, the amplitudes over quency hair cells with ϳ20 synapses, thus requiring at the very which this variance occurred also were quite broad, with least 20 vesicles per synapse per second. At these rates low- minimum values between 20 and 70 pA. The mechanism frequency dense bodies (with ϳ70 vesicles each) would be underlying this almost fourfold range is not clear. It is possible depleted in Ͻ4 s and the rapidly releasable pool in Ͻ1s. that this is a postsynaptic effect with glutamate receptor num- However, including recruitment vesicles within 700 nm of the bers and/or densities varying between fibers. It is also possible dense body would allow release to continue for Ͼ70 s (Schnee that the concentration of glutamate per vesicle varies (Wu et al. et al. 2005). Therefore, postsynaptic measurements are in 2007). Alternatively, the mean vesicle size in each ribbon accord with presynaptic capacitance measurements, attesting to synapse could be variable, as has been reported in CNS the ability of these ribbon synapses to recruit vesicles rapidly synapses (Hu et al. 2008; Qu et al. 2009), and different EPSC to the synapse and supporting previous arguments that release amplitudes could simply correspond to the release of vesicle and not vesicle recruitment to the ribbon is rate limiting pools of different sizes. Further work is needed to delineate (Schnee et al. 2005, 2011). between possibilities. In summary, the data presented show a broad range of Complex events were defined as those either with a non- EPSCs that can in part be accounted for by the range of vesicle monotonic rise time or with decays that did not reach baseline sizes but also are in part due to synchronized fusion of vesicles. prior to the next event initiation (the latter being the most Complex events that underlie bursts are argued to be due to

J Neurophysiol • doi:10.1152/jn.00121.2013 • www.jn.org SYNCHRONOUS RELEASE AT AUDITORY HAIR CELL-AFFERENT FIBER SYNAPSE 219 temporal summation between synapses. Temporal summation Grant L, Yi E, Goutman JD, Glowatzki E. Postsynaptic recordings at results in much more efﬁcient generation of APs and is pos- afferent dendrites contacting cochlear inner hair cells: monitoring multivesicular release at a ribbon synapse. J Vis Exp 2011: 2442, 2011. tulated to be the means of ensuring spike timing in these ϩ Graydon CW, Cho S, Li GL, Kachar B, von Gersdorff H. Sharp Ca2 calyceal types of endings. nanodomains beneath the ribbon promote highly synchronous multivesicular release at hair cell synapses. J Neurosci 31: 16637–16650, 2011. Hu Y, Qu L, Schikorski T. Mean synaptic vesicle size varies among ACKNOWLEDGMENTS individual excitatory hippocampal synapses. Synapse 62: 953–957, 2008. Our thanks to Mamiko Niwa, Connie Tsai, Anthony Peng, and Thomas Keen EC, Hudspeth AJ. Transfer characteristics of the hair cell’s afferent Effertz for comments on the manuscript. synapse. Proc Natl Acad Sci USA 103: 5537–5542, 2006. Li GL, Keen E, Andor-Ardo D, Hudspeth AJ, von Gersdorff H. The unitary event underlying multiquantal EPSCs at a hair cell’s ribbon synapse. GRANTS J Neurosci 29: 7558–7568, 2009. Liberman LD, Wang H, Liberman MC. Opposing gradients of ribbon size This work was supported by National Institute on Deafness and Other and AMPA receptor expression underlie sensitivity differences among Communication Disorders Grant RO1 DC-009913 to A. J. Ricci and Core cochlear-nerve/hair-cell synapses. J Neurosci 31: 801–808, 2011. Grant P30 44992, as well as a Dean’s fellowship and a Cajamadrid foundation

Martinez-Dunst C, Michaels RL, Fuchs PA. Downloaded from fellowship to M. Castellano-Muñoz. Release sites and calcium channels in hair cells of the chick’s cochlea. J Neurosci 17: 9133–9144, 1997. DISCLOSURES Matthews G, Fuchs P. The diverse roles of ribbon synapses in sensory neurotransmission. Nat Rev Neurosci 11: 812–822, 2010. No conflicts of interest, financial or otherwise, are declared by the author(s). Matthews G, Sterling P. Evidence that vesicles undergo compound fusion on the synaptic ribbon. J Neurosci 28: 5403–5411, 2008. Mehta B, Snellman J, Chen S, Li W, Zenisek D. Synaptic ribbons influence

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