Figures and data
Paired IHC-bouton patch-clamp recordings to study the release properties of individual IHC ribbon synapses as a function of synapse position
A. Differential Interference Contrast image of an explanted murine organ of Corti. In this example, supporting cells from the modiolar side were removed to gain access to the IHCs and their contacting boutons. The recorded boutons were classified based on their position (△ pillar or ❍ modiolar) and on their spontaneous rate (SR) (Low SR < 1 sEPSC/s vs High SR > 1 sEPSC/s). Scale bar: 10 µm. B. Spontaneous release was recorded in absence of stimulation (i.e., IHC holding potential = −58 mV; Supplementary Table 1; dashed line represents the threshold for sEPSC detection). sEPSCs were classified as monophasic (or compact: a steady rise to peak and monoexponential decay) or as multiphasic (or non-compact: multiple inflections and slowed raising and decaying kinetics). C. Evoked release: depolarizing pulses (black trace) were used to trigger whole IHC Ca2+ influx (ICa, blue trace) and ensuing release of neurotransmitter that evoked eEPSCs, light orange trace). Ca2+ charge and eEPSC charge were estimated by taking the integral of the currents (shaded light blue and light orange areas).
Synapses with high spontaneous release have larger and more compact sEPSCs A.
Spontaneous EPSCs recorded in the absence of stimulation (i.e. IHC holding potential = −58 mV) from two exemplary paired recordings with different spontaneous rate (SR: grey for low SR, orange for high SR). ‘Pair #’ identifies individual paired recordings. Insets show the selected sEPSCs in an expanded time scale. (a,b) correspond to multiphasic sEPSCs, while (c) represents a typical monophasic sEPSC. B. Cumulative sEPSC amplitude plots for 23 paired synapses that had spontaneous release. C-D. Average sEPSC amplitude (C) and charge (D) from individual synapses recorded from the pillar or modiolar side of the IHC. E-F. Pooled sEPSC amplitude (E) and charge (F) distributions show a distinct peak at −40 pA and 40 pC, respectively. G. Percentage of monophasic sEPSCs in pillar and modiolar synapses. H. Cumulative fraction (left axis) and normalized histogram (right axis) of the spontaneous rate (bin size is 1 sEPSC/s) of 33 pairs. I. Pillar synapses had higher rates of sEPSCs (p = 0.0311, Mann-Whitney U test). J-L. High SR synapses had significantly larger sEPSC amplitudes (J; p = 0.0042, unpaired t-test), a tendency to bigger sEPSC charges (K; p = 0.0527, unpaired t-test) and higher percentages of monophasic sEPSCs (p = 0.0185, Mann-Whitney U test).
Whisker plots represent the 25th, 50th and 75th percentiles with the individual data points overlaid. Synapses were classified as △ pillar or ❍ modiolar, and as Low SR < 1 sEPSC/s or High SR > 1 sEPSC/s.
Pillar and high SR synapses have sEPSCs with faster rising times.
A. Kinetics of sEPSCs, such as amplitude, 10-90% rise time, time constant of decay (τdecay) and full-width half-maximum (FWHM), were calculated with Neuromatic (Rothman and Silver, 2018). Bi-Bii. Cumulative fraction of amplitude and charge of sEPSCs from low and high SR synapses. C-E. Pillar synapses had faster 10-90% rise times (C) than modiolar synapses (p = 0.0111, unpaired t-test), while their τdecay (D) and FWHM (E) were comparable. F-H. High SR synapses had faster 10-90% rise times (F) than modiolar synapses (p = 0.0420, unpaired t-test), while their τdecay (G) and FWHM (H) were comparable.
Whisker plots represent the 25th, 50th and 75th percentiles with the individual data points overlaid. Synapses were classified as △ pillar or ❍ modiolar, and as Low SR < 1 sEPSC/s or High SR > 1 sEPSC/s.
High SR synapses have a more hyperpolarized voltage dependence and tighter Ca2+ channel coupling of synaptic release
A. IHC Ca2+ current (blue and gray) and eEPSCs (orange and gray) of a high and a low SR pair, respectively, in response to 10 ms depolarizations to different potentials ranging from −58 to −18 mV in 5 mV steps (upper left panel). The upper right panel shows the current-voltage relationships for the two pairs. Bi-Bii. The peak of whole-cell Ca2+ current (Bi) and the voltage eliciting maximum Ca2+ current (Bii) of IHCs were comparable between high and low SR synapses. C. Upper panel: Fractional activation of the Ca2+ channels (blue and gray data points from the examples shown in A) was obtained from the normalized chord conductance. Voltage of half-maximal activation (Vhalf Ca; dotted line) and sensitivity of the voltage dependence (slope k) were determined using a Boltzmann fit (black trace) to the activation curve. Lower panel: Release-intensity curve (orange and gray data points from the examples shown in A) was obtained from the QEPSC for each depolarization step. A sigmoidal function (black trace) was fitted to obtain the voltage of half-maximal synaptic release (Vhalf QEPSC; dotted line) and the voltage sensitivity of the release (slope), as well as the dynamic range for which the exocytosis changes from 10-90% (gray area). Di-Dii. Voltage dependence of whole-cell Ca2+ channel activation (activation curve; Di) and fits to release-intensity curves (Dii) for 31 synapses of low (< 1 sEPSC/s, gray) and high SR (≥ 1 sEPSC/s, orange). Averages (thick lines) and individual curves (thin lines) are overlaid. The release-intensity curve of two low SR pairs could not be fitted (grey dotted lines). Ei-Fi. The threshold of Ca2+ influx (Ei) and Vhalf ICa (Fi) did not differ between low and high SR synapses. Eii-Fii. Voltage of 10% maximum release (Q10 EPSC, Eii; p = 0.0001, Mann-Whitney U test) and Vhalf QEPSC (Fii; p = 0.0021, unpaired t-test) were significantly more hyperpolarized in high SR synapses. G. Dynamic range of release was comparable between low and high SR synapses. H. Ca2+ cooperativity (m) estimated from fitting a power function to the QEPSC – QCa relationship for each individual synapse was significantly lower in high SR synapses (p = 0.0016, Mann-Whitney U test). I. Scatter plot of normalized QEPSC versus the corresponding normalized QCa. The solid lines is a least-squares fit of a power function (QEPSC = a(QCa)m) to the data yielding mhigh SR of 0.8 (orange line) and mlow SR of 1.4 (black line).
Whisker plots represent the 25th, 50th and 75th percentiles with the individual data points overlaid. Synapses were classified as △ pillar or ❍ modiolar, and as Low SR < 1 sEPSC/s or High SR > 1 sEPSC/s.
The voltage dependence of synaptic release does not differ significantly between modiolar and pillar synapses, but the Ca2+ dependence does
A, B. The reversal potential (A) and the voltage sensitivity of ICa (B) were comparable between low and high SR synapses. C. Triggered single active zone eEPSCs (release intensity curves) of 31 pairs of low and high SR. Averages (thick lines) and individual curves (thin lines) are overlaid. The release-intensity curve of two low SR pairs could not be fitted with the sigmoidal function (grey dotted lines). D-E. Q90 EPSC (D) and voltage sensitivity of the release (E) were comparable between high and low SR synapses. Fi-Fii. The peak of Ca2+ current (Fi) and the voltage eliciting maximum Ca2+ current (Fii) were comparable between modiolar and pillar synapses. Gi-Ji. The threshold of Ca2+ influx (Gi), Vhalf ICa (Hi), reversal potential of ICa (Ii) and voltage sensitivity of ICa (Ji) did not differ between modiolar and pillar synapses. Gii-Jii. Q10 EPSC (Gii), Vhalf QEPSC (Hii), Q90 EPSC (Iii) and voltage sensitivity of the release (Jii) were comparable between modiolar and pillar synapses. K. Dynamic range of release was comparable between modiolar and pillar synapse. L. Ca2+ cooperativity (m) estimated for each individual synapse was significantly lower in pillar synapses (p = 0.0202, Mann-Whitney U test).
Whisker plots represent the 25th, 50th and 75th percentiles with the individual data points overlaid. Synapses were classified as △ pillar or ❍ modiolar, and as Low SR < 1 sEPSC/s or High SR > 1 sEPSC/s.
Apparent Ca2+ dependence of neurotransmitter release at individual synapses in the range of IHC receptor potentials
Scatter plots of the EPSC charges (QEPSC) vs. the corresponding Ca2+ current integrals (QCa) for each individual synapse in response to 10 ms depolarizations from −58 to −18 mV. The solid line is a least-squares fit of a power function (QEPSC = a(QCa)m) to each pair data.
High SR synapses have shorter synaptic delay and higher initial release rates
A. Representative Ca2+ currents (blue), eEPSCs (light orange) and eEPSC charges (QEPSC, green) to the “forward masking” protocols used to study depletion and recovery of RRP. The stimulus (top panel) consists of two sequential voltage steps (“masker” and “probe”) separated by different interstimulus intervals (ISI in ms). B. Latencies of the evoked EPSCs (EPSConset - Maskeronset) were significantly shorter in high SR than low SR synapses (p = 0.0221, Mann-Whitney U test). C. High SR synapses also had less latency jitter (p = 0.0012, Mann-Whitney U test). D. Presynaptic IHC Ca2+ charge (QCa) during the masker stimuli was comparable regardless the spontaneous rate of the recorded postsynaptic bouton. E. Pool depletion dynamics were studied by fitting the sum of a single exponential and a line function (black discontinuous line) to the first 50 ms of the average QEPSC trace in response to the masker stimulus. F-J. RRP, time constant (τ) of depletion, initial release rate and sustained release were calculated from the fits and the mean QsEPSC for each pair. High SR synapses depleted the RRP with faster time constants (G; p = 0.0140, Mann-Whitney U test) and reached higher initial release rates (H; p = 0.0472, unpaired t-test) followed by a stronger adaptation (J; p = 0.0058, unpaired t-test). K. Recovery from RRP depletion shown as ratio of QEPSC probe and QEPSC masker (mean ± sem) during the first 10 ms of the stimulus. L. Single exponential fits from 16 ms to 20000 ms (black dotted lines) to estimate the recovery kinetics from RRP depletion. M. Time constant of recovery from RRP depletion obtained from fits in L.
Whisker plots represent the 25th, 50th and 75th percentiles with the individual data points overlaid.Synapses were classified as △ pillar or ❍ modiolar, and as Low SR < 1 sEPSC/s or High SR > 1 sEPSC/s.
Parameters of synaptic vesicle pool dynamics in forward masking protocols
A-C. Presynaptic peak (A), initial (B) and final (C) Ca2+ current (ICa) during the masker stimuli were comparable regardless the SR of postsynaptic bouton. D. Latencies of the evoked EPSCs were also significantly shorter in high SR than low SR synapses when compared in a bigger sample size (31 pairs from Fig. 4; p = 0.0101, Mann-Whitney U test). E-F. High SR synapses had significantly larger amplitudes of evoked EPSCs in response to the masker stimulus (E; p = 0.0147, unpaired t-test), while the EPSC charge (QEPSC) did not differ (F). G. Exemplary average QEPSC response of a high SR (orange) and a low SR (gray) synapse to the masker stimuli. We fitted an exponential plus line function to the first 50 ms of the response (discontinuous lines) to study SV pool depletion dynamics. From these fits, we can retrieve information about RRP size (amplitude of the exponential component, A1), RRP depletion time constant (τ of the exponential component) and sustained release (linear slope of the line component). H-I. Amplitude (H) and linear slope (I) obtained from the fits of the exponential plus line function to the QEPSC from individual pairs (Fig. 4E). F. The spontaneous release after the probe offset recovered slower in high SR synapses.
Whisker plots represent the 25th, 50th and 75th percentiles with the individual data points overlaid. Synapses were classified as △ pillar or ❍ modiolar, and as Low SR < 1 sEPSC/s or High SR > 1 sEPSC/s.
Spontaneous activity (SR) was calculated from time windows without stimulation with the IHC held at −58 mV (Total time for SR calculation). This total time was calculated from the cumulative recording time of either from 5 – 10 s recordings and/or from the segments before and after a depolarizing pulse.
The kinetics of sEPSCs are comparable between low and high SR synapses
A. Kinetics of sEPSCs, such as amplitude, 10-90% rise time, time constant of decay (τdecay) and full-width half-maximum (FWHM), were calculated with Neuromatic (Rothman and Silver, 2018). Bi-Bii. Cumulative fraction of amplitude and charge of sEPSCs from low and high SR synapses. C-E. Pillar synapses had faster 10-90% rise times (C) than modiolar synapses (p = 0.0426, unpaired t-test), while their τdecay (D) and FWHM (E) were comparable. F-H. Low and high SR synapses had similar 10-90% rise times (F), τdecay (G) and FWHM (H).
Scatter plots represent the 25th, 50th and 75th percentiles with the individual data points overlaid. Synapses were classified as △ pillar or ❍ modiolar, and as Low SR < 1 sEPSC/s or High SR > 1 sEPSC/s.
The voltage dependence of synaptic release does not differ between modiolar and pillar synapses
A. The reversal potential of Ca2+ influx was comparable between low and high SR synapses. B. Triggered single active zone eEPSCs (release intensity curves) of 29 pairs of low and high SR. Averages (thick lines) and individual curves (thin lines) are overlaid. The release-intensity curve of two pairs could not be fitted with the sigmoidal function (grey dotted lines). C. High SR synapses had more hyperpolarized voltages of 90% maximum release (Q90 EPSC, p = 0.0396, unpaired t test). D. Q90 EPSC did not differ between modiolar and pillar synapses. Ei-Eii. The peak of Ca2+ current (Ei) and the voltage eliciting maximum Ca2+ current (Eii) were comparable between modiolar and pillar synapses. Fi-Hi. The threshold of Ca2+ influx (Ei), Vhalf ICa (Fi) and voltage sensitivity of ICa (Gi) did not differ between modiolar and pillar synapses. Fii-Hii. Q10 EPSC (Fii), Vhalf QEPSC (Gii) and voltage sensitivity of the release were comparable between modiolar and pillar synapses. I. The reversal potential of Ca2+ influx was comparable between modiolar and pillar synapses. J. Dynamic range of release was comparable between modiolar and pillar synapse.
Scatter plots represent the 25th, 50th and 75th percentiles with the individual data points overlaid. Synapses were classified as △ pillar or ❍ modiolar, and as Low SR < 1 sEPSC/s or High SR > 1 sEPSC/s.
Voltage dependence of IHC Ca2+ influx and synaptic release
A-C. Presynaptic peak (A), initial (B) and final (C) Ca2+ current (ICa) during the masker stimuli were comparable regardless the SR of postsynaptic bouton. D-E. High SR synapses had significantly larger amplitudes of evoked EPSCs in response to the masker stimulus (D; p = 0.0147, unpaired t-test), while the EPSC charge (QEPSC) did not differ (E). F. The spontaneous release after the probe offset recovered slower in high SR synapses. G. Exemplary average QEPSC response of a high SR (orange) and a low SR (gray) synapse to the masker stimuli. We fitted an exponential plus line function to the first 50 ms of the response (discontinuous lines) to study SV pool depletion dynamics. From these fits, we can retrieve information about RRP size (amplitude of the exponential component, A1), RRP depletion time constant (τ of the exponential component) and sustained release (linear slope of the line component). H-I. Amplitude (H) and linear slope (I) obtained from the fits of the exponential plus line function to the QEPSC from individual pairs (Fig. 4E).
Scatter plots represent the 25th, 50th and 75th percentiles with the individual data points overlaid. Synapses were classified as △ pillar or ❍ modiolar, and as Low SR < 1 sEPSC/s or High SR > 1 sEPSC/s.
