Two modes of release shape the postsynaptic response at the inner hair cell ribbon synapse

Abstract

Cochlear inner hair cells (IHCs) convert sounds into receptor potentials and via their ribbon synapses into firing rates in auditory nerve fibers. Multivesicular release at individual IHC ribbon synapses activates AMPA-mediated EPSCs with widely ranging amplitudes. The underlying mechanisms and specific role for multivesicular release in encoding sound are not well understood. Here we characterize the waveforms of individual EPSCs recorded from afferent boutons contacting IHCs and compare their characteristics in immature rats (postnatal days 8-11) and hearing rats (postnatal days 19-21). Two types of EPSC waveforms were found in every recording: monophasic EPSCs, with sharp rising phases and monoexponential decays, and multiphasic EPSCs, exhibiting inflections on rising and decaying phases. Multiphasic EPSCs exhibited slower rise times and smaller amplitudes than monophasic EPSCs. Both types of EPSCs had comparable charge transfers, suggesting that they were activated by the release of similar numbers of vesicles, which for multiphasic EPSCs occurred in a less coordinated manner. On average, a higher proportion of larger, monophasic EPSCs was found in hearing compared to immature rats. In addition, EPSCs became significantly faster with age. The developmental increase in size and speed could improve auditory signaling acuity. Multiphasic EPSCs persisted in hearing animals, in some fibers constituting half of the EPSCs. The proportion of monophasic versus multiphasic EPSCs varied widely across fibers, resulting in marked heterogeneity of amplitude distributions. We propose that the relative contribution of two modes of multivesicular release, generating monophasic and multiphasic EPSCs, may underlie fundamental characteristics of auditory nerve fibers.

Figure 1.

EPSCs in IHC afferent fibers are mediated by AMPA receptors in hearing animals. A, Image of a preparation from a P19 rat cochlea (apical turn) using a microscope with DIC optics showing the basal portion of six IHCs. Beneath the level of the IHC nuclei are a number of fibers innervating the region, asterisks indicate three afferent boutons. B, Exemplar voltage-clamp recording from a P19 afferent bouton showing EPSCs in 40 mm extracellular K⁺, holding potential −94 mV. Near-complete block of synaptic activity in the same cell is shown in the presence of 10 μm NBQX. C, Averaged monophasic EPSC waveform from a P20 afferent with 40 mm extracellular K⁺. τ decay significantly slowed from 0.58 ms to 2.19 ms in the presence of 100 μm cyclothiazide (CTZ) (n = 2314 control; 1172 CTZ; p < 0.0001). D, Averaged EPSCs (n = 25–59) recorded at different holding potentials, indicated for each trace. E, Current voltage relation plotted from EPSCs of the afferent fiber in D. The IV is nonrectifying and reverses at +1 mV.

Figure 2.

Afferent recordings from hearing rats exhibit on average more monophasic EPSCs compared to immature rats. A, B, Exemplar EPSCs from afferent boutons of a P10, a P20, and a P60 rat. EPSCs with a steady rising phase and monoexponential decay were classified as monophasic (A). EPSCs with inflections on rising and/or decaying phases were classified as multiphasic (B). C, Quantification of the mean percentage of monophasic EPSCs in afferent fibers of P8–11 and P19–21 rat cochleae (9289 EPSCs in 12 fibers of P8–11 rats and 10,500 EPSCs from 14 fibers of P19–21 rats). The percentage of monophasic EPSCs increased significantly from 56% at P8–11 to 71% at P19–21 (p = 0.024). D, The percentage of monophasic EPSCs in individual fibers from P8–11; P19–21 and P60 cochleae ranked from lowest to highest percentage in each age group. Two of 12 afferents from P8–11 rats had >75% monophasic EPSCs, whereas 9 of 14 afferents from P19–21 rats had >75% monophasic EPSCs. Note that four fibers from P19–21 rats had relatively low percentages of monophasic EPSCs. In two recordings from afferent boutons of P60 rat cochleae monophasic EPSCs were 52% and 74% (6170 EPSCs total).

Figure 3.

EPSC kinetics are significantly faster in afferents from hearing versus immature rat cochleae. A, B, Selection of monophasic EPSCs recorded from a P8 (A) and a P20 (B) afferent at −94 mV with 5.8 mm K⁺ extracellularly. C, Normalized average EPSC waveforms for the two cells presented in A and B (EPSCs averaged: P8 n = 336, green; P20 n = 216, black). D, Average time to peak and τ decay presented for monophasic EPSCs from 12 boutons of P8–11 (time to peak: 0.58 ± 0.1 ms; decay: 1.4 ± 0.6 ms; 5078 EPSCs), 14 boutons of P19–21 (time to peak: 0.29 ± 0.1 ms; decay 0.5 ± 0.2 ms; 6782 EPSCs), and 2 boutons of P60 (time to peak: 0.28 ± 0.002 ms; decay: 0.37 ± 0.002 ms; 3796 EPSCs) rats. EPSC kinetics were significantly faster in boutons from hearing rats (time to peak and τ decay p < 0.0001; comparing P8–11 and P19–21 datasets). E, Plot of τ decay against EPSC amplitude for every monophasic EPSC from one P8 afferent (green squares) and one P20 afferent (black squares), same fibers as in A and B. EPSCs in hearing afferents were significantly faster across the entire amplitude range. Monophasic EPSC waveforms were more uniform in boutons from hearing animals illustrated by the less variable decay time constant for the P20 afferent. F, Average time to peak and half-width for multiphasic EPSCs from cochleae of 12 P8–11 (time to peak: 1.1 ± 0.2 ms; half-width: 1.4 ± 0.6 ms; 4616 EPSCs), 14 P19–21 (time to peak: 0.6 ± 0.1 ms; half-width: 0.7 ± 0.3 ms; 2525 EPSCs), and 2 P60 (time to peak: 0.8 ± 0.003 ms; half-width: 0.6 ± 0.1; 2374 EPSCs) rats. Multiphasic EPSCs were significantly faster in boutons from hearing rats (time to peak: p < 0.0001; half-width: p = 0.0005, comparing P8–11 and P19–21 datasets). G, Plot of half-width against EPSC amplitude for every multiphasic EPSC from one P8 afferent (green squares) and one P20 afferent (black squares), same fibers as in A and B. Multiphasic EPSCs were significantly faster across the entire amplitude range.

Figure 4.

On average, EPSC amplitudes in afferent fibers shift to higher values in hearing animals. A, B, Pooled amplitude distributions for EPSC amplitudes from all P8–11 afferents (A; n = 12 cells 9289 EPSCs) and P19–21 afferents (B; n = 14 cells 10,500 EPSCs). For multiphasic EPSCs amplitude was measured from baseline to the maximal amplitude (see H). A shift in the modal EPSC amplitude is apparent from ∼30 pA in P8–11 afferents to ∼375 pA in P19–21 afferents. C, Cumulative amplitude distribution for EPSCs from all P8–11 and all P19–21 afferents pooled. D–G, Amplitude distribution for only multiphasic (D, E) or only monophasic (F, G) EPSCs from all P8–11 afferent fibers or from all P19–21 fibers. Bin width for all histograms is 3 pA. Arrowheads and numbers indicate amplitudes for peaks of distributions. H, Examples of how EPSC amplitudes were measured for multiphasic EPSCs, dashed lines indicate EPSC amplitude (a). I, J, Cumulative EPSC amplitude plots for multiphasic and monophasic EPSCs from all P8–11 (I) and all P19–21 (J) fibers pooled. K, L, Cumulative area plots for multiphasic and monophasic EPSCs from all P8–11 (K) and all P19–21 (L) fibers pooled.

Figure 5.

Monophasic and multiphasic EPSCs are distributed randomly in time. A, B, Interevent intervals plotted for all EPSCs from one P11 fiber and one P20 fiber both recorded with 5.8 mm K⁺ extracellularly. Individual points are numbers of EPSCs in 10 ms bins. C, D, Intervals preceding multiphasic and monophasic EPSCs shown separately for the same cells depicted in A and B. Data are fitted with single exponentials (τ from fits: C (P11): 249 ms monophasic, 233 ms multiphasic; D (P20): 80 ms monophasic, 63 ms multiphasic). Intervals preceding multiphasic and monophasic EPSCs were not significantly different [comparing median intervals or τ from exponential fits, median intervals: P8–11, 181 ± 125 ms (monophasic) versus 170 ± 117 (multiphasic); P19–21, 98 ± 101 ms (monophasic) versus 92 ± 136 ms (multiphasic)].

Figure 6.

EPSC amplitude distributions for individual fibers are more diverse for hearing rats compared to immature rats. A, B, Cumulative EPSC amplitude plots for individual fibers from cochleae of P8–11 (A) and P19–21 (B) rats. Fibers were classified as low amplitude or high amplitude by their median amplitude (<250 = low; >250 = high). Representative fibers from each class are highlighted in the cumulative plots (blue = low amplitude, red = high amplitude). C, D, Individual amplitude histograms for exemplar low-amplitude and high-amplitude fibers, chosen from a P11 and a P20 rat respectively. Beneath each amplitude histogram, multiphasic and monophasic EPSC amplitude distributions are plotted separately. E, Number of fibers classed as low and high amplitude from P8–11 and P19–21 rats. F, Percentage of monophasic EPSCs in fibers categorized as low or high amplitude from P8–11 and P19–21 rats. At P19–21 where there were sufficient fibers in each category for statistical analysis there were significantly higher percentages of monophasic EPSCs in high-amplitude fibers compared to low-amplitude fibers [percentage monophasic: 63 ± 7 (3 low-amplitude fibers) versus 82 ± 7 (8 high-amplitude fibers); p = 0.003].

Figure 7.

Fiber types do not change with the IHC at rest or depolarized. A, Cumulative plots of EPSC amplitude for three afferent fibers from P19–21 hearing rats (1 low- and 2 high-amplitude fibers). Plots are shown for EPSCs from each cell recorded with 5.8 mm K⁺ (black lines) and 40 mm K⁺ (gray lines) externally. EPSC amplitude distributions were similar in 5.8 mm K⁺ versus 40 mm K⁺ for all of these fibers. B, C, EPSC amplitude distribution plots for one fiber shown in A, with 5.8 mm K⁺ extracellularly (B) and 40 mm K⁺ extracellularly (C). D, Cumulative EPSC plots for one fiber from a P60 rat with 5.8 and 40 mm K⁺ externally, with individual EPSC amplitude distributions shown in E and F. There was no change in median EPSC amplitude or percentage of monophasic EPSCs for these four afferents with IHC depolarization (median amplitudes: 97 versus 74 pA, 344 versus 319 pA, 474 versus 469 pA, 300 versus 237 pA, 5.8 versus 40 K⁺, respectively; percentage of monophasic EPSCs in 5.8 K⁺ versus 40 K⁺: 66 versus 67%, 75 versus 88%, 86 versus 93%, and 52 versus 32%).