Time course and calcium dependence of transmitter release at a single ribbon synapse

Abstract

At the first synapse in the auditory pathway, the receptor potential of mechanosensory hair cells is converted into a firing pattern in auditory nerve fibers. For the accurate coding of timing and intensity of sound signals, transmitter release at this synapse must occur with the highest precision. To measure directly the transfer characteristics of the hair cell afferent synapse, we implemented simultaneous whole-cell recordings from mammalian inner hair cells (IHCs) and auditory nerve fiber terminals that typically receive input from a single ribbon synapse. During a 1-s IHC depolarization, the synaptic response depressed >90%, representing the main source for adaptation in the auditory nerve. Synaptic depression was slightly affected by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor desensitization; however, it was mostly caused by reduced vesicular release. When the transfer function between transmitter release and Ca(2+) influx was tested at constant open probability for Ca(2+) channels (potentials >0 mV), a super linear relation was found. This relation is presumed to result from the cooperative binding of three to four Ca(2+) ions at the Ca(2+) sensor. However, in the physiological range for receptor potentials (-50 to -30 mV), the relation between Ca(2+) influx and afferent activity was linear, assuring minimal distortion in the coding of sound intensity. Changes in Ca(2+) influx caused an increase in release probability, but not in the average size of multivesicular synaptic events. By varying Ca(2+) buffering in the IHC, we further investigate how Ca(2+) channel and Ca(2+) sensor at this synapse might relate.

Fig. 1.

Depression of AF activity during IHC depolarization mostly depends on a presynaptic mechanism. (A) Differential interference contrast image of the excised organ of Corti preparation; with patch pipette on the left approaching an (outlined) IHC, pipette on the right, an afferent bouton (indicated by arrow). (B and C) Simultaneous recording R6 of IHC and AF with and without 100 μM CTZ. IHC Ca²⁺ buffer contained 1 mM EGTA. (B1 and C1) IHC voltage command (V_c) was applied every 30 s for 1 s and isolated IHC Ca²⁺ current. (B2 and C2) AF single responses, holding potential −84 mV. (B2 Inset) Peak AF response and single EPSCs later in the AF response. (Scale bars: 2 ms, 50 pA.) (B3 and C3) AF average responses to 13 (Control) and 12 (CTZ) depolarizations. Peak amplitudes: control, 314 ± 27 pA; CTZ, 618 ± 25 pA. Red trace, double exponential fit, τ₁ = 7.4 ms, τ₂ = 135 ms. (C3 Inset) Average EPSCs for R6. Control: amplitude 139 ± 7 pA, τ_decay 1.1 ± 0.1 ms, n = 69. CTZ: amplitude 152 ± 10 pA, τ_decay 3.9 ± 0.1 ms, n = 36. (D) Deconvolution (average of six recordings, 96 IHC depolarizations) of average AF responses with average EPSCs. (E) Integral of deconvolution in D.

Fig. 2.

Voltage and Ca²⁺ dependence of the AF response. EPSC rate, but not EPSC amplitude, is Ca²⁺-dependent. (A1) IHC voltage steps protocol: −89 to +31 mV, 10-mV steps, for 200 ms and IHC Ca²⁺ currents. (Scale bar: 50 ms, 100 pA.) (A2) AF single responses at selected holding potentials. (Scale bars: 50 ms, 200 pA.) (B1 and B2) Voltage dependence of simultaneous recordings for IHC Ca²⁺ currents (B1) and average AF responses (B2) (four repetitions per voltage, peak, or integral). (B3) Voltage dependence of EPSC rate and EPSC amplitude (Inset) for the last 100 ms of stimulation. Number of EPSCs per V_c is indicated.

Fig. 3.

The Ca²⁺ dependence of release is linear for negative IHC membrane potentials and super linear for positive IHC membrane potentials. Transfer functions between IHC Ca²⁺ current and postsynaptic response for a negative (A1, A2, and C) and a positive (B1, B2, and D) voltage range. (A1 and B1) IHC Ca²⁺ current. (A2) AF peak response. (B2) AF integral response. (C and D) Ca²⁺ dependence of release for both voltage ranges; the normalized AF response was plotted versus the normalized Ca²⁺ current. Red line: fit with power function. (C) Symbols represent data from three cell pairs (R6, R18, and R19); five to seven voltage steps per recording, two to four repetitions per step. (D) Normalized integral of AF response vs. normalized Ca²⁺ current for the positive voltage range was tested in two different buffer conditions: 1 mM EGTA (filled symbols, n = 3 cell pairs, R4, R8, R20; five to seven voltage steps, two to four repetitions) and 5 mM EGTA (open symbols, n = 3 cell pairs, R10, R12, R9; five to nine steps, three to four repetitions).

Fig. 4.

The EPSC steady-state rate is sensitive to fast and slow Ca²⁺ buffering in the IHC. (A1–A3) Simultaneous recordings of IHCs and AFs using different IHC Ca²⁺ buffers; in 100 μM CTZ, 1-s IHC depolarization, V_c: −89 to −29 mV. (B) For different buffer conditions, EPSC steady-state rates were significantly different (#, P = 0.01), whereas EPSC amplitudes were not (1 mM EGTA: 253.9 ± 35.1 pA, n = 6, 179 EPSCs; 5 mM EGTA: 315 ± 51.8 pA, n = 4, 87 EPSCs; 5 mM BAPTA: 220 ± 27.8 pA, n = 3, 67 EPSCs, ANOVA, P = 0.35, mean values and SE). Ca²⁺ current amplitudes were not significantly different (1 mM EGTA: 160.4 ± 13.5 pA, n = 6, 96 depolarizations; 5 mM EGTA: 171.2 ± 4.7 pA, n = 4, 66 depolarizations; 5 mM BAPTA: 165.1 ± 25.3 pA, n = 4, 93 depolarizations, ANOVA, P = 0.89).

Fig. 5.

Fast and slow components of release are sensitive to IHC Ca²⁺ buffering with elevated EGTA and BAPTA. (A1) Average AF responses in 1 mM EGTA (n = 6, 96 depolarizations), 5 mM EGTA (n = 4, 66 depolarizations), and 5 mM BAPTA (n = 3, 150 depolarizations, magnified in Inset). (A2) Peaks of average AF response in A1 on extended time scale. (B) Deconvolution of the average AF responses in 1 and 5 mM EGTA with first 40 ms shown. (C) Integrals of the deconvolutions in B.