Coronal slices (180 μm) containing the rostral anteroventral cochlear nucleus (AVCN) were cut from P22-P30 gerbils of either sex. The brainstem was sliced with a vibratome in low-calcium artificial cerebrospinal fluid (ACSF) solution containing (in mM): 125 NaCl, 2.5 KCl, 0.1 CaCl₂, 3 MgCl₂, 1.25 NaH₂PO₄, 25 NaHCO₃, 25 glucose, 2 sodium pyruvate, 3 myo-inositol, 0.5 ascorbic acid, continuously bubbled with 5% CO₂ and 95% O₂, pH 7.4. Slices were incubated in the standard recording solution (ACSF same as for slicing, except CaCl₂ and MgCl₂ were changed to 2 mM and 1 mM, respectively) for 30 min at 37°C and stored at room temperature until recording. Experiments were performed at nearly physiological temperature (33.5 ± 0.5°C).

Whole-cell patch clamp recordings were performed on SBCs in the rostral pole of the AVCN. Due to their large soma size and localization in the low-frequency area of the gerbil AVCN, these neurons can be visually distinguished from globular bushy cells. Morphological verification of SBCs was done during the recording by intracellular labeling with ATTO 488 and visualization by a CCD camera (IMAGO Typ VGA; TILL Photonics). The pipettes had resistances of 3–4 MΩ when filled with (mM): 125 CsMeSO₃, 18 TEA-Cl, 3 MgCl₂, 10 HEPES, 0.1 EGTA, 4.5 QX-314-Cl, 5 phosphocreatine, 2 ATP disodium salt, 0.3 GTP disodium salt, and 50 μM ATTO 488 (pH 7.3 with CsOH). The resulting inward currents with larger amplitudes enabled more accurate analyses compared to the small events occurring with physiological [Cl⁻]_pip. Consistent with slow inhibitory kinetics in the present study, the synaptically evoked hyperpolarizations acquired with gramicidin perforated patch recordings also exhibited slow activity-dependent synaptic decays (gramicidin perforated patch: t_wd single: 18.5 ± 4.0 ms, 100 Hz 10th IPSC: 42.6 ± 4.1 ms, n = 6, see Nerlich et al., 2014). IPSCs were evoked by electrical stimulation of afferent fibers through a bipolar theta glass electrode (Science Products, tip Ø 5 μm) filled with bath solution and placed at distances of 30–60 μm from the cell. Pulse stimuli (100 μs, 15–90 V) were generated by a stimulator (Master 8) and delivered via a stimulus isolation unit (AMPI Iso-flex) to evoke either single events or train-responses at different frequencies. Voltage clamp measurements were done from V_hold = −71 mV, (Nerlich et al., 2014), except in cases where the amplitudes of the small asynchronous events were increased for precise event detection by holding the cell at −81 mV (Figure 5). To isolate glycine- and GABA_A receptor mediated signals, a pharmacological inhibition of glutamate (50 μM AP-5, 10 μM NBQX) and GABA_Breceptors (3 μM CGP 55845) was performed in all experiments. Offline correction of voltages was done for junction potentials of 11 mV. For some experiments, the extracellular calcium concentration in the ACSF was reduced to 1.2 mM in order to decrease the release probability. In such cases, the magnesium concentration was simultaneously increased to 1.8 mM to maintain the concentration of divalent cations.

The recordings were acquired using a Multiclamp 700B amplifier (Molecular Devices). The mean capacitance of the cells was 23.15 ± 4.34 pF (mean ± SD, n = 52). The average series resistance was 11.25 ± 1.56 MΩ (n = 52), which was compensated by 50% to a remaining Rs of 3–7 MΩ. During experiments the series resistance changed on average by 2.6% (n = 52). Cells with series resistance changes >10% were excluded from analysis. There was no correlation between the IPCS amplitudes and decay time constants (r_s = 0.17, p = 0.22, n = 53). Together with the previously reported lack of correlation between the IPSC amplitudes and rise times (r_s = 0.04, p = 0.78, n = 43, Nerlich et al., 2014), these data rule out the possible contribution of series resistance error to decay time constant measurements. Recorded signals were digitized at 50 kHz and filtered with a 6 kHz Bessel low-pass filter. Data were examined with pClamp 10 software (Molecular Devices) followed by analyses using custom-written Matlab routines (version 8.3, Mathworks, Natick). Mean amplitudes, 10–90% rise times and decay time constants of IPSCs were analyzed from averaged traces (>5 repetitions). For detection of asynchronous release events, the current traces were band-pass filtered between 50 and 1500 Hz using a zero-phase forward and reverse digital IIR filter to remove the decay component (see Figure 5A). Statistical analysis by means of z-test revealed events with current amplitudes exceeding 1.96 times (p < 0.05) the standard deviation of the baseline. To avoid multiple triggers of the same event, all triggers occurring within 5 ms after the preceding event were excluded from the analysis. The results were visually controlled to ensure the correct detection of spontaneous events. The events occurring prior to electrical stimulation (spontaneous IPSCs) were quantified and compared to events emerging after the end of synaptic stimulation of inhibitory inputs. Asynchronous events were clearly visible during the decay phase of the last evoked IPSC. Events detected by the software during the first 20 ms of the current decay were not included into the analysis to avoid false positive results. Data from 3 repetitions for each condition were pooled and presented for each cell.

IPSC decay phase was fitted with mono- or bi-exponential functions based on an increase in adjusted R² values. The weighted τ decay was calculated as τ_wd = (A_fast × τ_fast + A_slow × τ_slow )/(A_fast + A_slow), where A_fast and A_slow are amplitudes at t = 0 and τ_fast and τ_slow are the fast and slow time constants, respectively. In cases of mono-exponential fits, one exponential component was set to 0.

Data sets were compared with the appropriate t-test or analysis of variance (ANOVA). The p-values of multiple comparisons were adjusted by the Dunn-Šidak procedure. Within-subject comparisons were performed by repeated-measures (RM) ANOVA after testing for sphericity using Mauchly test, and Greenhouse-Geissner correction applied as appropriate. ANOVA was used to test for effects of drugs and possible interactions of input frequency and drug superfusion. The changes in asynchronous release evoked by different input frequencies were tested for significance by a z-test, z = (A−BL)/SD_BL, with A being the average number of events/bin 20–60 ms after stimulation, BL the mean of the baseline (1 s prior to stimulation = spontaneous IPSC level) and SD_BL the standard deviation of the baseline. All data are reported as mean ± SEM, unless otherwise stated.

SBCs exhibit slow inhibitory synaptic decays (Figure 1A) mediated by GlyR and GABA_AR (gerbil: spontaneous IPSCs: τ_wd = 16.7 ± 1.2 ms, synaptically evoked IPSCs τ_wd = 23.7 ± 5.3 ms, Nerlich et al., 2014; mouse: spontaneous IPSCs: τ_wd = 8.75 ± 0.6 ms, synaptically evoked IPSCs τ_wd = 11.1 ± 0.8 ms, Xie and Manis, 2013). Both the glycinergic and the GABAergic components were observed in all pharmacology experiments acquiring IPSCs with suprathreshold stimulation to evoke reliable responses (see also Nerlich et al., 2014). Notably, the pharmacologically isolated glycinergic and GABAergic single events were previously shown to have similar kinetics (Nerlich et al., 2014), thus arguing against the hypothesis that in SBCs the fast decay component can be accounted to glycine and the slow component to GABA. In the present study we investigated the underlying mechanism determining such inhibitory current kinetics. The decay phase of synaptically evoked single IPSCs was best fit with a bi-exponential function with a fast decay time constant τ_fast = 8.7 ± 2.1 ms, a slow decay time constant τ_slow = 38.9 ± 7.2 ms and their respective relative amplitudes A_fast = 0.47 ± 0.06 and A_slow = 0.53 ± 0.06. The resulting average weighted decay time constant for 20 SBCs was τ_wd = 24.7 ± 5.4 ms (mean ± SD). The synaptic current decays were progressively slower after repetitive stimulation of inputs and with increasing input frequencies (Figures 1B,D). Also, the relative amplitude of the slow exponential component increased with input rates (Figure 1C; A_slow single = 0.53 ± 0.06, 100 Hz = 0.56 ± 0.03; 333 Hz = 0.95 ± 0.03, single vs. 100 Hz p < 0.05; 100 Hz vs. 333 Hz p < 0.001, n = 20, RM ANOVA). Complementary decrease in amplitude of the fast component occurred over the same range of stimuli (A_fast single = 0.47 ± 0.06, 100 Hz = 0.44 ± 0.03, 333 Hz = 0.05 ± 0.03). The slow decay time constant was prolonged substantially at 100 Hz and 333 Hz (Figure 1D; τ_slow single = 38.9 ± 1.6 ms, 100 Hz = 87.1 ± 5.0 ms, 333 Hz = 76.3 ± 3.3 ms, n = 20, p < 0.001 for single vs. 100 Hz and 333 Hz, RM ANOVA). At 333 Hz, the decay phase was best fit with a mono-exponential function excluding the fast exponential component in 75% of recorded SBCs (in these cells A_fast was set to 0). Due to the rare occurrence of τ_fast at 333 Hz (only in 5 out of 20 cells), this parameter was statistically compared only for the single and 100 Hz stimulation. The τ_fast was prolonged at 100 Hz compared to single pulse stimulation (τ_fast single = 7.1 ± 0.6, 100 Hz = 21.4 ± 3.0, n = 20, single vs. 100 Hz p < 0.001, paired t-test). Activity-dependent change of τ_fast, τ_slow and the respective amplitudes resulted in a prolongation of τ_wd with increasing input frequency (Figure 1D; τ_wd single = 24.7 ± 1.2 ms, 100 Hz = 58.8 ± 2.8 ms, 333 Hz = 74.2 ± 3.5 ms, n = 20, p < 0.001 for all comparisons, RM ANOVA). However, the longer decays were not associated with larger IPSC amplitudes (τ_wd single = 22.8 ± 0.8 ms, 50th 100 Hz = 61.8 ± 2.5 ms, n = 25, p < 0.001, paired t-test; IPSC amplitude single = 0.56 ± 0.5 nA, 50th 100 Hz = 0.55 ± 0.04 nA, n = 25, p = 0.81, paired t-test), thus suggesting that activity-dependent synaptic mechanisms, rather than series resistance errors cause the decay time prolongation. Together, these data indicate that the increase in input rates increases the transmitter quantity in the cleft which particularly prolongs the τ_slow through transmitter rebinding.

Studies at the calyx of Held-MNTB principal neuron synapse revealed that in vivo release probability may be approximated in slice recordings by using 1.2 mM Ca²⁺ in the extracellular solution (Borst, 2010). The dependance of the IPSC decay times on the release probability was shown at other auditory synapses (Balakrishnan et al., 2009; Tang and Lu, 2012). Although the in vivo release probability for the endbulb of Held-SBC synapse remains to be elucidated, we still addressed the question, whether the slow decay of inhibitory currents measured in SBCs is due to an increased release probability in the slice recordings. For this, the weighted decay time constant was measured from the last event within trains consisting of different numbers of pulses and the results were compared for 1.2 mM and 2 mM external calcium concentration (Figure 1F). Single IPSCs and short trains (10 pulses at 100 Hz) revealed faster decay time constants under the lower release probability condition. However, with increasing stimulus number and frequency, the weighted decay time constant of the last pulse in the train was similar between the two calcium conditions, (Figure 1F; main effect calcium p = 0.04, 1.2 vs. 2 mM: single, 100 Hz 10th IPSC p < 0.05, 100 Hz 50th IPSC p = 0.72, 333 Hz 10th p = 0.27 and 50th IPSC p = 0.33, n = 11, RM ANOVA). The slower IPSC at the higher release probability could either be due to transmitter pooling or to multivesicular release. The latter mechanism could explain the data for the single and short IPSC trains. The reduction of extracellular calcium to 1.2 mM decreased the IPSCs amplitudes, especially at the onset of a 100 Hz train (50 pulses) (Figure 1E). However, at the end of the train the baseline IPSC amplitudes were similar to those measured under 2 mM [Ca²⁺]_o (IPSC amplitudes 2 mM vs. 1.2 mM: onset = 1st IPSC, p < 0.001; maximum = mean 6–8th IPSC vs. mean 10–12th IPSC, p < 0.01; end = mean 48–50th IPSC, p = 0.88, n = 6, RM ANOVA). Thus, the release probability is unlikely to play a role for longer trains of stimuli or high frequency stimulation pointing to transmitter pooling as a mechanism contributing to slow decays.

Recruitment of nearby inputs can lead to spillover of transmitter to remote synaptic or extrasynaptic sites, and thereby to intersynaptic pooling. Thus, it is conceivable that the slow IPSC decay in SBCs is caused by ongoing rebinding of transmitter and activation of extrasynaptic receptors, as shown earlier (Balakrishnan et al., 2009; Tang and Lu, 2012). To determine whether synaptic recruitment influences the kinetics of inhibition in SBCs, IPSCs were evoked with increasing stimulus intensities (Figure 2A). The low intensity was set slightly above the threshold with IPSC amplitudes reaching 38.5 ± 16% (mean ± SD, n = 7) of the maximal amplitude evoked by high stimulation intensity. With an increase in stimulus intensity, the amplitude of a single evoked IPSC and the last IPSC in a 100 Hz train increased threefold (Figure 2B; low vs. high intensity: single, n = 7, p < 0.01; 100 Hz 10th IPSC, n = 5, p < 0.01, paired t-test). Furthermore, the weighted decay time constants of single and repetitive IPSCs were prolonged at high stimulus intensities (Figure 2C; τ_wd single: low = 21.6 ± 0.6 ms, high = 26.3 ± 0.6 ms, n = 7, p < 0.01, 100 Hz 10th IPSC: low = 35.4 ± 0.8 ms, high = 46.0 ± 0.8 ms, n = 5, p < 0.01, paired t-tests). On the other hand, the rise time of single IPSCs was not dependent on stimulus intensity, suggesting the recruitment of nearby synaptic inputs (Figure 2D; low = 0.44 ± 0.03 ms, high = 0.46 ± 0.03 ms, n = 7, p = 0.79, paired t-test). As we neither observed a build-up, nor a run-down of IPSC amplitudes during repetitions at a given stimulus intensity, it is unlikely that the number of activated inputs changed. Therefore, these data suggest that the transmitter quantity substantially contributes to slow inhibitory kinetics.

If transmitter spillover and its clearance from the synaptic cleft shape the IPSC decay in an activity-dependent manner, the postsynaptic currents should be sensitive to a blockade of re-uptake transporters (Otis et al., 1996; Balakrishnan et al., 2009; Tang and Lu, 2012). Bath application of the glycine transporter 2 (GlyT2) inhibitor ORG 25543 (20 μM, Bradaïa et al., 2004; Balakrishnan et al., 2009) slowed down the decay of both single and train IPSCs (Figures 3A,B; n = 6, RM ANOVA). The fast decay time constant, the respective amplitudes of the exponential components and the IPSC rise time were not affected by the glycine re-uptake block (n = 6, control vs. +ORG, τ_fast: p = 0.15; A_slow and A_fast: p = 0.37, RM ANOVA, rise time: p = 0.99, paired t-test). Therefore, it can be concluded that the τ_wd prolongation (Figure 3B) is caused by the significantly longer τ_slow (Figure 3B). Given the similar IPSC decays of the isolated glycinergic and GABAergic single events (Nerlich et al., 2014), the τ_fast probably describes the intrinsic kinetics of receptor activation and de-activation, whereas the transmitter clearance, relief from saturation and delayed release determine τ_slow.

In addition to the sensitivity to glycine re-uptake, the IPSC decay phase was also shaped by GABA clearance. Simultaneous inhibition of the respective neuronal- and glial-GABA transporters with 20 μM NO 711 (Szabadics et al., 2007; Tang and Lu, 2012) and 100 μM SNAP 5114 (Keros and Hablitz, 2005; Song et al., 2013) extended the weighted decay time constant of the last IPSC in 100 Hz trains. Again, the prolongation of τ_slow during re-uptake blockade underlie the slowing of train IPSCs (Figures 3C,D; n = 8, control vs. +NO/SNAP, τ_slow: p < 0.001, τ_wd: p < 0.01, RM ANOVA). Notably, the GABA uptake is apparently only contributing to the decay during ongoing activity, as single evoked IPSC decays were not affected (p > 0.45) (Figure 3D). Similar to the effects seen after blockade of glycine re-uptake, GABA re-uptake inhibition did not change the fast decay time constants, the respective amplitudes of the exponential components and the IPSC rise times (n = 8, control vs. +NO/SNAP, τ_fast: p = 0.11, A_slow and A_fast: p = 0.43, RM ANOVA, rise time: p = 0.40, paired t-test). Together, these data suggest that glycine rebinding caused by transmitter pooling due to slow clearance is an important factor in shaping the inhibitory current profile. On the other hand, the GABA re-uptake only contributes at high stimulus input rates to the decay kinetics, when it presumably accumulates in the synaptic cleft.

To investigate transmitter rebinding and the possible contribution of spillover to inhibitory kinetics, we assessed the effects of low affinity GlyR and GABA_AR antagonists. The rationale to use weak competitive antagonists to probe for putative remote synapses arises from studies showing that the receptors distant to the release site are likely to encounter a lower transmitter concentration during a synaptic event (Diamond, 2001; Chen and Diamond, 2002). Hence, the low-affinity competitive antagonists are progressively more effective at receptors facing low transmitter concentrations through spillover (Overstreet and Westbrook, 2003; Szabadics et al., 2007; Balakrishnan et al., 2009; Tang and Lu, 2012). Bath application of a high concentration of SR 95531 (200 μM), employed as a low affinity antagonist of GlyR (Wang and Slaughter, 2005; Beato, 2008; Balakrishnan et al., 2009), reduced the IPSC amplitudes considerably and accelerated the weighted decay time constant of single and repetitive IPSCs (Figures 4A–C; n = 6, RM ANOVA). Low concentrations of SR 95531 (10 and 20 μM) specifically blocked GABA_A receptors on SBCs and reduced the IPSC amplitudes by 18 ± 2% and 16 ± 3%, respectively (Nerlich et al., 2014). At the concentration of 200 μM, a 76 ± 5% reduction of the IPSC amplitude was observed (Figure 4D; 200 μM vs. 10 μM (n = 6), p < 0.001; 200 μM (n = 6) vs. 20 μM (n = 11), p < 0.001, ANOVA). This result is consistent with an additional antagonistic action of a high SR 95531 concentration at GlyR, as opposed to specific GABA_AR blockade at concentrations of 10 and 20 μM (Nerlich et al., 2014). To further confirm the competitive antagonistic action of SR 95531 at GlyR, the glycine concentration in the cleft was increased by co-applying the glycine re-uptake inhibitor ORG 25543. This partially reversed the long τ_wd confirming that the current decay is regulated by the amount of available glycine (Figure 4D; change of τ_wd: +20 μM SR = −8.1 ± 2.4%, n = 13; +200 μM SR = −35.3 ± 2.5%, n = 6; +200 μM SR+ORG = −21.2 ± 5.5%, n = 6; 20 μM vs. 200 μM SR p < 0.001, 200 μM SR vs. 200 μM SR+ORG p = 0.01, ANOVA). As the uptake blockers reveal transmitter spillover by increasing its quantity and enabling distant receptors to encounter a lower concentration of an agonist (Chen and Diamond, 2002; Thomas et al., 2011), our data showing a decay time prolongation under ORG 25543 are consistent with glycine spillover.

Inhibitory currents in SBCs show an activity-dependent depression of the IPSC peak amplitudes (I_peak, Figure 4E). Yet, under the low affinity GlyR antagonist (200 μM SR 95531), the peak amplitudes showed a weaker depression which in some cells even changed into facilitation (Figures 4E,F). This result is consistent with a relief from receptor saturation and/or desensitization (Chanda and Xu-Friedman, 2010). Thus, the peak IPSC depression at high input rates is presumably caused by postsynaptic receptor saturation and/or desensitization, rather than presynaptic short-term plasticity.

Contrary to glycine signaling, GABA is apparently not activating nearby synapses. Even at high stimulation rates, the low affinity GABA_AR antagonist TPMPA (Szabadics et al., 2007; Tang and Lu, 2012) neither affected the IPSC decay nor the amplitudes, suggesting that the postsynaptic GABA_AR could be tightly coupled to release sites of the transmitter and, thus, encounter consistently high GABA concentration (Figures 4G–I; control vs. TPMPA τ_wd: single p = 0.97; 100 Hz 10th IPSC p = 0.62; IPSC amplitude: single p = 0.67; 100 Hz 10th IPSC p = 0.19; n = 8, RM ANOVA). In summary, the apparently low uptake capacity of glycine and GABA transporters slows the decay of IPSCs, thereby enabling spillover of glycine. While we found no evidence for spillover of GABA, the transmitter quantity and intrinsic receptor properties probably account for the slow GABAergic transmission.

A further mechanism putatively contributing to IPSC decay time in SBCs could be an activity-induced desynchronization of vesicle release, as shown for several inhibitory synapses (Lu and Trussell, 2000; Hefft and Jonas, 2005; Tang and Lu, 2012). To test this hypothesis, high frequency stimulation (100 and 333 Hz) of inhibitory inputs to SBCs was employed to evoke small asynchronous events in the decay phase following the IPSC trains (Figure 5A). The quantity of asynchronous IPSCs increased with stimulus duration (Figures 5B,E) and frequency (Figures 5C,F). To compare the spontaneously occurring IPSCs (sIPSC, without input stimulation) and delayed IPSCs following repetitive input stimulation, the events were detected during 1 s before synaptic stimulation and 2 s after the last evoked IPSC (see methods). The number of asynchronous events increased significantly after the 333 Hz stimulation (Figure 5E summary data for 4 cells and respective presentation of 10, 25 and 50 stimuli at 333 Hz z values > 10.1, p < 0.001, z-test), compared to the rate of sIPSC before stimulation (Figure 5D; mean # of sIPSCs/20 ms bin = 0.56 ± 0.08, n = 4). This result suggests the occurrence of activity induced asynchronous events in the decay phase of IPSC trains. Detailed analyses revealed the time course of incidence of asynchronous events which was best fit with a mono-exponential function relaxing back to the resting state. The following parameters were used to quantify the delayed release: initial amplitude (peak incidence of asynchronous events (A), an exponential time constant (τ) and a steady state value (c) (Figure 5E; fit comparison 10 vs. 25 vs. 50 stimuli p < 0.001, F-test). The peak incidence of asynchronous events after stimulation at 333 Hz increased almost 2-fold by extending the train from 10 to 50 pulses (A_#eIPSC 95% CI [lower, upper]: A₁₀ = 10.6 [9.1, 12.2] events, A₂₅ = 12.9 [11.7, 14.0] events, A₅₀ = 18.8 [17.3, 20.3] events, 10 vs. 25 vs. 50 stimuli p < 0.05). The exponential time constant was also prolonged (τ_#eIPSC 95% CI [lower, upper]: τ₁₀ = 26.5 [20.5, 37.4] ms, τ₂₅ = 54.1 [46.8, 64.4] ms, τ₅₀ = 79.0 [69.7, 91.1] ms, 10 vs. 25 vs. 50 stimuli p < 0.05), whereas the steady state values were similar to the mean sIPSC levels 1 s before stimulation (c_{# eIPSC} 95% CI [lower, upper]: c₁₀ = 0.63 [0.52, 0.75] events/bin, c₂₅ = 0.43 [0.33, 0.53] events/bin, c₅₀ = 0.73 [0.59, 0.87] events/bin; sIPSCs ± SEM: prior to 10 eIPSCs = 0.54 ± 0.12 sIPSCs/bin, prior to 25 eIPSCs = 0.48 ± 0.10 sIPSCs/bin, prior to 50 eIPSCs = 0.74 ± 0.10 sIPSCs/bin, p > 0.05).

The rate of delayed release was not only dependent on the stimulus duration, but also on the stimulation frequency (Figures 5C,F summary data for 6 cells). Figure 5F, shows that presynaptic activity determines the quantity and duration of asynchronous release (A or τ, 95% CI [lower, upper]: A_100Hz = 12.4 [10.8, 13.9] events, A_333Hz = 28.3 [26.0, 30.5] events, p < 0.05; τ_100Hz = 58.6 [48.3, 74.7] ms, τ_333Hz = 78.5 [69.3, 90.7] ms, p < 0.05, n = 6, fit comparison 100 Hz vs. 333 Hz p < 0.001, F-test).

The putative cause for the delayed asynchronous transmitter release is the accumulation of presynaptic calcium during high frequency IPSC trains. This can be experimentally enhanced by replacing the extracellular calcium with strontium, or alternatively, the effect can be reduced by applying a cell permeable calcium chelator that accelerates the decay of presynaptic calcium transients (Goda and Stevens, 1994; Atluri and Regehr, 1996, 1998; Lu and Trussell, 2000; Xu-Friedman and Regehr, 2000; Hefft and Jonas, 2005; Best and Regehr, 2009; Tang and Lu, 2012). To test whether the delayed asynchronous events can prolong the IPSCs decay times, their incidence was increased by replacing 2 mM extracellular calcium by 8 mM strontium (Figures 6A–C). In addition to a higher frequency of asynchronous events (A) shortly after the 100 Hz stimulation (10 eIPSCs), strontium also caused a prolongation of the time course (τ) of the last IPSC (Figures 6B,C; n = 6, mono-exponential fit comparison: p < 0.001, F-Test; A or τ 95 % CI [lower, upper]: A_Ca²⁺ = 6.8 [5.7, 7.9] events, A_Sr²⁺ = 21.2 [19.4, 23.0] events; τ_Ca²⁺ = 13.5 [9.4, 23.9] ms, τ_Sr²⁺ = 47.2 [40.9, 55.8] ms, Ca²⁺ vs. Sr²⁺ p < 0.05). Consistent with the observation that asynchronous release strongly depends on synaptic activity (Figure 5), 8 mM strontium was more potent in prolonging the IPSC τ_wd following higher stimulation rate and longer stimulation (Figure 6C; Ca²⁺ vs. Sr²⁺ 100 Hz 10th p < 0.05; 333 Hz 50th p < 0.01, n = 6, RM ANOVA). Notably, single IPSC decays were not affected by the Ca²⁺ replacement (p = 0.8). These results suggest that the slow decay of IPSCs is indeed shaped by the asynchronous release which in turn is determined by the rate of presynaptic activity.

To further address this issue, the contribution of the delayed vesicle release to IPSC decay times was determined through reduction of presynaptic calcium with the calcium chelator EGTA-AM (100 μM) (Figures 6D–G). The IPSC amplitudes strongly decreased under EGTA-AM by 55 ± 8% for single IPSC, 71 ± 4% for the 10th IPSC at 100 Hz and 55 ± 10% for 50th IPSC at 333 Hz, presumably due to a reduction in the peak Ca²⁺ concentration in the presynaptic terminal (Atluri and Regehr, 1996). The significant increase of asynchronous events observed in control condition after 333 Hz stimulation (50 IPSCs) vanished completely under EGTA-AM (Figure 6E; change of asynchronous events after stimulation compared to baseline level before stimulation: control z = 29.2, p < 0.001; +EGTA-AM z = −1.01, p = 0.16, z-test). Moreover, the weighted decay time constant was shortened by 30 ± 3% regardless of stimulus condition (Figure 6F; EGTA-AM effect vs. frequency p = 0.9, n = 5, RM ANOVA) and transmitter type (glycine, GABA or both) (Figure 6G; EGTA-AM effect vs. transmission type p = 0.59, n = 5 for each condition, ANOVA). Together, these data suggest that both glycine and GABA contribute to the delayed release and thereby shape the inhibitory current profile in an activity-dependent manner.

The SBCs of the cochlear nucleus are the first synaptic center where primary auditory input from the cochlea is integrated with a higher-order, acoustically-evoked inhibition to preserve or improve temporal precision on the sub-millisecond scale (Gai and Carney, 2008; Dehmel et al., 2010; Kuenzel et al., 2011). Such synaptic inhibition is likely to contribute to the temporal synchronization of AP discharges to a particular phase of a low frequency tone burst (Joris et al., 1994; Paolini et al., 2001; Dehmel et al., 2010). Saying this, it should not be disregarded that also other previously discussed mechanisms (Nerlich et al., 2014), such as coincidence of presynaptic inputs also add to the synchronicity of postsynaptic SBC discharges (Kuhlmann et al., 2002; Xu-Friedman and Regehr, 2005). With respect to the inhibitory effects, it was shown that an activity-dependent regulation of inhibitory strength mediated trough a slow glycine-GABA transmission adjusts the fidelity at the endbulb of Held synapse towards fast rising and large EPSPs (Kuenzel et al., 2011; Xie and Manis, 2013; Nerlich et al., 2014). The present data further demonstrate that the inhibitory strength crucially depends on the presynaptic firing rate, which is of particular physiological importance given the weak inhibition at lower sound intensities, i.e., low firing rates (Kuenzel et al., 2011). Following strong inhibitory stimulation, slow glycine and GABA clearance from the synaptic cleft appears to enable ongoing rebinding of transmitters and intersynaptic pooling of glycine. A similar mechanism was previously shown to mediate prolonged inhibition of granule cells in the rat DCN (Balakrishnan et al., 2009) and of the nucleus laminaris neurons in the chick auditory brainstem (Tang and Lu, 2012). Several lines of evidence suggest that both the GlyT2 and the GABA transporters GAT1/3 contribute to the kinetics of activity-dependent inhibition at SBCs. Particularly, the slow decay phase (τ_slow) was affected by increasing stimulus frequencies, suggesting longer availability of transmitters. Similar τ_slow prolongation was also observed in the presence of glycine and GABA transporter antagonists. As the respective slowly decaying current is most likely due to slow transmitter clearance (Otis et al., 1996; Williams et al., 1998), our data are consistent with transporter saturation during higher neuronal activity. In line with this, the vesicular inhibitory amino acid transporter (VGAT), which depends on the supply of cytosolic transmitter to enable synaptic corelease of glycine and GABA (Wojcik et al., 2006), is apparently not the rate limiting factor for efficient refilling of inhibitory vesicles (Apostolides and Trussell, 2013).

At mixed glycine-GABA synapses, several factors determine the characteristics of the postsynaptic response: (i) the glycine/GABA ratio in synaptic vesicles which is not only determined by the higher VGAT affinity for glycine than for GABA (McIntire et al., 1997; Bedet et al., 2000); but also crucially depends on the availability of glycine through GlyT2 activity (Rousseau et al., 2008); and (ii) the postsynaptic receptor expression levels, clustering, and localizations (Todd et al., 1996; Dugué et al., 2005). In our recent study, we described a relative increase of the GABAergic component from 5 to 12% of the mixed IPSC amplitude during ongoing synaptic activity (Nerlich et al., 2014). When glycine and GABA were puff-applied (equimolar concentrations of 500 μM), the current amplitude ratio was ~3:2 (1.7:1.2 nA), indicating that GABA_AR availability is probably not the rate limiting factor for restricted GABAergic contribution to the IPSC. Presently, we found no evidence for GABA spillover to distant receptors that could account for the activity-dependent increase in the GABAergic proportion. Thus, the slow glycine clearance by GlyT2 and its low intracellular availability may possibly explain the progressively higher GABAergic signaling at in vivo-like firing rates.

Compared to the events elicited in 1.2 mM [Ca²⁺]_o, IPSCs measured under the standard 2 mM [Ca²⁺]_o condition were found to be slower after shorter stimulus trains or at lower input rates, possibly indicating a contribution of multivesicular release. However, longer or high frequency stimulation (50 pulses at 100 Hz, 10 or 50 pulses at 333 Hz) evoked IPSC of comparable amplitudes towards the end of the train and the similar decay kinetics of the last event. This suggests that prolonged neuronal activity at physiological-like rates leads to a steady state transmitter level in the cleft, independent of the initial release probability. The data also argue against the prominent receptor desensitization, because its effect would render IPSCs faster with increasing transmitter concentrations at higher rates (Jones and Westbrook, 1996), which was not observed in our experiments.

Glycine receptor saturation and spillover onto nearby or distant receptors probably shapes the IPSC kinetics at physiologically relevant firing rates. Due to the lack of effect of the low-affinity GABA_A antagonist, we conclude that the respective receptors are probably tightly coupled to the release sites and unlikely to saturate. In line with this notion, is the lack of GABA_A α6 and δ subunit expression on SBCs (Campos et al., 2001) which were shown to constitute the extrasynaptic receptors (Nusser et al., 1998; Kullmann et al., 2005). The slow IPSC kinetics measured at SBCs contrast with respective data for other inhibitory auditory synapses with decay time constants <5 ms, in which GABA is either not engaged or plays a minor role in determining the response decay time (Awatramani et al., 2004; Magnusson et al., 2005; Chirila et al., 2007; Couchman et al., 2010). As the SBC IPSCs progressively resembled the kinetics of pharmacologically isolated GABAergic events at increasing input frequencies, the mechanisms engaged with GABA release are likely to regulate the duration of inhibition at physiologically relevant rates (Nerlich et al., 2014). One possible contributing mechanism, though not a focus of the present study, is the kinetics of the GABA_A receptor itself. The α3 subunit, linked to slow kinetics, deactivation- and desensitization rates (Verdoorn, 1994; Gingrich et al., 1995) is coexpressed with the subunits α1, α5, β3 and γ2L in the rat AVCN (Campos et al., 2001). In olfactory bulb neurons of juvenile rats, the deactivation kinetics of GABA-evoked mIPSCs can span a τ_wd range of 3–30 ms through the expression of different compositions of the fast α1 and the slow α3 subunits in the receptor heteromers (Eyre et al., 2012). Therefore, it is conceivable that the specific composition of GABA_A subunits in SBCs partially contributes to an increase in τ_wd which ranged between 20–90 ms in an activity-dependent manner. Notably, even the isolated glycinergic spontaneous- and evoked-IPSCs exhibit slow synaptic decays (Xie and Manis, 2013, 2014; Nerlich et al., 2014). This may be surprising, given the general developmental down-regulation of the GlyR α2 subunit, associated with slower receptor kinetics (Veruki et al., 2007) in the cochlear nucleus and in the superior olivary compex (Sato et al., 1995; Friauf et al., 1997). However, the developmental replacement by the α1 subunit in the AVCN seems in the AVCN, as low levels of α2 mRNA were found up to the third postnatal week (Piechotta et al., 2001). Our data substantiate the hypothesis of a persistent α2 subunit expression throughout adulthood by showing that single pulse stimulation evokes IPSCs of similar τ_wd for pharmacologically isolated glycinergic, GABAergic, and mixed glycine-GABA events (Nerlich et al., 2014). Here, further corroboration is provided by showing comparable slow kinetics of the spontaneous IPSCs and IPSCs synaptically elicited in low release probability conditions. In both cases, the amount of released transmitters is presumed low, thus reducing the contribution of activity-dependent mechanisms. Although our results argue for a role of slow GlyR and GABA_AR, this still seems to be only an additional mechanism shaping the overall kinetic profile.

Asynchronous (or delayed) transmitter release is another mechanism to generate a long-long lasting inhibition in the brain (Lu and Trussell, 2000; Hefft and Jonas, 2005; Tang and Lu, 2012). The ongoing synaptic activity can lead to Ca²⁺ accumulation in the presynaptic terminal, thereby causing the loss of coupling between the presynaptic AP and the release machinery (Chen and Regehr, 1999). Our data from the present and previous study (Nerlich et al., 2014) consistently show the activity-dependent change of release mode. The individual IPSCs were clearly segregated at 100 Hz, indicating synchronized quantal release, whereas the 333 Hz stimulation evoked a large plateau current caused by asynchronous release. Notably, the delayed asynchronous release events, mediated by both glycine and GABA, were observed at both frequencies during the decay phase of the last IPSC. The prominent effect of the slow calcium chelator EGTA-AM in our experiments, suggests a loose coupling between presynaptic Ca²⁺ channels and the Ca²⁺ sensor, possibly in a form of microdomains (Meinrenken et al., 2002; Eggermann et al., 2012; Vyleta and Jonas, 2014). The Ca²⁺ dependance of release machinery was, however, not in focus of the present study. Alongside transmitter rebinding and spillover, asynchronous release is a major mechanism contributing to activity-dependent changes in inhibitory duration.

Neurons in the auditory brainstem generate APs with an extraordinary temporal accuracy required for the computation of sound source location (for review see, Grothe et al., 2010). In the binaural nuclei of the superior olivary complex, the fast and mainly glycinergic inhibition may contribute by providing high precision phasic suppression of excitatory conductance (Awatramani et al., 2004; Magnusson et al., 2005; Chirila et al., 2007; Couchman et al., 2010; Roberts et al., 2013, 2014; Myoga et al., 2014). The monaural neurons in the mammalian cochlear nucleus, such as bushy cells of the AVCN and the granule cells of the DCN, on the other hand, utilize slow inhibition characterized by an activity-dependent conductance build-up (Balakrishnan et al., 2009; Xie and Manis, 2013). In vivo, the onset of acoustically evoked inhibition on SBCs is delayed compared to the excitation (Kuenzel et al., 2011; Nerlich et al., 2014) which cannot be solely explained by the polysynaptic inhibitory pathway causing a delay of just a few milliseconds (Smith and Rhode, 1989; Wickesberg and Oertel, 1990; Saint Marie et al., 1991; Ostapoff et al., 1997; Campagnola and Manis, 2014). Rather, the new data suggest that the slow time course of inhibition is predominantly determined by the receptor kinetics and transmitter clearance during short stimuli, whereas long duration or high frequency stimulation additionally engage spillover of glycine and asynchronous release of both glycine and GABA to prolong IPSCs. The contribution of these synaptic mechanisms can explain the slow onset of inhibition observed in vivo. With increasing sound intensity, the dynamic adjustment of inhibitory potency through synergistic glycine-GABA signaling (Nerlich et al., 2014) would ensure effective integration with strong and coincident excitatory inputs leading to non-monotonic rate-level functions and improved temporal precision of the SBC output (Kopp-Scheinpflug et al., 2002; Dehmel et al., 2010; Kuenzel et al., 2011). Consistent with this hypothesis, a modeling study of SBC inhibition with fast IPSC kinetics as those measured in AVCN T-stellate cells impaired the temporal precision of spiking (Xie and Manis, 2013). Thus, the activity-dependance of slow inhibition seems to be a critical factor for precise temporal processing in SBCs.