DL-TBOA induces membrane depolarization and inward current mediated by ionotropic glutamate receptors.

A. Representative current-clamp recording from a T-stellate cell during bath application of DL-TBOA (200 μM) showing rapid onset of spontaneous firing followed by depolarization block. B. Summary plot of the membrane depolarization induced by DL-TBOA (25.5 ± 4.1 mV, n = 5). C. Representative voltage-clamp experiment showing a large inward shift in holding current induced by DL-TBOA. Subsequent application of ionotropic glutamate receptor antagonists (GYKI 53655, 50 μM; MK-801, 5 μM) significantly reduced this holding current. The addition of group I mGluR antagonists (JNJ and MPEP) produced no further reduction. D. Summary of holding current under various conditions (in pA: DL-TBOA, –250.80 ± 65.10; GYKI + MK-801, –73.25 ± 27.10; JNJ+MPEP, –26.37 ± 8.301; DL-TBOA vs GYKI + MK-801, p = 0.022, n = 8). E. Percentage block of DL-TBOA induced holding current by ionotropic glutamate receptor. AMPARs account for the majority of the inward current as mGluR inhibition provided no significant additional block (in %: GYKI + MK-801, 76.50 ± 7.47; JNJ + MPEP, 80.98 ± 7.72, p > 0.05, n = 6). F. Representative voltage-clamp recording in which DL-TBOA was applied in the presence of GYKI and MK-801. Prior application of receptor blockers prevented a current response upon DL-TBOA application. Subsequent application of JNJ + MPEP produced no additional change in holding current. G. Summary of holding current measured in the presence of GYKI + MK-801 during DL-TBOA application (–21.22 ± 6.93 pA) and after addition of JNJ + MPEP (–22.38 ± 6.16 pA, p >0.05, n = 8).

T-stellate cells reliably encode stimulus intensity and presynaptic firing rate.

A, B. Representative current-clamp recordings from a T-stellate cell showing sustained firing during a 600-ms depolarizing current injection of +150 pA (A) and +250 pA (B). C. Frequency-intensity (F–I) plot showing that firing rate increases linearly with depolarizing current injection (n = 16). D, E. Representative current-clamp recordings from a T-stellate cell in response to a train of AN stimulation at 10 Hz (D) and 200 Hz (E). F. Plot showing the linear input-output relationship between presynaptic stimuli and spike output across stimulus frequencies (n = 6). Inhibitory glycinergic and GABAA receptors were blocked using selective antagonists in all recordings.

Glutamate transporters are required for precise encoding of auditory nerve activity in T-stellate cells.

A, B. Representative current-clamp recordings from a T-stellate cell in response to a 25-pulse train AN stimulation at 10 Hz under control conditions (A) and in the presence of DL-TBOA (25–50 μM). Raster plots below show spike timing across trials. C. Cumulative spike output at 10 Hz for the same cell in A and B. Application of DL-TBOA increased spike output compared to control. D, E. Representative current-clamp recordings and raster plots of a T-stellate cell in response to a 25-pulse, 200 Hz AN stimulation under control conditions (D) and in DL-TBOA (E). DL-TBOA led to prolonged spiking following AN stimulation. F. Cumulative spike output at 200 Hz for the same cell in D and E. Application of DL-TBOA increased spike output compared to control. G, H. Summary of cumulative spike output plot at 10 Hz (G) and 200 Hz (H) (n = 6). I. Plot of spike output/input ratio across frequencies. Partial transporter blockade significantly increases this ratio across all tested frequencies (p < 0.05, n = 6). J, K. Summary plot showing total spikes during the stimulus train (J) and after the stimulus train (delayed spikes, K). DL-TBOA significantly increased spiking both during and after the stimulus (10 Hz, control, 0 ± 0, TBOA, 1.12 ± 0.62; 30 Hz, control, 1.33 ± 0.42, TBOA, 24.10 ± 9.46; 100 Hz, control, 0.42 ± 0.16, TBOA, 55.24 ± 17.51; 200 Hz, control, 0.89 ± 0.33, TBOA, 67.06 ± 19.99; 300 Hz, control, 0.87 ± 0.43, TBOA, 64.34 ± 21.46, n = 6, paired-t-test, *p < 0.05).

Recruitment of auditory nerve (AN) inputs to T-stellate cells.

Shocks of increasing duration delivered to AN root evoked excitatory postsynaptic currents (EPSCs) that increased in a graded manner with stimulus duration. A. Representative voltage-clamp recording of EPSCs in a T-stellate cell evoked by AN root stimulation. EPSC amplitudes increased in a graded manner with increasing stimulus duration. B. Total synaptic charge plotted as a function of stimulus duration for the cell shown in A. Discrete increments in synaptic charge reflect the recruitment of additional AN fiber inputs.

Glutamate transporter block enhances the excitability of T-stellate cells to auditory nerve input and prolongs delayed firing.

A, B. Example current-clamp recordings from T-stellate cells during AN root stimulation (25 pulses, 200 Hz) with increasing shock duration. A. Under control conditions, brief shocks (50-75 μs) evoked mostly subthreshold excitatory EPSPs, while longer duration shocks (200 μs) generated suprathreshold responses and spikes largely confined to the period of the stimulus train. B. In the presence of DL-TBOA (25-50 μM), even brief shocks reliably evoked spikes, and longer stimuli generated more spikes during the train and for hundreds of milliseconds after the end of the stimulation. C. Total spikes generated during the stimulus plotted as a function of stimulus duration. DL-TBOA shifted the input-output curve to the left, reducing the stimulus duration required for half-maximal firing (ctrl, duration at half-maximal stimulus: control, 0.094 ms; DL-TBOA, 0.071 ms; n = 6 cells, p = 0.0035, paired t-test). D. Number of spikes occurring after the stimulus train (delayed spikes). Post-train spiking was minimal in control but increased significantly in DL-TBOA, especially at longer stimulus durations. E. Duration of delayed activity following AN stimulation. Transporter blockade produced prolonged firing that persisted for hundreds of milliseconds beyond the stimulus period. (n = 6, *p < 0.05).

Glutamate transporters prevent glutamate accumulation during high-frequency AN activity.

A. Example voltage-clamp recordings of excitatory postsynaptic currents (EPSCs) from a T-stellate cell evoked by a single AN shock followed by a 45-pulse train at 200 Hz. Under control conditions (black), electrical stimulation evoked a fast single EPSC (inset: middle) and a train response with minimal tonic current buildup during the stimulus (inset: right). Moreover, the train EPSC decayed to baseline within milliseconds (C). In the presence of DL-TBOA (red), the tonic current progressively built up throughout the stimulation (inset: right) and the decay of the EPSC train was markedly prolonged (*). These effects were blocked by AMPA receptor antagonist, NBQX (blue). B. Tonic current amplitude measured immediately before each EPSC, plotted as a function pulse number. Under control conditions (open circles), the tonic current reached a steady state, whereas in DL-TBOA, the tonic current progressively increased throughout the train. C. Summary plot showing weighted decay time constant of EPSC train as a function of stimulus number. DL-TBOA significantly slowed the decay time as the number of stimulations in a train increased. D. Total synaptic charge after the train across different conditions. (p * < 0.05, ** < 0.005, *** < 0.0005).

Glutamate does not spillover between auditory nerve inputs.

A. Example voltage-clamp recordings of excitatory postsynaptic currents (EPSCs) from a T-stellate cell evoked by a single AN shock followed by a 45-pulse train at 200 Hz, delivered at weak (minimal) or strong (maximal) stimulus pulse durations before and after DL-TBOA application. Insets show single EPSC (left) and normalized EPSC (right). DL-TBOA did not alter peak amplitude of single EPSCs for a given stimulus duration. When peak-normalized all single EPSCs had similar decay times. B. Stimulus trains with weak (short duration) stimuli showing that enhancement of tonic current and slowed decay time after the train in DL-TBOA. C. As in B, but for strong (long duration) stimuli. D. Tonic current amplitude was normalized to the peak of the first EPSC (EPSC1) in a train, and plotted against pulse number during the train. While DL-TBOA enhanced tonic current, the relative enhancement was not dependent on stimulus strength. Color legend as in panel A. E. The ratio of EPSC1 for strong vs weak stimuli was not changed by DL-TBOA (control, 5.40 ± 1.28; DL-TBOA, 5.77 ± 1.55; p = 0.86, n = 5). F. Decay time (weighted tau) after stimulus trains were prolonged equally by DL-TBOA, regardless of stimulus strength.

Glial and neuronal EAATs clear synaptic glutamate.

A. Voltage-clamp recordings of response to AN stimulus train in control (black), UCPH-101 (30 μM) and DHK (200 μM) (pink), and following the addition of high-affinity blocker TFB-TBOA (2 μM). B. Fold increase in weighted tau after stimulus train ended, for UCPH-101 and DHK and for the subsequent addition of TFB-TBOA (UCPH-101 and DHK, 4.33 ± 1.19, TFB-TBOA, 23.17 ± 4.11, n = 3, p = 0.037). C. Fold change in total charge during the decay phase across these two different conditions (UCPH-101 and DHK, 9.46 ± 5.31, TFB-TBOA, 48.53 ± 4.70, n = 3, p = 0.006)

Synaptic efficacy of EPSPs in VCN bushy cells are not impacted by EAAT block.

A, B. Current-clamp recordings of the response to 100 Hz AN stimulus trains, in control and DL-TBOA solutions, respectively. Spike raster plots beneath traces indicate consistent suprathreshold transmission. C, D. As in A and B, but for 300 Hz stimulus trains. EPSPs later in the trains were subthreshold but again DL-TBOA had little impact on EPSP efficacy. E. Cumulative spike number during 100 Hz train shows no effect of DL-TBOA. No additional spikes occurred after the train ended. F. As in E, but for 300-Hz stimuli. G. Ratio of emitted spikes in bushy cells to number of presynaptic stimuli at 100, 200, and 300 Hz showed no effect of DL-TBOA (100 Hz, control, 0.98 ± 0.01, TBOA, 0.97 ± 0.03; 200 Hz, control 0.87 ± 0.09, TBOA, 0.856 ± 0.11; 300 Hz, control 0.68 ± 0.11, TBOA, 0.68 ± 0.12, n = 8, p > 0.5).

Effects of DL-TBOA on synaptic currents in bushy cells.

A. Voltage-clamp traces of EPSC trains in a bushy cell, with and without DL-TBOA. Asterisk highlights longer decay of current in DL-TBOA. B. Comparison of weighted decay tau after EPSC train for bushy cells (BC) and T-stellate cells (T-st) with and without DL-TBOA. Increase in decay time in bushy cell with DL-TBOA did not reach significance. C. As in B, but for total charge during current decay phase ((p ** < 0.005, *** < 0.0005).