Acid-Sensing Ion Channels Activated by Evoked Released Protons Modulate Synaptic Transmission at the Mouse Calyx of Held Synapse

Abstract

Acid-sensing ion channels (ASICs) regulate synaptic activities and play important roles in neurodegenerative diseases. We found that these channels can be activated in neurons of the medial nucleus of the trapezoid body (MNTB) of the auditory system in the CNS. A drop in extracellular pH induces transient inward ASIC currents (I_ASICs) in postsynaptic MNTB neurons from wild-type mice. The inhibition of I_ASICs by psalmotoxin-1 (PcTx1) and the absence of these currents in knock-out mice for ASIC-1a subunit (ASIC1a^-/-) suggest that homomeric ASIC-1as are mediating these currents in MNTB neurons. Furthermore, we detect ASIC1a-dependent currents during synaptic transmission, suggesting an acidification of the synaptic cleft due to the corelease of neurotransmitter and H⁺ from synaptic vesicles. These currents are capable of eliciting action potentials in the absence of glutamatergic currents. A significant characteristic of these homomeric ASIC-1as is their permeability to Ca²⁺ Activation of ASIC-1a in MNTB neurons by exogenous H⁺ induces an increase in intracellular Ca²⁺ Furthermore, the activation of postsynaptic ASIC-1as during high-frequency stimulation (HFS) of the presynaptic nerve terminal leads to a PcTx1-sensitive increase in intracellular Ca²⁺ in MNTB neurons, which is independent of glutamate receptors and is absent in neurons from ASIC1a^-/- mice. During HFS, the lack of functional ASICs in synaptic transmission results in an enhanced short-term depression of glutamatergic EPSCs. These results strongly support the hypothesis of protons as neurotransmitters and demonstrate that presynaptic released protons modulate synaptic transmission by activating ASIC-1as at the calyx of Held-MNTB synapse.SIGNIFICANCE STATEMENT The manuscript demonstrates that postsynaptic neurons of the medial nucleus of the trapezoid body at the mouse calyx of Held synapse express functional homomeric Acid-sensing ion channel-1a (ASIC-1as) that can be activated by protons (coreleased with neurotransmitter from acidified synaptic vesicles). These ASIC-1as contribute to the generation of postsynaptic currents and, more relevant, to calcium influx, which could be involved in the modulation of presynaptic transmitter release. Inhibition or deletion of ASIC-1a leads to enhanced short-term depression, demonstrating that they are concerned with short-term plasticity of the synapse. ASICs represent a widespread communication system with unique properties. We expect that our experiments will have an impact in the neurobiology field and will spread in areas related to neuronal plasticity.

Keywords: ASIC-1a; calyx of Held; glutamatergic EPSCs; protons; short-term depression; synaptic plasticity.

Figure 1.

ASIC-1as are activated by a decrease of extracellular pH in MNTB neuron. a, H⁺-gated I_ASICs from MNTB neurons in WT mice during transient acidification (4 s) of the extracellular media from pH 7.3 to 5.5 (mean peak amplitude, 2.5 ± 0.5 nA; n = 25), pH 6.0 (mean peak amplitude, 0.94 ± 0.22 nA; n = 20), or pH 6.5 (mean peak amplitude, 0.32 ± 0.04 nA; n = 6). MNTB neurons were whole-cell patch clamped at a holding potential of −70 mV. b, Acidification of the extracellular media induced no current in ASIC1a^−/− mice. Please note the different magnitude in the current calibration bar. c, Activation of ASICs by a drop in pH from 7.3 to 6.0 induced membrane depolarization and triggered action potentials (shown in an expanded timescale in the inset at the right) in MNTB neurons from WT mice. d, In the presence of amiloride (150 μm), a nonspecific ASIC blocker, the amplitudes of I_ASICs triggered by dropping the pH from 7.4 to 6.0 were reduced in 82 ± 4% in WT mice (n = 12). The effect was reversible after washout. e, Effect of PcTx1 (10 nm), a specific inhibitor of ASIC-1a homomers, on ASIC currents. The mean inhibition of I_ASIC peak value (n = 7) was 90 ± 3%.

Figure 2.

Desensitization of ASIC-1as. a, Representative ASIC currents activated by successive 3-s-long acidic puffs of a pH 6.0 solution at 10 s intervals, showing ASIC desensitization (transient ASIC-1a currents with progressively lower peak amplitudes). b, ASIC current amplitudes as a function of time during repetitive activation of ASICs, normalized to the first evoked I_ASIC. Desensitization time constant (τ_D) calculated by fitting to a single-exponential equation was τ_D = 5.7 ± 0.8 s (n = 6). c, After the desensitization of ASIC currents by three successive 3-s-long acidic puffs of pH 6.0 (10 s interval), recovery was determined by returning the pH of the extracellular solution to 7.4 for different time intervals (from 20 to 120 s) and then applying a test 3-s-long puff at pH 6.0. Recovery was quantified as the ratio between the peak current amplitude in response to the test pH 6.0 puff and the first peak current amplitude before desensitization (control). d, Time course of recovery of ASIC current amplitudes after desensitization, fitted by a sigmoid function: I = 1/[1 + exp((t − τ_R)/k)]. The time for half-recovery (τ_R) was 39.5 ± 0.7 s, and the slope k was 10.8 ± 0.7 s (n = 6).

Figure 3.

Voltage dependence of ASIC currents and ASIC permeability to Ca²⁺. a, Representative traces showing I_ASICs activated by a 3-s-long pH drop from 7.3 to 6.5 while MNTB neurons were whole-cell patch clamped at different holding potentials. After returning to pH 7.3, cells were allowed to recover for 2 min. b, Mean I–V plot for ASIC currents activated by a 3-s-long pH drop from 7.3 to 6.5 (n = 6). The detection of calcium transients evoked during the activation of ASIC-1as by H⁺ injection. c, Representative traces of ASIC currents evoked by H⁺ iontophoresis in WT mice. The bar indicates the positive current pulse (2 nA, 3 s-duration) applied to a micropipette filled with HCl 0.1 m through a monopolar filament. d, Changes in calcium-sensitive indicator ΔF/F₀ as a function of time. The arrow indicates the time of iontophoretic injection of H⁺. In MNTB neurons from WT mice, the Ca²⁺-dependent fluorescence rises up to 15 ± 3% (filled squares, n = 9), indicating that Ca²⁺ enters into the MNTB neuron through ASIC-1as when these are activated by H⁺. This increment in [Ca²⁺] is abolished when PcTx1 is applied to the bath solution (open squares; n = 8) and was not observed in MNTB neurons from ASIC1a^−/− mice (filled triangles; n = 4).

Figure 4.

Blockage of glutamatergic EPSCs reveals that protons released from presynaptic vesicles elicit postsynaptic ASIC-mediated currents and action potentials in MNTB neurons from WT mice. aI, Representative traces of EPSCs before (gray) and after (black) blocking AMPA, NMDA, GABA, and glycine receptors with CNQX (40 μm), d-APV (50 μm), bicuculline (20 μm), and strychnine (2 μm) in an MNTB neuron from a WT mouse [postnatal day 15 (P15)]. aII, Higher magnification of the ASIC-1a-mediated currents insensitive to CNQX, APV, bicuculline, and strychnine (black). Mean amplitudes were 46 ± 3 pA (n = 27). These ASIC-1a-mediated currents were highly reduced by amiloride (top, gray trace; n = 15) and were inhibited by PcTx1 (bottom, gray trace; n = 4). aIII, Top, Representative traces of EPSCs before (gray) and after (black) blocking AMPA, NMDA, GABA, and glycine receptors with CNQX (40 μm), d-APV (50 μm), bicuculline (20 μm), and strychnine (2 μm) in an MNTB neuron from ASIC1a^−/− mouse (P16). Bottom, Higher-magnification image showing the absence of any current after postsynaptic receptor inhibition. b, Mean half-width and decay time of amiloride and PcTx1-sensitive ASIC-1a-mediated currents compared with glutamatergic EPSCs (HW: 1.31 ± 0.1 ms vs 0.59 ± 0.03 ms; DT: 3.0 ± 0.2 ms vs 1.18 ± 0.05 ms; n = 27; Student's t test, p < 0.001). c, Sample traces of ASIC-1a currents at different membrane potentials in WT mouse (P14). d, Increasing pH buffering using a 10 mm HEPES-based extracellular solution, pH 7.3, the ASIC-1a-mediated currents in MNTB neurons from WT mice were highly reduced (traces from a P15 mouse). e, ASIC-1a-mediated currents were highly diminished when the HEPES-based extracellular solution was maintained at pH 6.0 due to the desensitization of ASICs (traces from a P17 mouse). f, ASIC-1a-mediated currents (top, black trace) evoked by 100 Hz stimulation of presynaptic axons after the blockage of postsynaptic receptors with NBQX (a higher-affinity AMPA receptor antagonist, 10 μm) plus d-APV (50 μm), bicuculline (20 μm), and strychnine (2 μm) in a P15 WT mouse. Mean amplitudes were 38 ± 8 pA (HW, 1.1 ± 0.2 ms; DT, 2.7 ± 0.3 ms; n = 7). These currents were able to evoke APs (measured in current-clamp configuration at the resting potential of the MNTB neurons) whose amplitudes and kinetics did not differ from those evoked by glutamatergic currents (bottom, black trace; current-clamp recording). PcTx1 inhibited both ASIC-1a-mediated currents and evoked APs (gray traces).

Figure 5.

ASIC-1a-dependent increase of intracellular Ca²⁺ in MNTB neurons during HFS. a, Time course of the Fluo 8 fluorescence emission ratio (ΔF/F₀) in postsynaptic MNTB neurons from WT mice (n = 9) during presynaptic HFS (150 Hz, 0.4 s; indicated by gray bar) in normal bicarbonate-based aCSF. Images were taken every 140 ms (first six images before stimulation served as the baseline). Left, During HFS, a maximum increase in intracellular Ca²⁺ of 28 ± 5% (n = 9) is observed (filled squares; control). After AMPA/kainite glutamate receptor inhibition with 40 μm CNQX (open squares, CNQX), a small component of the fluorescence increment (peak, 3.8 ± 0.9%; n = 9) remained during 150 Hz stimulation. PcTx1 inhibited this residual fluorescence (circles, + PcTx1; p < 0.05, one-way repeated-measure ANOVA). Right, Applying drugs in reverse order, PcTx1 reduced the maximum increase in intracellular Ca²⁺ of 29 ± 2% in control conditions (n = 10; filled squares, control) to 24 ± 3% (circles, PcTx1), while the addition of CNQX abolished any increase in fluorescence during 150 Hz stimulation (open squares, CNQX). b, Changing the pH buffer capacity of the aCSF alters ASIC-1a-dependent Ca²⁺ entry in MNTB neurons from WT mice. Plot of ΔF/F₀ versus time during HFS in a low-pH buffer capacity aCSF before (filled squares, HEPES 1 mm; peak increase, 28 ± 8%; n = 9) and after the inhibition of glutamate AMPA and NMDA receptors (open squares, HEPES 1 mm plus CNQX+APV; peak increment, 3.2 ± 0.8%, n = 9). This remaining fluorescence component that may be attributed to Ca²⁺ influx through ASIC-1as was eliminated when a high-pH buffer capacity aCSF inhibits acidification (circles, HEPES 10 mm; n = 9; p < 0.05, one-way repeated-measures ANOVA). c, Time course of ΔF/F₀ in postsynaptic MNTB neurons from ASIC1a^−/− mice during HFS (150 Hz, 0.4 s) in normal bicarbonate-based aCSF (filled triangles, control; n = 8). The maximum increase in [Ca²⁺] through AMPA receptors (26 ± 5%) is not statistically different from that observed in WT mice (p > 0.05, Student's t test, WT vs ASIC-1a^−/−). After glutamate AMPA and NMDA receptor inhibition, the increase in fluorescence is completely eliminated (open triangles, CNQX+APV; n = 8).

Figure 6.

Enhanced STD at excitatory synaptic transmission in ASIC1a^−/− mice compared with WT mice. a, Representative recording of AP-evoked EPSCs in WT and ASIC1a^−/− MNTB neurons during 300 Hz stimulation. b, Time course of EPSC peak amplitudes (normalized to the first EPSC amplitude in the train) during 0.7 s stimulation at 300 Hz, fitted to a single exponential decay function, with a mean decay time constant τ = 7.0 ± 0.5 ms in WT mice (n = 42) and τ = 5.0 ± 0.6 ms in ASIC1a^−/− mice (n = 21; p = 0.01, Student's t test). The EPSC amplitudes at the end of the stimuli reach a steady-state value of 9.3 ± 0.2% and 6.2 ± 0.2%, respectively, of the first EPSC amplitude in the train, in WT and ASIC1a^−/− mice (p = 0.04, Student's t test). *p < 0.05, one-way repeated-measures ANOVA. c, Effect of PcTx1 on STD in WT mice. Normalized EPSC amplitudes as a function of time recorded in WT MNTB neurons during 300 Hz stimulation in bicarbonate-based aCSF at pH 7.3 before (filled squares) and during (open squares) the application of PcTx1 in the external solution. Data fitted to a single exponential decay function show an enhanced STD when ASIC-1as are blocked (mean decay time constants: before application of PcTx1, τ = 7.3 ± 0.6 ms; after application of PcTx1, τ = 5.96 ± 0.53 ms; n = 9). Inset, Individual and mean τ values, p = 4 × 10⁻⁵, paired student′s t test). Steady-state values of EPSC amplitudes at the end of the stimuli are also statistically different (before application of PcTx1, 9.4 ± 0.7% of the first EPSC amplitude in the train; after application of PcTx1, 8.8 ± 0.8% of the first EPSC amplitude in the train; p = 0.002, paired Student's t test). d, Effect of pH buffering on STD in WT mice. Time course of normalized EPSC amplitudes during 300 Hz stimulation recorded sequentially in a 1 mm (filled squares) and 10 mm (open squares) HEPES/MES-based aCSF (n = 8), fitted by single exponential decay functions. When acidification is inhibited by a high-pH buffer capacity solution, STD is increased (mean decay time constants: HEPES 1 mm: τ = 6.4 ± 0.7 ms; HEPES 10 mm: τ = 4.9 ± 0.6 ms). Inset, Individual τ values and mean (n = 8, p = 6 × 10⁻⁴, paired Student's t test). Steady-state values of EPSC amplitudes at the end of the stimuli are 9.5 ± 0.9% vs 8.3 ± 0.9% of the first EPSC amplitude in the train, in HEPES 1 mm and HEPES 10 mm, respectively (p = 0.005, paired Student's t test).