Protons are a neurotransmitter that regulates synaptic plasticity in the lateral amygdala

Abstract

Stimulating presynaptic terminals can increase the proton concentration in synapses. Potential receptors for protons are acid-sensing ion channels (ASICs), Na(+)- and Ca(2+)-permeable channels that are activated by extracellular acidosis. Those observations suggest that protons might be a neurotransmitter. We found that presynaptic stimulation transiently reduced extracellular pH in the amygdala. The protons activated ASICs in lateral amygdala pyramidal neurons, generating excitatory postsynaptic currents. Moreover, both protons and ASICs were required for synaptic plasticity in lateral amygdala neurons. The results identify protons as a neurotransmitter, and they establish ASICs as the postsynaptic receptor. They also indicate that protons and ASICs are a neurotransmitter/receptor pair critical for amygdala-dependent learning and memory.

Keywords: PcTX1; acid sensing ion channel; long-term potentiation.

Fig. 1.

Stimulating presynaptic neurons elicits postsynaptic ASIC currents in lateral amygdala pyramidal neurons. (A) Schematic showing lateral amygdala, acid injection micropipette, stimulating electrode, and whole-cell patch-clamp recording electrode. (B) Representative ASIC currents elicited by acid injections in WT and ASIC1a^−/− neurons. Holding potential, −70 mV. (C) pH-dependent activation of ASIC currents. Best-fit yielded EC₅₀ pH of 6.1 ± 0.1 (n = 6–10 cells in 4 mice). (D) Left, representative traces of EPSCs in WT and ASIC2^−/− neurons. Right, mean ± SEM of best-fit EPSC decay times. P < 0.05 (Student t test; n = 10 cells in 4 mice). (E and F) EPSC recordings in lateral amygdala brain slices from WT and ASIC1a^−/− mice. To induce EPSCs, test pulses (100 µS, 0.05 or 0.1 Hz) were delivered through extracellular bipolar electrodes placed on cortical inputs. Slices were perfused with 25 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (an AMPA receptor antagonist) plus 50 µM (2R)-amino-5-phosphonovaleric acid (D-APV) (an NMDA receptor antagonist) and with 200 µM amiloride during times indicated. Data are from single slices. (Inset) EPSCs with expanded y axis. (G) EPSC amplitudes as recorded in D and E. **P < 0.01 (Student t test; n = 20 cells in 10 mice). Representative EPSC traces are shown at top.

Fig. 2.

Presynaptic stimulation reduces extracellular pH. (A) Schematic of pHluorin linked to syndecan 2 to assay changes in extracellular pH. (B) Biolistic transfection of pyramidal neurons. (Left) Syndecan 2-pHluorin. (Middle) mCherry as a control fluorescent indicator. (Right) Merged image. (Bottom) Enlarged image of selected area from upper image. Note syndecan 2-pHlourin expression in spines. (C) Representative traces of fluorescence with stimulation at indicated frequencies for 1 s. (D) Ratio of change in fluorescence (acidic ∆F1, alkaline ∆F2) to basal fluorescence (F₀) at indicated stimulation frequencies (n = 7).

Fig. 3.

Changing pH buffer capacity alters ASIC-dependent EPSCs. (A, Left) Example of ASIC-dependent EPSCs in lateral amygdala neuron sequentially perfused with 25 mM HCO₃⁻/5% CO₂ ACSF, 10 mM HCO₃⁻/2% CO₂ ACSF, and 90 mM HCO₃⁻/15% CO₂ ACSF, all at pH 7.4. In addition, 25 µM CNQX and 50 µM D-APV were present throughout. Representative traces are shown at top. (Right) EPSC amplitude in indicated buffers, as described for left. Each set of connected points is from a different cell: 25 mM HCO₃⁻ group, −7.0 ± 0.6 pA; 10 mM HCO₃⁻, −13.5 ± 1.5 pA; 90 mM HCO₃⁻, −4.0 ± 0.4 pA (n = 10 cells in 4 mice); one-way ANOVA with Tukey’s post hoc multiple comparison test. *P < 0.05. (B, Left) ASIC-dependent EPSCs in lateral amygdala neuron in ASIC2^−/− brain slice perfused with 25 mM HCO₃⁻/5% CO₂ ACSF and then 10 mM HCO₃⁻/2% CO₂ ACSF, both at pH 7.4. PcTX1 (100 nM) was present during time indicated. (Right) ASIC-dependent EPSC amplitude in buffers described for left (n = 8); one-way ANOVA with Tukey’s post hoc multiple comparison test. *P < 0.001.

Fig. 4.

Protons and ASIC1a contribute to synaptic plasticity in the lateral amygdala. (A) EPSPs were induced by test pulses (100 µS, 0.05 or 0.1 Hz) delivered to cortical inputs. HFS (100 Hz, 1 s) was used to induce LTP. EPSPs were recorded before and after HFS in WT (blue, 135 ± 3% of baseline; P < 0.001; n = 12 cells in 6 mice) and ASIC1a^−/− (red, 100 ± 2% of baseline, no significant difference; n = 12/6 mice) lateral amygdala pyramidal neurons. Solution was 25 mM HCO₃⁻/5% CO₂ at pH 7.4 ACSF. Representative EPSP traces at top were recorded under basal conditions and 50 min after HFS. WT differed from ASIC1a^−/−. P < 0.01 (Student t test). (B) EPSPs before and after HFS (as in A) in WT lateral amygdala neurons. ACSF contained 25 mM HCO₃⁻/5% CO₂ at pH 7.4 (blue), 10 mM HCO₃⁻/2% CO₂ at pH 7.4 (green, 180 ± 7% of baseline; P < 0.01; n = 9/5 mice), or 90 mM HCO₃⁻/15% CO₂ at pH 7.4 (gray, 99 ± 5% of baseline; no significant difference; n = 7 cells in 4 mice). Data with 25 mM HCO₃⁻ differed from 10 mM HCO₃⁻ and 90 mM HCO₃⁻. P < 0.01 (one-way ANOVA; Tukey’s post hoc multiple comparison). Representative EPSP traces at top were recorded before and 50 min after HFS.

Fig. 5.

Extracellular application of an acidic solution to lateral amygdala pyramidal neurons induces LTP. (A, Left) Representative traces of acid-generated action potentials. (Right) average number of action potentials generated at indicated pH (n = 10 for each pH; 4 mice). pH 6.8 induced the greatest number of action potentials. (B) EPSPs before and after three puffs of pH 6.8 ACSF were delivered to the target neuron in slices from WT (blue, 145 ± 6% of baseline; P < 0.001; n = 6 cells in 4 mice) and ASIC1a^−/− (red, 101 ± 2% of baseline; no significant difference; n = 8 cells in 4 mice) mice. A test pulse was delivered 50 ms before the end of each puff of pH 6.8 ACSF. Representative EPSP traces from each group are at the top. WT differed from ASIC1a^−/− (P < 0.01). Control data with test pulses are the same in B–D. Statistical analysis was one-way ANOVA; Tukey’s post hoc multiple comparison for B–D. (C) pH 6.8 application in slices treated with 50 µM D-APV (green, 116 ± 5% of baseline; P < 0.05; n = 6 cells in 4 mice) or vehicle control (blue). Control differed from 50 µM D-APV (P < 0.01). (D) Application of pH 6.8 ACSF to lateral amygdala pyramidal neurons in absence (gray, 99 ± 3% of baseline; no significant difference; n = 9 cells in 5 mice) or presence (blue) of test pulses during application of pH 6.8 solution. Control differed from slices without test pulses (P < 0.01).