Dual action of hydrogen peroxide on synaptic transmission at the frog neuromuscular junction

Abstract

There is evidence that reactive oxygen species (ROS) are produced and released during neuromuscular activity, but their role in synaptic transmission is not known. Using a two-electrode voltage-clamp technique, at frog neuromuscular junctions, the action H2O2 on end-plate currents (EPC) was studied to determine the targets for this membrane-permeable ROS. In curarized or cut muscles, micromolar concentrations of H2O2 increased the amplitude of EPCs. Higher (> 30 microM) doses inhibited EPCs and prolonged current decay. These effects were presynaptic since H2O2 did not change the amplitude or duration of miniature EPCs (although it reduced the rate of spontaneous release at high concentrations). Quantal analysis and deconvolution methods showed that facilitation of EPCs was due to increased quantal release, while depression was accompanied by temporal dispersion of evoked release. Extracellular recordings revealed prolonged presynaptic Ca2+ entry in the presence of high H2O2. Both low and high H2O2 increased presynaptic potentiation during high-frequency stimulation. Pro-oxidant Fe2+ did not affect facilitation by low doses of H2O2 but augmented the inhibition of EPCs by high H2O2, indicating involvement of hydroxyl radicals. High Mg2+ and the ROS scavenger N-acetylcysteine eliminated both the facilitatory and depressant effects of H2O2. The facilitatory effect of H2O2 was prevented by protein kinase C (PKC) inhibitors and 4beta-phorbol 12-myristate, 13-acetate (PMA), an activator of PKC. PKC inhibitors but not PMA also abolished the depressant effect of H2O2. Our data suggest complex presynaptic actions of H2O2, which could serve as a fast feedback modulator of intense neuromuscular transmission.

Figure 1. Opposite effects of low and high concentrations of H₂O₂ on multiquantal end-plate currents (EPCs)

A, facilitating effect of 3 μM H₂O₂ on multiquantal EPCs. Aa, representative EPCs in control, in the presence of 3 μM H₂O₂ and after washout. Ab, histograms showing the action of 3 μM H₂O₂ on peak amplitude of EPCs (A_EPC), decay time constant of EPCs (τ_EPC), amplitude of MEPCs (A_MEPC) and quantal content estimated either as the ratio of peak current amplitudes of averaged EPCs and MEPCs (QC-1) or as the ratio of current integrals (QC-2) on cut preparation (* P < 0.05; n = 6). Data in this and subsequent histograms are presented as the mean ± s.e.m. (n = number of synapses). B, depressant action of 300 μM H₂O₂ on multiquantal EPCs. Ba, EPCs in control, in the presence of 300 μM H₂O₂ and after washout. Bb, histograms showing the action of 300 μM H₂O₂ on the same parameters of EPCs and MEPCs as in Ab (* P < 0.05; n = 6). Note that depression of QC-1 was stronger (P < 0.05) than that of QC-2 (P > 0.05).

Figure 3. The action of H₂O₂ on short-term plasticity

A, peak amplitudes of 20 successive EPCs during 60 Hz motor nerve stimulation of a cut preparation in control (•), and in the presence of 3 μM (○) or 300 μM (▴; n = 5-7) H₂O₂. Note the significantly enhanced potentiation of successive EPCs in the presence of both concentrations of H₂O₂.

Figure 2. Concentration dependence of H₂O₂ action on EPCs

A, effect of various H₂O₂ concentrations on peak EPC amplitude in a cut preparation. B, effect of H₂O₂ on the decay time constant of EPCs. C, the action of H₂O₂ on the current integrals. (* P < 0.05; n = 4-8 synapses). Note the biphasic action of H₂O₂ on the amplitude and area of EPCs and monophasic action of H₂O₂ on the time course of EPCs.

Figure 4. Effect of H₂O₂ on the time course of transmitter release

A, examples of averaged MEPCs (n = 120) or averaged multiquantal EPCs (n = 10) used for deconvolution in control and after 300 μM H₂O₂. All signals were normalized for comparison. Note that H₂O₂ selectively prolonged the decay of EPCs, while in the presence of 300 μM H₂O₂ the shape of MEPCs was the same as in control. B illustrates the results of deconvolution of the averaged EPC from A (transmitter release functions) with the fitted MEPCs (for detail see Methods). Deconvolution revealed two exponential components of transmitter release with decay time constants of 0.18 ± 0.04 and 1.02 ± 0.11 ms, respectively. Transmitter release functions were obtained by averaging data from 6 endplates. Note that 300 μM H₂O₂ increased the relative contribution of the slow phase of transmitter release function. C, histograms showing that 300 μM H₂O₂ significantly increased the decay time constant of the slow phase of the release function (τ₂; * P < 0.05; n = 6), while it did not affect the fast phase (τ₁; P > 0.05; n = 6) or the ratio of the amplitudes of the two components (A₁/A₂; P > 0.05; n = 6).

Figure 5. Effect of H₂O₂ on the presynaptic Ca²⁺ current

A, presynaptic action potential recorded by a microelectrode inserted into the perineurium adjacent to the nerve ending. The large initial downward part of the action potential represents the initial Na⁺ current while the late upward part represents the Ca²⁺ current. Each signal was obtained by averaging 24 records evoked at 0.05 Hz. The postsynaptic response was blocked by a high dose of (+)-tubocurarine (for details, see Methods). Low (3 μM) H₂O₂ did not change the Na⁺ or Ca²⁺ phase of the presynaptic signal, while high (100 μM) H₂O₂ slowed the decay of the Ca²⁺ current without producing significant changes in the Na⁺ phase. B and C, histograms showing the action of 3 or 300 μM H₂O₂ on the amplitude (% change vs. control) or the time constant of Ca²⁺ current decay. * P < 0.05, n = 6 for both H₂O₂ concentrations.

Figure 6. Modification by Mg²⁺ of H₂O₂ action on EPCs

A, time profile for the facilitatory effect of 3 μM H₂O₂ on EPCs in control (•; [Ca²⁺]_o = 1.8 mM) and in a magnesium preparation (○; [Mg²⁺]_o = 6 mM; [Ca²⁺]_o = 0.9 mM). * P < 0.05; n = 5. B, time profile for the depressant effect of 300 μM H₂O₂ on EPCs in control (•) and in a magnesium preparation (○; * P < 0.05; n = 5).

Figure 7. Fe²⁺ selectively promoted the depressant effect of 300 μM H₂O₂

Aa, effect of 3 μM H₂O₂ in the presence of 100 μM Fe²⁺ on multiquantal EPCs in a cut preparation. Ab, histograms showing the facilitatory action of 3 μM H₂O₂ on peak EPC amplitude or current area. Note that the facilitatory action of a low dose of H₂O₂ was not changed by Fe²⁺ (26 ± 5 % enhancement in Fe²⁺; * P < 0.05; n = 5; compare with 28 ± 7 % in control). Ba, the depressant action of 300 μM H₂O₂ on EPCs was strongly enhanced by 100 μM Fe²⁺. Bb, histograms showing the action of 300 μM H₂O₂ on peak EPC amplitude or current area in the presence of 100 μM Fe²⁺ (** P < 0.01; significantly different from the action of 300 μM H₂O₂ in control; n = 5).

Figure 8. Testing the role of PKC in the action of H₂O₂

A, histograms showing the action of 3 μM H₂O₂ on the peak amplitude of EPCs (□) and the current area (▪) in control (* P < 0.05; n = 5), in the presence of 0.5 μM staurosporine (P > 0.05; n = 5), 10 μM chelerythrine (P > 0.05; n = 5), and 0.5 μM PMA (P > 0.05; n = 5). B, the same data for the action of 300 μM H₂O₂ (n = 4-7). Note, that unlike the facilitatory action of 3 μM H₂O₂, the depressant effect of 300 μM H₂O₂ was not only preserved in the presence of PMA but even enhanced compared with control (P < 0.05).