Notice the reduced number of active zone and synaptic vesicles and compromised mitochondria at different levels (muscle, nerve and Schwann cells) generating increased level of ROS. H2O2-induced presynaptic depressant effect. Thus, at the mammalian neuromuscular junction HCY largely increases the inhibitory effect of oxidative stress on transmitter release, via NMDA receptors activation. This combined effect of HCY and local oxidative stress can specifically contribute to the damage of presynaptic terminals in neurodegenerative motoneuron diseases, including ALS. = number of synapses with indication of number of animals used). Statistical significance was assessed by using Students 0.05. Results We first tested the action of acute oxidative stress on transmitter release in control conditions. MEPPs representing spontaneous release of ACh quanta from nerve terminals occurred at a mean frequency of 0.60 0.05 s?1 and an amplitude of 0.52 0.02 mV (= 46 synapses/6 mice). These quantal events are classical readouts of the functional state of the motor synapse when the frequency of MEPPs reflects the presynaptic function whereas the amplitude of MEPPs characterizes the functional state of the postsynaptic membrane (Fatt and Katz, 1952). To model acute oxidative stress we used the moderate oxidant H2O2 in concentration of 300 M. Based on our previous experience (Giniatullin et al., 2006) we considered this concentration efficient and safe. H2O2 applied via bath perfusion significantly reduced the frequency of MEPPs (Figures 1A,C,D). After 30C35 min exposure to Esm1 H2O2 the mean frequency of MEPPs decreased by 32.3 8.6% (= 47 synapses/6 mice, 0.05 by Mann-Whitney test) regarding control untreated preparation. No changes were observed in MEPP amplitudes (47 synapses/6 mice, 0.05; Figures 1ACD) indicating a real presynaptic effect of H2O2. Open in a separate windows Physique 1 H2O2 inhibitory action on spontaneous acetylcholine (ACh) release from nerve endings at intact and homocysteine (HCY) pre-treated mice diaphragms. (A) Representative traces of MEPPs in control, after 300 M H2O2 application at intact and pre-treated by HCY (500 M, 2 h) diaphragm muscles. (B) MEPP shapes before, 30C35 min after application of 300 M H2O2 in intact and pre-incubated with HCY (500 M, for 2 h) preparations. Average of 25C30 MEPPs. (C) Cumulative curves of MEPP frequencies and amplitudes in control and after H2O2 application at intact and HCY pre-treated muscles. (D) The top panelmean MEPP frequencies in the presence of 300 M H2O2 at intact (= 47 synapses/6 mice), and HCY-pre-incubated (= 55 synapses/7 mice) muscles compared to the frequency level before H2O2 application (taken as 100%), * 0.05; the bottom panelmean MEPP amplitudes (mV) before, 30C35 min after application of 300 M H2O2 in intact (= 47 synapses/6 mice) and pre-incubated with HCY (500 M, for 2 h; = 55 synapses/7 mice) preparations, ns = non significant. The temporal characteristics of MEPP were not changed by H2O2. Thus, the rise-time in control was 0.20 0.01 ms (= 47 synapses/6 mice) and the decay time constant of MEPP was 3.18 0.28 ms (= 44 synapses/5 mice). After H2O2 treatment these parameters remained unchanged (95 7% and 94 8% from control values, respectively, = 44 synapses/5 mice, 0.05). Next, we tested the action of HCY on spontaneous ACh release and whether this exposure changed the inhibitory effect of H2O2. As we intended to model naturally long-lasting hHCY in the short time window we selected the relatively large concentration of HCY of 500 M which nevertheless corresponds the severe hHCY in humans (Kang et al., 1992; Stanger et al., 2009). Notably, as we used the racemic D, L-form of HCY, the effective concentration of the naturally occurring L-form was actually only half of the total (Lipton et al., 1997). Nevertheless, after incubation of the neuromuscular preparations for 2 h in solution containing 500 M HCY, the mean frequency of MEPPs was the same as in untreated preparations (+5.8 9.0% change comparing with untreated samples; = 56 synapses/6 mice). No changes were observed in the amplitudes of MEPPs (Figures 1ACD) and in the time-course of MEPPs (Figure ?(Figure1B)1B) after HCY exposure (rise-time = 94 5% and the decay time constant = 95 7%, 0.05, = 49 synapses/6 mice). However, after HCY pre-treatment (500 M, 2 h) the inhibitory potency.Thus, the depressant effect of H2O2 on spontaneous ACh release was significantly ( 0.05 by Mann-Whitney test) more (almost two times) efficient after muscles exposure to HCY compared to the action of H2O2 alone. quantum release from nerve terminals (measured as the frequency of miniature endplate potentials, MEPPs) without changes in the amplitude of MEPPs, indicating a presynaptic effect. Pre-treatment with HCY for 2 h only slightly affected both amplitude and frequency of MEPPs but increased the inhibitory potency of H2O2 almost two fold. As HCY can activate certain subtypes of glutamate N-methyl D-aspartate (NMDA) receptors we tested the role of NMDA receptors in the sensitizing action of HCY. Remarkably, the selective blocker of NMDA receptors, AP-5 completely removed the sensitizing effect of HCY on the H2O2-induced presynaptic depressant effect. Thus, at the mammalian neuromuscular junction HCY largely increases the inhibitory effect of oxidative stress on transmitter release, via NMDA receptors activation. This combined effect of HCY and local oxidative stress can specifically contribute to the damage of presynaptic terminals in neurodegenerative motoneuron diseases, including ALS. = number of synapses with indication of number of animals used). Statistical significance was assessed by using Students 0.05. Results We first tested the action of acute oxidative stress on transmitter release in control conditions. MEPPs representing spontaneous release of ACh quanta from nerve terminals occurred at a mean frequency of 0.60 0.05 s?1 and an amplitude of 0.52 0.02 mV (= 46 synapses/6 mice). These quantal events are classical readouts of the functional state of the motor synapse when the frequency of MEPPs reflects the presynaptic function whereas the amplitude of MEPPs characterizes the functional state of the postsynaptic membrane (Fatt and Katz, 1952). To model acute oxidative stress we used the mild oxidant H2O2 in concentration of 300 M. Based on our previous experience (Giniatullin et al., 2006) we considered this concentration efficient and safe. H2O2 applied via bath perfusion significantly reduced the frequency of MEPPs (Figures 1A,C,D). After 30C35 min exposure to H2O2 the mean frequency of MEPPs decreased by 32.3 8.6% (= 47 synapses/6 mice, 0.05 by Mann-Whitney test) regarding control untreated preparation. No changes were observed in MEPP amplitudes (47 synapses/6 mice, 0.05; Figures 1ACD) indicating a pure presynaptic effect of H2O2. Open in a separate window Figure 1 H2O2 inhibitory action on spontaneous acetylcholine (ACh) release from nerve endings at intact and homocysteine (HCY) pre-treated mice diaphragms. (A) Representative traces of MEPPs in control, after 300 M H2O2 application at intact and pre-treated by HCY (500 M, 2 h) diaphragm muscles. (B) MEPP shapes before, 30C35 min after application of 300 M H2O2 in intact and pre-incubated with HCY (500 M, for 2 h) preparations. Average of 25C30 MEPPs. (C) Cumulative curves of MEPP frequencies and amplitudes in control and after H2O2 application at intact and HCY pre-treated muscles. (D) The top panelmean MEPP frequencies in the presence of 300 M H2O2 at intact (= 47 synapses/6 mice), and HCY-pre-incubated (= 55 synapses/7 mice) IRAK-1-4 Inhibitor I muscles compared to the frequency level before H2O2 application (taken as 100%), * 0.05; the bottom panelmean MEPP amplitudes (mV) before, 30C35 min after application of 300 M H2O2 in intact (= 47 synapses/6 mice) and pre-incubated IRAK-1-4 Inhibitor I with HCY (500 M, for 2 h; = 55 synapses/7 mice) preparations, ns = non significant. The temporal characteristics of MEPP were not changed by H2O2. Thus, the rise-time in control was 0.20 0.01 ms (= 47 synapses/6 mice) and the decay time constant of MEPP was 3.18 0.28 ms (= 44 synapses/5 mice). After H2O2 treatment these parameters remained unchanged (95 7% and 94 8% from control values, respectively, = 44 synapses/5 mice, 0.05). Next, we tested the action of HCY on spontaneous ACh release and whether this exposure changed the inhibitory effect of H2O2. As we intended to model naturally long-lasting hHCY in the short time window we selected the relatively large concentration of IRAK-1-4 Inhibitor I HCY of 500 M which nevertheless corresponds the severe hHCY in humans (Kang et al., 1992; Stanger et al., 2009). Notably, as we used the racemic D, L-form of HCY, the effective concentration of the naturally occurring L-form was actually only half of the total (Lipton et al., 1997). Nevertheless, after incubation of the neuromuscular preparations for 2 h in solution containing 500 M HCY, the mean frequency of MEPPs was the same as in untreated preparations (+5.8 9.0% change comparing with untreated samples; = 56 synapses/6 mice). No changes were observed in the amplitudes of MEPPs (Figures 1ACD) and in the time-course of MEPPs (Figure ?(Figure1B)1B) after HCY exposure (rise-time = 94 5% and the decay time constant = 95 7%, 0.05, = 49 synapses/6 mice). However, after HCY pre-treatment (500 M, 2 h) the inhibitory potency of H2O2 was largely increased. MEPPs frequency decreased by 60.0 3.6% from the level in the.