Ryanodine and IP3 Receptors Modulate Facilitation and Tetanic Depression at the Frog Neuromuscular Junction.
Priscila E. Silveira, Ph.D1, Ricardo F. Lima, Ph.D 1,2, Jennifer D. S. Guimarães1, Jordi Molgó Ph.D3, Ligia A. Naves, Ph.D.1, Christopher Kushmerick, Ph.D.1
1-Departamento de Fisiologia e Biofísica, ICB, UFMG, Belo Horizonte, Brazil. 2 – Present address, Dept. Fisiologia e Farmacologia, UFC, Fortaleza, Brazil 3- C.N.R.S., Institut Fédératif de Neurobiologie Alfred Fessard Laboratoire de Neurobiologie et Dévelopement, CNRS, 91198-Gif sur Yvette, France. Correspondence to: Christopher Kushmerick Departamento de Fisiologia e Biofísica Instituto de Ciências Biológicas Universidade Federal de Minas Gerais Av Antonio Carlos 6627 Belo Horizonte, MG, 31270-901, Brazil [email protected] Tel +55 31 3409-2512
Acknowledgments Funded by grants from the Brazilian agencies CNPq and FAPEMIG. PES, RFL, and J.D.S.G were recipients of student fellowships from CAPES and CNPq. Keywords: Neuromuscular Junction, Ryanodine receptor, IP3 Receptor, Short-term plasticity, Intracellular Ca2+ stores. Running title: Ryanodine and IP3 receptors
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/mus.24571 This article is protected by copyright. All rights reserved.
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Abstract Introduction: Short-term plasticity of synaptic function is an important physiological control of transmitter release. Short-term plasticity can be regulated by intracellular calcium released by ryanodine and inositol tri-phosphate (IP3) receptors, but the role of these receptors at the neuromuscular junction is understood incompletely. Methods: We measured short-term plasticity of evoked endplate potential (EPP) amplitudes from frog neuromuscular junctions treated with ryanodine , 2-Aminoethoxydiphenylborane (2-APB), or 1-[6-[[(17β)-3-Methoxyestra-1,3,5(10)-trien-17yl]amino]hexyl]-1H-pyrrole-2,5-dione (U-73122). Results: Ryanodine decreases paired-pulse facilitation for intervals < 20 ms and markedly decreases tetanic depression. Treatment with 2-APB reduces EPP amplitude, increases paired-pulse facilitation for intervals < 20 ms, and significantly reduces tetanic depression. U-73122 decreases EPP amplitude and decreases paired pulse depression for intervals < 20 ms. Conclusions: Ryanodine, IP3 receptors, and phopholipase C modulate short-term plasticity of transmitter release at the neuromuscular junction. These results suggest possible targets to improve the safety factor for neuromuscular transmission during repetitive activity of the neuromuscular junction.
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Introduction Short-term synaptic plasticity refers to activity-dependent modification of the strength or efficacy of transmission at preexisting synapses. Although post-synaptic mechanisms such as receptor desensitization can contribute to short-term plasticity, expression of 2 well-studied forms of plasticity, paired-pulse facilitation and tetanic depression, largely reflect changes in transmitter release, which depend on both the number of available quanta of neurotransmitter and their release probability.1 Both of these parameters may change during expression of short-term plasticity, but the mechanisms that underlie these changes in the release process are understood incompletely. Several lines of evidence suggest that expression of short-term plasticity at the neuromuscular junction is associated with alterations in presynaptic Ca2+ dynamics during repetitive high-frequency stimulation. These include the following observations: 1) increasing the ratio of Mg2+/Ca2+ in the bathing solution reduces Ca2+ entry through voltage-gated channels, reduces release probability, and consequently shifts the response to high-frequency stimulation from depression to facilitation;2 2) loading nerve terminals with the Ca2+ chelator 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) inhibits facilitation;3 and 3) block of Ca2+ uptake into or release from mitochondria inhibits the post-tetanic rise in intracellular Ca2+ and post-tetanic potentiation of quantal content.4,5 Thus, manipulations that interfere with Ca2+ dynamics affect short-term plasticity. The endoplasmic reticulum (ER) is another well-characterized source of intracellular Ca2+, and Ca2+ release from the ER via ryanodine receptors or IP3 receptors plays an important role in cellular responses to both electrical and chemical signals.6 However, compared to the role of Ca2+ entry, Ca2+ clearance, and calcium uptake and release by mitochondria, the role of Ca2+ released from ER in evoked neurotransmitter release and its short-term plasticity at the neuromuscular junction is less explored. Previous studies indicate that Ca2+ released from the ER modulates transmitter release at the neuromuscular junction. Ca2+ released from internal stores by caffeine promotes spontaneous synaptic John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.
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vesicle exocytosis7,8. During nerve stimulation under conditions of low-Ca2+/high Mg2+, Ca2+-induced Ca2+-release underlies a form of short-term plasticity mediated by ryanodine receptors.9 Stimulation of the neuromuscular junction with exogenous IP3 applied via liposomes increases quantal transmitter release,10 suggesting the presence of IP3 receptors at the nerve terminal. IP3 can be formed by Gprotein coupled receptors that activate phospholipase C, and there is evidence that this pathway may be active in neuromuscular junctions, since activation of phospholipase C and IP3 receptors are necessary to mediate neurotrophin-3-induced potentiation of synaptic transmission in motoneuron/muscle fiber co-cultures.11 Nonetheless, it is still unknown how Ca2+ release from the ER contributes to short-term plasticity in the intact neuromuscular junction when the extracellular Ca2+ concentration is at normal levels. In addition, release of Ca2+ via activation of ryanodine and IP3 receptors has been implicated in certain neuromuscular junction diseases including the alterations of neurotransmitter release evoked by Amyotrophic Lateral Sclerosis IgG12 and the facilitation of spontaneous release by beta bungarotoxin13. Therefore, knowledge on the role of these receptors in synapse function will also be relevant to understanding certain pathological states. In an attempt to clarify the role of ryanodine and IP3 receptors in synaptic function and plasticity, we blocked these receptors with ryanodine or 2-APB, or blocked the phospholipase that generates IP3 with U-73122 and investigated the effects of these treatments on evoked quantal transmitter release during low-frequency stimulation during application of paired pulses to measure facilitation and during tetanic depression.
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Methods Preparation and solutions Experiments were performed at room temperature using the cutaneous pectoris nerve-muscle preparations from the frog Rana temporaria, Rana esculenta, or Rana catesbeiana weighing 20-70 g. The animals were sacrificed by double pithing according to local animal care guidelines, and the muscle with attached nerve was pinned onto a bed of Sylgard in a 3-4 ml chamber. The chamber was filled with Ringer contained (in mM): NaCl, 115; KCl, 2.5; CaCl2, 2; and N-[2Hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid] (HEPES), 5; pH 7.4 (adjusted with NaOH). For experiments employing nerve stimulation, tubocurarine (6-8 x 10-6 M) was added to reduce the EPP amplitudes to below threshold and thus block muscle contraction. Electrophysiological recordings EPP amplitudes were recorded with intracellular microelectrodes fabricated from borosilicate glass with resistances of 5-15 M-Ohm when filled with 3 M KCl and measured while immersed in the Ringer solution. Recordings were obtained using a Dagan 8500 amplifier (Dagan Co., Minneapolis, USA) or an Axoclamp-2A amplifier (Molecular Devices, CA, USA). The signals were further amplified 50 - 500 fold by an Ectron 750 amplifier (Ectron Co., San Diego, USA) or Grass AC/DC strain gauge amplifier (Astro-Med Inc., W. Warnick, USA) and then fed to an A/D converter controlled by a PC computer. Data acquisition was controlled using the Strathclyde Electrophysiology Software suite (University of Strathclyde, Glasgow, Scotland). The resting membrane potential was monitored continuously during the measurements, and EPP amplitudes were corrected for changes in resting potential (using -90 mV as a standard resting potential) and for non-linear summation assuming a reversal potential of -15 mV.14 The nerve stump was drawn into a suction electrode and stimulated using 100 µs supramaximal stimuli applied at 0.2 Hz using a Grass SD9 or S48 stimulator (Astro-Med Inc., W. Warnick, USA).
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Minature EPP (MEPP) amplitudes were measured in the same fiber in control conditions (normal frog Ringer) and 15 and 30 minutes after addition of drugs or vehicle and were corrected as described above for EPP amplitudes. To measure facilitation, EPP amplitudes were measured when the nerve was stimulated by 30 pairs of supramaximal stimuli separated by intervals ranging from 5 to 60 msec and delivered every 5 sec. For the shortest intervals, the tail of the first EPP overlapped the peak of the second. In these cases we calculated the amplitude of the second EPP as the difference of the peak potential and the projected tail of the preceding EPP. The amount of facilitation (f) in any given response was defined by the quantity f= (EPP2 – EPP1)/EPP1, where EPP1 and EPP2 are the mean amplitudes of the first and second EPPs, corrected for resting potential and non-linear summation (see above). To measure depression, we stimulated the nerve at 0.2 Hz to measure the baseline EPP amplitude. After 30 stimuli at 0.2 Hz, we increased the stimulation frequency to 20 Hz for 60 s to generate EPP depression and then returned to 0.2 Hz for an additional 5 min to measure recovery from depression. Drugs and toxins All chemicals were ACS grade. Ryanodine and 2-APB were obtained from Sigma (Saint Louis, USA), U-73122 was obtained from Calbiochem (San Diego, CA, USA), and U-73343 was obtained from Tocris (UK). Drugs were added directly to the bath from ethanol, methanol, or DMSO stock solution to the final concentrations given in the text. Control experiments were performed separately with the drug vehicles, and differences were analyzed using an unpaired t-test. Data analysis Data were analyzed by custom-written software within Igor 6 (Wavemetrics, Lake Oswego, USA). EPP amplitudes during tetanic trains were normalized to their amplitudes during the preceding baseline period of low-frequency stimulation. Normalized EPP amplitude changes during tetanic stimulation occurred in 3 phases, an initial facilitation followed by a fast phase of depression that gave way to a John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.
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slower, more linear phase of depression that continued until the end of the 60 s tetanic train. Facilitation during the train was defined as the largest normalized EPP amplitude after the onset of the train. Slow depression was the slope of a line fit to the last 15 s of EPP amplitude data before the end of the train. Fast depression was the difference between the EPP amplitude at peak train facilitation and the extrapolation of slow depression back to the point of maximum facilitation, expressed as the fraction (%) of maximum facilitation. EPP amplitudes in nerve-muscle preparations treated with drugs were compared with preparations treated with the corresponding drug vehicle. MEPP amplitudes and frequency were measured in the same fiber in normal frog Ringer solution, and after 30 min treatment with either drugs or vehicle, and are expressed normalized to their values in control Ringer solution. Statistical analysis of data was performed using the Student t-test. Values are expressed as mean ± 95% confidence intervals, and error bars in figures represent the 95% confidence intervals about the mean, calculated using the Student tdistribution. Differences in mean values given in the results were designated as statistically significant when P < 0.05.
Results Calcium-induced calcium release Previous studies indicate that blockade of ryanodine receptors reduces frequency facilitation when low Ca2+ and high Mg2+ are used to block muscle contraction.9 In order to evaluate the role of ryanodine receptors in evoked quantal transmitter release under more physiological conditions, we used normal frog Ringer solution and blocked contractions with tubocurarine. Under these conditions, ryanodine (10-5 M) did not significantly change the amplitude of EPPs evoked at 0.2 Hz compared to vehicle control (ethanol 0.1%; Fig. 1A). In separate experiments without tubocurarine, we observed no significant effect of these concentrations of ryanodine or ethanol on the amplitude or frequency of John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.
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MEPPs (Fig. 1B). Based on the lack of effect of ryanodine on EPP and MEPP amplitudes, we conclude that ryanodine does not alter quantal content during low-frequency stimulation. We next examined the effects of ryanodine on paired pulse facilitation with intervals ranging from 5-60 ms and observed that ryanodine reduced facilitation at short intervals (< 20 ms), but not at longer intervals (Fig. 1C). During prolonged 20 Hz stimulation, EPP amplitudes from control fibers exhibited fast and slow components of depression (Fig. 1D, see Methods for definitions). In preparations treated with ryanodine (10-5 M) the fast phase of depression was reduced markedly (Fig 1E). Although there was some reduction in the slow phase of depression, this difference was not statistically significant (Fig. 1F). After stimulating at 20 Hz for 50 s, EPP amplitudes were reduced to 37 ± 18 % of their initial values (n = 15), while in preparations treated with ryanodine they were sustained, remaining at 96 ± 33% of their initial values (n = 12). (Here and below, mean values are given ± their 95% confidence intervals). Thus, for 20 Hz stimulation, preparations treated with ryanodine (10-5 M) exhibited much less EPP depression. High concentrations of ryanodine block the ryanodine receptor, whereas lower concentrations may facilitate channel opening.15 We thus repeated our measurements in muscles treated with 10-7 M ryanodine. In these experiments, ryanodine (10-7 M) did not significantly affect EPP amplitudes recorded at low frequency (control: 2.7 ± 1.1 mV, n = 25 vs RY 1.9 ± 0.44 mV, n = 22; P=0.15, t-test), paired pulse facilitation, or the fast and slow components of depression (Fig. 1 G-J).
IP3 Receptors We compared responses in preparations treated with 10-5 M 2-APB, an antagonist of IP3 receptors, with vehicle controls (0.002% methanol, Fig 2). In fibers treated with 2-APB, EPP amplitudes were smaller than controls, but not significantly (control 2.54 ± 0.67 mV, n=14 vs. APB 1.81 ± 0.49 mV, n=16, P=0.064, t-test). Neither 2-APB nor its vehicle significantly affected MEPP amplitudes or frequency. John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.
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Paired-pulse facilitation was increased by 2-APB for intervals < 20 ms. During tetanic high-frequency stimulation, fibers treated with 2-APB exhibited less fast depression than controls. IP3 is formed from PIP2 in a reaction catalyzed by phopholipase-C. We therefore tested the effect of an inhibitor of this enzyme, U-73122 (5 x 10-6 M, Fig. 3). EPP amplitudes in fibers treated with U-73122 were significantly smaller than vehicle controls (DMSO 0.05%, 4.29 ± 1.1 mV, n = 15 vs U-73122 2.57 ± 0.69 mV, n = 20, P = 0.009, t-test). In separate experiments, U-73122 caused a 30 ± 23% increase in MEPP amplitudes (n=4, P=0.02, paired t-test) that was not observed in DMSO controls (n = 5). Pairedpulse facilitation was reduced by U-73122 for intervals < 30 ms, whereas fast depression was increased. The compound 1-[6-[[(17β)-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrrolidinedione (U-73433) is structurally very similar to U-73122 but is reported to be inactive on PLC. 16. To determine the specificity of the effects of U-73122 described above we therefore tested U-73433 (5x10-6 M; Fig 3 G-J). Unlike U-73122, U-73433 did not reduce EPP amplitudes (control: 1.86 ± 0.38 mV, n. = 24 vs. U-73433: 1.71 ± 0.22 mV, n = 24, P=0.46, t-test) and had no effect on paired-pulse facilitation (Fig 3G). However, the increase in fast depression by U-73122 was also observed with U-73433 (Fig 3 H-J). Discussion Release of Ca2+ from intracellular stores via ryanodine and IP3 receptors plays a role in synaptic plasticity at several synapses17–19. Here, we examined the role of these 2 receptors at the neuromuscular junction during low- and high-frequency stimulation at a physiological concentration of extracellular Ca2+. Initial EPP amplitudes and recovery after tetanic trains were measured during stimulation at 0.2 Hz, a frequency that generated little short-term plasticity. For tetanic trains, we employed relatively long stimulus trains at 20 Hz. Long trains were necessary to measure accurately the slow phase of depression. The choice of 20 Hz for tetanic stimulation was based on the observation that in control John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.
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conditions it generated EPP depression of roughly 50% and thus allowed us to test for either an increase or a decrease in depression. Previous studies demonstrated a role for ryanodine receptors and calcium-induced calcium release in the release of neurotransmitter at the neuromuscular junction. 9 Treatment with ryanodine reduces paired-pulse facilitation for short intervals (< 20 ms, corresponding to stimulation frequencies > 50 Hz). On the other hand, we observed an increase in quantal output during 20 Hz stimulation when ryanodine receptors (and, presumably, calcium-induced calcium release) were blocked with ryanodine. Simple depletion models with mono-exponential replenishment predict more rather than less depression if frequency facilitation is blocked.1 Therefore, it seems that treatment with ryanodine accelerates the rate at which the vesicle pool is replenished during high-frequency stimulation. Although in central nervous system synapses replenishment of the vesicle pool is accelerated by activity in a Ca2+-dependent manner 20, our data suggest that Ca2+ release by ryanodine receptors at the neuromuscular junction has the opposite effect and limits release of neurotransmitter during tetanic trains. Given the neuromuscular junction safety margin 21–23, this may be an efficient way to allocate resources. Previous studies have shown that exogenous IP3 introduced into the frog neuromuscular nerve terminal increased the quantal content of EPPs, indicating that IP3 receptors are coupled to transmitter release. 10
Blocking IP3 receptors with 2-APB (10-5 M) had no effect on MEPP frequency. It increased
facilitation and reduced depression during high-frequency stimulation. This suggests that IP3 receptors are activated by nerve stimulation and contribute to the expression of short-term plasticity. Paired pulse facilitation in the controls for the 2-APB group was lower than for the controls of the other groups. This may be a vehicle effect or a seasonal variation, but all results are reported versus control experiments realized in the same period and in the presence of drug vehicle. What might be the source of IP3 during nerve stimulation? At the central nervous system, presynaptic auto- or heteroreceptor
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activation stimulates phospolipase C and IP3 formation. 24,25 Previous studies indicate a role for IP3 in the presynaptic receptor-mediated modulation of spontaneous release from cultured Xenopus motoneurons 11 and in evoked release at the crayfish neuromuscular junction. 26 The frog neuromuscular junction expresses several G protein coupled receptors including muscarinic M1 autoreceptors. 27 In neuronal cell lines, activated M1 receptors couple though Gq/11 and phospholipase C to produced IP3 28, suggesting a possible route for IP3 formation via autoreceptor activation. Our finding of reduced EPP amplitude at low frequency when IP3 receptors are blocked with 2-APB is consistent with previous reports that activation of M1 autoreceptors at the frog neuromuscular junction facilitate release27,29 and suggest that endogenously formed IP3 or some other mechanism of activation of IP3 receptors affects release even at low stimulation frequencies. In addition, the reduction in tetanic depression by APB suggests that IP3 receptors contribute to a reduction in quantal output during highfrequency stimulation. Treatment with U-73122, an inhibitor of the G-protein coupled phospholipase that produces IP3, but not its inactive analog U-73433 reduced EPP amplitude, like 2-APB. U-73122, but not U-73433, also reduced paired-pulse facilitation, an effect that was not observed for 2-APB. In addition to IP3, cleavage of PIP2 forms DAG, an activator of protein kinase C that regulates neurotransmitter release 30. Since inhibition of phospholipase C will affect both pathways, it is not surprising that drugs that affect this enzyme have effects that are more complex than that of APB. Both U-73122 and U-73433 increased tetanic depression, suggesting that this effect is unrelated to block of phospholipase C. Previous studies have demonstrated a use-dependent increase in dense-core vesicle mobility in Drosophila neuromuscular junctions via a process that can be blocked by Ryanodine and KN-93, an inhibitor of CaMKII31. In the frog neuromuscular junction, small synaptic vesicles can be separated into 2 pools based on their mobility, a relatively mobile recycling pool and a immobile reserve pool32. Nerve activity increases the mobility of the immobile reserve. Future studies might address whether
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Ryanodine affects this activity-dependent mobilization. Previous reports at the frog neuromuscular junction have shown that ryanodine receptors mediate Ca2+induced Ca2+ release that modulates transmitter release at the frog neuromuscular junction. 9,33,34 In these studies, the ryanodine-sensitive increase of release induced by a previous conditioning train did not change facilitation during brief trains. We observed, in the absence of conditioning trains, that ryanodine decreased facilitation. Two factors may account for these differences. Firstly, we observed effects of ryanodine on facilitation for intervals of 15 ms and below, shorter than the intervals used in the aforementioned study9. Secondly, we measured facilitation in Ringer solution containing 1.8 mM Ca2+ without Mg2+, and the relatively large influx of Ca2+ in these conditions may generate results that are different than are obtained in low Ca2+ / high Mg2+ Ringer solution. In conclusion, expression of 2 well-defined forms of synaptic plasticity, paired-pulse facilitation and tetanic depression, are regulated by IP3 receptors and ryanodine receptors at the frog neuromuscular junction. These data suggest that Ca2+-dependent second messenger pathways may play an important role in quantal release of transmitter at this synapse.
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Abbreviations 2-APB, 2-Aminoethoxydiphenylborane BAPTA , 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid EPP, evoked endplate potential ER, endoplasmic reticulum HEPES, N-[2-Hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]
IP3, inositol tri-phosphate MEPP, minature EPP U-73122, 1-[6-[[(17β)-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione U-73433, 1-[6-[[(17β)-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrrolidinedione
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Figure Legends Fig. 1. Reduced paired-pulse facilitation and tetanic depression in nerve-muscle preparations treated with Ryanodine (0.1 or 10 µM) or vehicle control (ethanol, 0.01%). A. EPP amplitudes recorded during low-frequency 0.2 Hz stimulation in control treated preparations (Ctrl) or preparations treated with ryanodine (Ry, 10 µM). B. MEPP amplitude and frequency after 30 min treatment with ryanodine (Ry, 10 µM), normalized to their values prior to treatment. C. Paired-pulse facilitation in control treated preparations (white circles) or preparations treated with ryanodine (10 µM , black circles). D. Normalized EPP amplitudes during low-frequency 0.2 Hz stimulation (time < 0 s), during highfrequency 20 Hz stimulation (indicated by the thick black line) and upon return to low-frequency stimulation (time > 60 s). Black traces, mean values. Gray traces, 95% confidence limits for the mean. The gap in the traces was introduced to re-align the recordings from individual preparations to the start of recovery from depression. E-F. Fast and slow components of depression in control or ryanodine (10 µM). G-J, same as for C-F except that the concentration of Ryanodine was 0.1 µM. In all panels, error bars represent 95% confidence intervals about the mean values. n=10-25 per group.
Fig. 2. Increased paired-pulse facilitation and reduced tetanic depression in nerve-muscle preparations treated with 2-APB (10 µM, n=15) or vehicle control (methanol, 0.002%, n=14). A. EPP amplitudes recorded during low-frequency 0.2 Hz stimulation in control treated preparations (Ctrl) or preparations treated with 2-APB. B. MEPP amplitude and frequency after 30 min treatment with methanol (ctrl) or 2-APB, normalized to their values prior to treatment. C. Paired-pulse facilitation in control treated preparations (white circles) or preparations treated with 2-APB (black circles). D. Normalized EPP amplitudes during low-frequency 0.2 Hz stimulation (time < 0 s), during high-frequency 20 Hz stimulation (indicated by the thick black line) and upon return to low-frequency stimulation (time > 60 s). Black traces, mean values. Gray traces, 95% confidence limits for the mean. The gap in the traces
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was introduced to re-align the recordings from individual preparations to the start of recovery from depression. E. Fast and slow components of depression in the two experimental conditions. In all panels, error bars represent 95% confidence intervals about the mean values.
Fig. 3. Reduction in basal quantal content, decreased paired-pulse facilitation, and increased tetanic depression in nerve-muscle preparations treated with U-73122 (5 µM, n = 20) or vehicle control (DMSO, 0.01%, n = 15). A. EPP amplitudes recorded during low-frequency 0.2 Hz stimulation in control treated preparations (Ctrl) or preparations treated with U-73122. B. MEPP amplitude and frequency after 30 min treatment with DMSO (Ctrl) or U-73122, normalized to their values prior to treatment. C. Paired-pulse facilitation in control treated preparations (white circles) or preparations treated with U-73122 (black circles). D. Normalized EPP amplitudes during low-frequency 0.2 Hz stimulation (time < 0 s), during high-frequency 20 Hz stimulation (indicated by the thick black line) and upon return to low-frequency stimulation (time > 60 s). Black traces, mean values. Gray traces, 95% confidence limits for the mean. The gap in the traces was introduced to re-align the recordings from individual preparations to the start of recovery from depression. E-F. Fast and slow components of depression in the two experimental conditions. G-J, same as for C-F except that the structurally related inactive analog, U-73433, was tested. Error bars represent 95% confidence intervals about the mean values.
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Fig. 1. Reduced paired-pulse facilitation and tetanic depression in nerve-muscle preparations treated with Ryanodine (0.1 or 10 µM) or vehicle control (ethanol, 0.01%). A. EPP amplitudes recorded during lowfrequency 0.2 Hz stimulation in control treated preparations (Ctrl) or preparations treated with ryanodine (Ry, 10 µM). B. MEPP amplitude and frequency after 30 min treatment with ryanodine (Ry, 10 µM), normalized to their values prior to treatment. C. Paired-pulse facilitation in control treated preparations (white circles) or preparations treated with ryanodine (10 µM , black circles). D. Normalized EPP amplitudes during low-frequency 0.2 Hz stimulation (time < 0 s), during high-frequency 20 Hz stimulation (indicated by the thick black line) and upon return to low-frequency stimulation (time > 60 s). Black traces, mean values. Gray traces, 95% confidence limits for the mean. The gap in the traces was introduced to re-align the recordings from individual preparations to the start of recovery from depression. E-F. Fast and slow components of depression in control or ryanodine (10 µM). G-J, same as for C-F except that the concentration of Ryanodine was 0.1 µM. In all panels, error bars represent 95% confidence intervals about the mean values. n=10-25 per group.
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Fig. 2. Increased paired-pulse facilitation and reduced tetanic depression in nerve-muscle preparations treated with 2-APB (10 µM, n=15) or vehicle control (methanol, 0.002%, n=14). A. EPP amplitudes recorded during low-frequency 0.2 Hz stimulation in control treated preparations (Ctrl) or preparations treated with 2APB. B. MEPP amplitude and frequency after 30 min treatment with methanol (ctrl) or 2-APB, normalized to their values prior to treatment. C. Paired-pulse facilitation in control treated preparations (white circles) or preparations treated with 2-APB (black circles). D. Normalized EPP amplitudes during low-frequency 0.2 Hz stimulation (time < 0 s), during high-frequency 20 Hz stimulation (indicated by the thick black line) and upon return to low-frequency stimulation (time > 60 s). Black traces, mean values. Gray traces, 95% confidence limits for the mean. The gap in the traces was introduced to re-align the recordings from individual preparations to the start of recovery from depression. E. Fast and slow components of depression in the two experimental conditions. In all panels, error bars represent 95% confidence intervals about the mean values. 108x217mm (300 x 300 DPI)
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Fig. 3. Reduction in basal quantal content, decreased paired-pulse facilitation, and increased tetanic depression in nerve-muscle preparations treated with U-73122 (5 µM, n = 20) or vehicle control (DMSO, 0.01%, n = 15). A. EPP amplitudes recorded during low-frequency 0.2 Hz stimulation in control treated preparations (Ctrl) or preparations treated with U-73122. B. MEPP amplitude and frequency after 30 min treatment with DMSO (Ctrl) or U-73122, normalized to their values prior to treatment. C. Paired-pulse facilitation in control treated preparations (white circles) or preparations treated with U-73122 (black circles). D. Normalized EPP amplitudes during low-frequency 0.2 Hz stimulation (time < 0 s), during highfrequency 20 Hz stimulation (indicated by the thick black line) and upon return to low-frequency stimulation (time > 60 s). Black traces, mean values. Gray traces, 95% confidence limits for the mean. The gap in the traces was introduced to re-align the recordings from individual preparations to the start of recovery from depression. E-F. Fast and slow components of depression in the two experimental conditions. G-J, same as for C-F except that the structurally related inactive analog, U-73433, was tested. Error bars represent 95% confidence intervals about the mean values.
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Priscila E. Silveira, Ph.D1, Ricardo F. Lima, Ph.D 1,2, Jennifer D. S. Guimarães1, Jordi Molgó Ph.D3, Ligia A. Naves, Ph.D.1, Christopher Kushmerick, Ph.D.1
1-Departamento de Fisiologia e Biofísica, ICB, UFMG, Belo Horizonte, Brazil. 2 – Present address, Dept. Fisiologia e Farmacologia, UFC, Fortaleza, Brazil 3- C.N.R.S., Institut Fédératif de Neurobiologie Alfred Fessard Laboratoire de Neurobiologie et Dévelopement, CNRS, 91198-Gif sur Yvette, France. Correspondence to: Christopher Kushmerick Departamento de Fisiologia e Biofísica Instituto de Ciências Biológicas Universidade Federal de Minas Gerais Av Antonio Carlos 6627 Belo Horizonte, MG, 31270-901, Brazil [email protected] Tel +55 31 3409-2512
Acknowledgments Funded by grants from the Brazilian agencies CNPq and FAPEMIG. PES, RFL, and J.D.S.G were recipients of student fellowships from CAPES and CNPq. Keywords: Neuromuscular Junction, Ryanodine receptor, IP3 Receptor, Short-term plasticity, Intracellular Ca2+ stores. Running title: Ryanodine and IP3 receptors
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/mus.24571 This article is protected by copyright. All rights reserved.
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Abstract Introduction: Short-term plasticity of synaptic function is an important physiological control of transmitter release. Short-term plasticity can be regulated by intracellular calcium released by ryanodine and inositol tri-phosphate (IP3) receptors, but the role of these receptors at the neuromuscular junction is understood incompletely. Methods: We measured short-term plasticity of evoked endplate potential (EPP) amplitudes from frog neuromuscular junctions treated with ryanodine , 2-Aminoethoxydiphenylborane (2-APB), or 1-[6-[[(17β)-3-Methoxyestra-1,3,5(10)-trien-17yl]amino]hexyl]-1H-pyrrole-2,5-dione (U-73122). Results: Ryanodine decreases paired-pulse facilitation for intervals < 20 ms and markedly decreases tetanic depression. Treatment with 2-APB reduces EPP amplitude, increases paired-pulse facilitation for intervals < 20 ms, and significantly reduces tetanic depression. U-73122 decreases EPP amplitude and decreases paired pulse depression for intervals < 20 ms. Conclusions: Ryanodine, IP3 receptors, and phopholipase C modulate short-term plasticity of transmitter release at the neuromuscular junction. These results suggest possible targets to improve the safety factor for neuromuscular transmission during repetitive activity of the neuromuscular junction.
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Introduction Short-term synaptic plasticity refers to activity-dependent modification of the strength or efficacy of transmission at preexisting synapses. Although post-synaptic mechanisms such as receptor desensitization can contribute to short-term plasticity, expression of 2 well-studied forms of plasticity, paired-pulse facilitation and tetanic depression, largely reflect changes in transmitter release, which depend on both the number of available quanta of neurotransmitter and their release probability.1 Both of these parameters may change during expression of short-term plasticity, but the mechanisms that underlie these changes in the release process are understood incompletely. Several lines of evidence suggest that expression of short-term plasticity at the neuromuscular junction is associated with alterations in presynaptic Ca2+ dynamics during repetitive high-frequency stimulation. These include the following observations: 1) increasing the ratio of Mg2+/Ca2+ in the bathing solution reduces Ca2+ entry through voltage-gated channels, reduces release probability, and consequently shifts the response to high-frequency stimulation from depression to facilitation;2 2) loading nerve terminals with the Ca2+ chelator 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) inhibits facilitation;3 and 3) block of Ca2+ uptake into or release from mitochondria inhibits the post-tetanic rise in intracellular Ca2+ and post-tetanic potentiation of quantal content.4,5 Thus, manipulations that interfere with Ca2+ dynamics affect short-term plasticity. The endoplasmic reticulum (ER) is another well-characterized source of intracellular Ca2+, and Ca2+ release from the ER via ryanodine receptors or IP3 receptors plays an important role in cellular responses to both electrical and chemical signals.6 However, compared to the role of Ca2+ entry, Ca2+ clearance, and calcium uptake and release by mitochondria, the role of Ca2+ released from ER in evoked neurotransmitter release and its short-term plasticity at the neuromuscular junction is less explored. Previous studies indicate that Ca2+ released from the ER modulates transmitter release at the neuromuscular junction. Ca2+ released from internal stores by caffeine promotes spontaneous synaptic John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.
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vesicle exocytosis7,8. During nerve stimulation under conditions of low-Ca2+/high Mg2+, Ca2+-induced Ca2+-release underlies a form of short-term plasticity mediated by ryanodine receptors.9 Stimulation of the neuromuscular junction with exogenous IP3 applied via liposomes increases quantal transmitter release,10 suggesting the presence of IP3 receptors at the nerve terminal. IP3 can be formed by Gprotein coupled receptors that activate phospholipase C, and there is evidence that this pathway may be active in neuromuscular junctions, since activation of phospholipase C and IP3 receptors are necessary to mediate neurotrophin-3-induced potentiation of synaptic transmission in motoneuron/muscle fiber co-cultures.11 Nonetheless, it is still unknown how Ca2+ release from the ER contributes to short-term plasticity in the intact neuromuscular junction when the extracellular Ca2+ concentration is at normal levels. In addition, release of Ca2+ via activation of ryanodine and IP3 receptors has been implicated in certain neuromuscular junction diseases including the alterations of neurotransmitter release evoked by Amyotrophic Lateral Sclerosis IgG12 and the facilitation of spontaneous release by beta bungarotoxin13. Therefore, knowledge on the role of these receptors in synapse function will also be relevant to understanding certain pathological states. In an attempt to clarify the role of ryanodine and IP3 receptors in synaptic function and plasticity, we blocked these receptors with ryanodine or 2-APB, or blocked the phospholipase that generates IP3 with U-73122 and investigated the effects of these treatments on evoked quantal transmitter release during low-frequency stimulation during application of paired pulses to measure facilitation and during tetanic depression.
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Methods Preparation and solutions Experiments were performed at room temperature using the cutaneous pectoris nerve-muscle preparations from the frog Rana temporaria, Rana esculenta, or Rana catesbeiana weighing 20-70 g. The animals were sacrificed by double pithing according to local animal care guidelines, and the muscle with attached nerve was pinned onto a bed of Sylgard in a 3-4 ml chamber. The chamber was filled with Ringer contained (in mM): NaCl, 115; KCl, 2.5; CaCl2, 2; and N-[2Hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid] (HEPES), 5; pH 7.4 (adjusted with NaOH). For experiments employing nerve stimulation, tubocurarine (6-8 x 10-6 M) was added to reduce the EPP amplitudes to below threshold and thus block muscle contraction. Electrophysiological recordings EPP amplitudes were recorded with intracellular microelectrodes fabricated from borosilicate glass with resistances of 5-15 M-Ohm when filled with 3 M KCl and measured while immersed in the Ringer solution. Recordings were obtained using a Dagan 8500 amplifier (Dagan Co., Minneapolis, USA) or an Axoclamp-2A amplifier (Molecular Devices, CA, USA). The signals were further amplified 50 - 500 fold by an Ectron 750 amplifier (Ectron Co., San Diego, USA) or Grass AC/DC strain gauge amplifier (Astro-Med Inc., W. Warnick, USA) and then fed to an A/D converter controlled by a PC computer. Data acquisition was controlled using the Strathclyde Electrophysiology Software suite (University of Strathclyde, Glasgow, Scotland). The resting membrane potential was monitored continuously during the measurements, and EPP amplitudes were corrected for changes in resting potential (using -90 mV as a standard resting potential) and for non-linear summation assuming a reversal potential of -15 mV.14 The nerve stump was drawn into a suction electrode and stimulated using 100 µs supramaximal stimuli applied at 0.2 Hz using a Grass SD9 or S48 stimulator (Astro-Med Inc., W. Warnick, USA).
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Minature EPP (MEPP) amplitudes were measured in the same fiber in control conditions (normal frog Ringer) and 15 and 30 minutes after addition of drugs or vehicle and were corrected as described above for EPP amplitudes. To measure facilitation, EPP amplitudes were measured when the nerve was stimulated by 30 pairs of supramaximal stimuli separated by intervals ranging from 5 to 60 msec and delivered every 5 sec. For the shortest intervals, the tail of the first EPP overlapped the peak of the second. In these cases we calculated the amplitude of the second EPP as the difference of the peak potential and the projected tail of the preceding EPP. The amount of facilitation (f) in any given response was defined by the quantity f= (EPP2 – EPP1)/EPP1, where EPP1 and EPP2 are the mean amplitudes of the first and second EPPs, corrected for resting potential and non-linear summation (see above). To measure depression, we stimulated the nerve at 0.2 Hz to measure the baseline EPP amplitude. After 30 stimuli at 0.2 Hz, we increased the stimulation frequency to 20 Hz for 60 s to generate EPP depression and then returned to 0.2 Hz for an additional 5 min to measure recovery from depression. Drugs and toxins All chemicals were ACS grade. Ryanodine and 2-APB were obtained from Sigma (Saint Louis, USA), U-73122 was obtained from Calbiochem (San Diego, CA, USA), and U-73343 was obtained from Tocris (UK). Drugs were added directly to the bath from ethanol, methanol, or DMSO stock solution to the final concentrations given in the text. Control experiments were performed separately with the drug vehicles, and differences were analyzed using an unpaired t-test. Data analysis Data were analyzed by custom-written software within Igor 6 (Wavemetrics, Lake Oswego, USA). EPP amplitudes during tetanic trains were normalized to their amplitudes during the preceding baseline period of low-frequency stimulation. Normalized EPP amplitude changes during tetanic stimulation occurred in 3 phases, an initial facilitation followed by a fast phase of depression that gave way to a John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.
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slower, more linear phase of depression that continued until the end of the 60 s tetanic train. Facilitation during the train was defined as the largest normalized EPP amplitude after the onset of the train. Slow depression was the slope of a line fit to the last 15 s of EPP amplitude data before the end of the train. Fast depression was the difference between the EPP amplitude at peak train facilitation and the extrapolation of slow depression back to the point of maximum facilitation, expressed as the fraction (%) of maximum facilitation. EPP amplitudes in nerve-muscle preparations treated with drugs were compared with preparations treated with the corresponding drug vehicle. MEPP amplitudes and frequency were measured in the same fiber in normal frog Ringer solution, and after 30 min treatment with either drugs or vehicle, and are expressed normalized to their values in control Ringer solution. Statistical analysis of data was performed using the Student t-test. Values are expressed as mean ± 95% confidence intervals, and error bars in figures represent the 95% confidence intervals about the mean, calculated using the Student tdistribution. Differences in mean values given in the results were designated as statistically significant when P < 0.05.
Results Calcium-induced calcium release Previous studies indicate that blockade of ryanodine receptors reduces frequency facilitation when low Ca2+ and high Mg2+ are used to block muscle contraction.9 In order to evaluate the role of ryanodine receptors in evoked quantal transmitter release under more physiological conditions, we used normal frog Ringer solution and blocked contractions with tubocurarine. Under these conditions, ryanodine (10-5 M) did not significantly change the amplitude of EPPs evoked at 0.2 Hz compared to vehicle control (ethanol 0.1%; Fig. 1A). In separate experiments without tubocurarine, we observed no significant effect of these concentrations of ryanodine or ethanol on the amplitude or frequency of John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.
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MEPPs (Fig. 1B). Based on the lack of effect of ryanodine on EPP and MEPP amplitudes, we conclude that ryanodine does not alter quantal content during low-frequency stimulation. We next examined the effects of ryanodine on paired pulse facilitation with intervals ranging from 5-60 ms and observed that ryanodine reduced facilitation at short intervals (< 20 ms), but not at longer intervals (Fig. 1C). During prolonged 20 Hz stimulation, EPP amplitudes from control fibers exhibited fast and slow components of depression (Fig. 1D, see Methods for definitions). In preparations treated with ryanodine (10-5 M) the fast phase of depression was reduced markedly (Fig 1E). Although there was some reduction in the slow phase of depression, this difference was not statistically significant (Fig. 1F). After stimulating at 20 Hz for 50 s, EPP amplitudes were reduced to 37 ± 18 % of their initial values (n = 15), while in preparations treated with ryanodine they were sustained, remaining at 96 ± 33% of their initial values (n = 12). (Here and below, mean values are given ± their 95% confidence intervals). Thus, for 20 Hz stimulation, preparations treated with ryanodine (10-5 M) exhibited much less EPP depression. High concentrations of ryanodine block the ryanodine receptor, whereas lower concentrations may facilitate channel opening.15 We thus repeated our measurements in muscles treated with 10-7 M ryanodine. In these experiments, ryanodine (10-7 M) did not significantly affect EPP amplitudes recorded at low frequency (control: 2.7 ± 1.1 mV, n = 25 vs RY 1.9 ± 0.44 mV, n = 22; P=0.15, t-test), paired pulse facilitation, or the fast and slow components of depression (Fig. 1 G-J).
IP3 Receptors We compared responses in preparations treated with 10-5 M 2-APB, an antagonist of IP3 receptors, with vehicle controls (0.002% methanol, Fig 2). In fibers treated with 2-APB, EPP amplitudes were smaller than controls, but not significantly (control 2.54 ± 0.67 mV, n=14 vs. APB 1.81 ± 0.49 mV, n=16, P=0.064, t-test). Neither 2-APB nor its vehicle significantly affected MEPP amplitudes or frequency. John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.
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Paired-pulse facilitation was increased by 2-APB for intervals < 20 ms. During tetanic high-frequency stimulation, fibers treated with 2-APB exhibited less fast depression than controls. IP3 is formed from PIP2 in a reaction catalyzed by phopholipase-C. We therefore tested the effect of an inhibitor of this enzyme, U-73122 (5 x 10-6 M, Fig. 3). EPP amplitudes in fibers treated with U-73122 were significantly smaller than vehicle controls (DMSO 0.05%, 4.29 ± 1.1 mV, n = 15 vs U-73122 2.57 ± 0.69 mV, n = 20, P = 0.009, t-test). In separate experiments, U-73122 caused a 30 ± 23% increase in MEPP amplitudes (n=4, P=0.02, paired t-test) that was not observed in DMSO controls (n = 5). Pairedpulse facilitation was reduced by U-73122 for intervals < 30 ms, whereas fast depression was increased. The compound 1-[6-[[(17β)-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrrolidinedione (U-73433) is structurally very similar to U-73122 but is reported to be inactive on PLC. 16. To determine the specificity of the effects of U-73122 described above we therefore tested U-73433 (5x10-6 M; Fig 3 G-J). Unlike U-73122, U-73433 did not reduce EPP amplitudes (control: 1.86 ± 0.38 mV, n. = 24 vs. U-73433: 1.71 ± 0.22 mV, n = 24, P=0.46, t-test) and had no effect on paired-pulse facilitation (Fig 3G). However, the increase in fast depression by U-73122 was also observed with U-73433 (Fig 3 H-J). Discussion Release of Ca2+ from intracellular stores via ryanodine and IP3 receptors plays a role in synaptic plasticity at several synapses17–19. Here, we examined the role of these 2 receptors at the neuromuscular junction during low- and high-frequency stimulation at a physiological concentration of extracellular Ca2+. Initial EPP amplitudes and recovery after tetanic trains were measured during stimulation at 0.2 Hz, a frequency that generated little short-term plasticity. For tetanic trains, we employed relatively long stimulus trains at 20 Hz. Long trains were necessary to measure accurately the slow phase of depression. The choice of 20 Hz for tetanic stimulation was based on the observation that in control John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.
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conditions it generated EPP depression of roughly 50% and thus allowed us to test for either an increase or a decrease in depression. Previous studies demonstrated a role for ryanodine receptors and calcium-induced calcium release in the release of neurotransmitter at the neuromuscular junction. 9 Treatment with ryanodine reduces paired-pulse facilitation for short intervals (< 20 ms, corresponding to stimulation frequencies > 50 Hz). On the other hand, we observed an increase in quantal output during 20 Hz stimulation when ryanodine receptors (and, presumably, calcium-induced calcium release) were blocked with ryanodine. Simple depletion models with mono-exponential replenishment predict more rather than less depression if frequency facilitation is blocked.1 Therefore, it seems that treatment with ryanodine accelerates the rate at which the vesicle pool is replenished during high-frequency stimulation. Although in central nervous system synapses replenishment of the vesicle pool is accelerated by activity in a Ca2+-dependent manner 20, our data suggest that Ca2+ release by ryanodine receptors at the neuromuscular junction has the opposite effect and limits release of neurotransmitter during tetanic trains. Given the neuromuscular junction safety margin 21–23, this may be an efficient way to allocate resources. Previous studies have shown that exogenous IP3 introduced into the frog neuromuscular nerve terminal increased the quantal content of EPPs, indicating that IP3 receptors are coupled to transmitter release. 10
Blocking IP3 receptors with 2-APB (10-5 M) had no effect on MEPP frequency. It increased
facilitation and reduced depression during high-frequency stimulation. This suggests that IP3 receptors are activated by nerve stimulation and contribute to the expression of short-term plasticity. Paired pulse facilitation in the controls for the 2-APB group was lower than for the controls of the other groups. This may be a vehicle effect or a seasonal variation, but all results are reported versus control experiments realized in the same period and in the presence of drug vehicle. What might be the source of IP3 during nerve stimulation? At the central nervous system, presynaptic auto- or heteroreceptor
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activation stimulates phospolipase C and IP3 formation. 24,25 Previous studies indicate a role for IP3 in the presynaptic receptor-mediated modulation of spontaneous release from cultured Xenopus motoneurons 11 and in evoked release at the crayfish neuromuscular junction. 26 The frog neuromuscular junction expresses several G protein coupled receptors including muscarinic M1 autoreceptors. 27 In neuronal cell lines, activated M1 receptors couple though Gq/11 and phospholipase C to produced IP3 28, suggesting a possible route for IP3 formation via autoreceptor activation. Our finding of reduced EPP amplitude at low frequency when IP3 receptors are blocked with 2-APB is consistent with previous reports that activation of M1 autoreceptors at the frog neuromuscular junction facilitate release27,29 and suggest that endogenously formed IP3 or some other mechanism of activation of IP3 receptors affects release even at low stimulation frequencies. In addition, the reduction in tetanic depression by APB suggests that IP3 receptors contribute to a reduction in quantal output during highfrequency stimulation. Treatment with U-73122, an inhibitor of the G-protein coupled phospholipase that produces IP3, but not its inactive analog U-73433 reduced EPP amplitude, like 2-APB. U-73122, but not U-73433, also reduced paired-pulse facilitation, an effect that was not observed for 2-APB. In addition to IP3, cleavage of PIP2 forms DAG, an activator of protein kinase C that regulates neurotransmitter release 30. Since inhibition of phospholipase C will affect both pathways, it is not surprising that drugs that affect this enzyme have effects that are more complex than that of APB. Both U-73122 and U-73433 increased tetanic depression, suggesting that this effect is unrelated to block of phospholipase C. Previous studies have demonstrated a use-dependent increase in dense-core vesicle mobility in Drosophila neuromuscular junctions via a process that can be blocked by Ryanodine and KN-93, an inhibitor of CaMKII31. In the frog neuromuscular junction, small synaptic vesicles can be separated into 2 pools based on their mobility, a relatively mobile recycling pool and a immobile reserve pool32. Nerve activity increases the mobility of the immobile reserve. Future studies might address whether
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Ryanodine affects this activity-dependent mobilization. Previous reports at the frog neuromuscular junction have shown that ryanodine receptors mediate Ca2+induced Ca2+ release that modulates transmitter release at the frog neuromuscular junction. 9,33,34 In these studies, the ryanodine-sensitive increase of release induced by a previous conditioning train did not change facilitation during brief trains. We observed, in the absence of conditioning trains, that ryanodine decreased facilitation. Two factors may account for these differences. Firstly, we observed effects of ryanodine on facilitation for intervals of 15 ms and below, shorter than the intervals used in the aforementioned study9. Secondly, we measured facilitation in Ringer solution containing 1.8 mM Ca2+ without Mg2+, and the relatively large influx of Ca2+ in these conditions may generate results that are different than are obtained in low Ca2+ / high Mg2+ Ringer solution. In conclusion, expression of 2 well-defined forms of synaptic plasticity, paired-pulse facilitation and tetanic depression, are regulated by IP3 receptors and ryanodine receptors at the frog neuromuscular junction. These data suggest that Ca2+-dependent second messenger pathways may play an important role in quantal release of transmitter at this synapse.
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Abbreviations 2-APB, 2-Aminoethoxydiphenylborane BAPTA , 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid EPP, evoked endplate potential ER, endoplasmic reticulum HEPES, N-[2-Hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]
IP3, inositol tri-phosphate MEPP, minature EPP U-73122, 1-[6-[[(17β)-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione U-73433, 1-[6-[[(17β)-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrrolidinedione
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Figure Legends Fig. 1. Reduced paired-pulse facilitation and tetanic depression in nerve-muscle preparations treated with Ryanodine (0.1 or 10 µM) or vehicle control (ethanol, 0.01%). A. EPP amplitudes recorded during low-frequency 0.2 Hz stimulation in control treated preparations (Ctrl) or preparations treated with ryanodine (Ry, 10 µM). B. MEPP amplitude and frequency after 30 min treatment with ryanodine (Ry, 10 µM), normalized to their values prior to treatment. C. Paired-pulse facilitation in control treated preparations (white circles) or preparations treated with ryanodine (10 µM , black circles). D. Normalized EPP amplitudes during low-frequency 0.2 Hz stimulation (time < 0 s), during highfrequency 20 Hz stimulation (indicated by the thick black line) and upon return to low-frequency stimulation (time > 60 s). Black traces, mean values. Gray traces, 95% confidence limits for the mean. The gap in the traces was introduced to re-align the recordings from individual preparations to the start of recovery from depression. E-F. Fast and slow components of depression in control or ryanodine (10 µM). G-J, same as for C-F except that the concentration of Ryanodine was 0.1 µM. In all panels, error bars represent 95% confidence intervals about the mean values. n=10-25 per group.
Fig. 2. Increased paired-pulse facilitation and reduced tetanic depression in nerve-muscle preparations treated with 2-APB (10 µM, n=15) or vehicle control (methanol, 0.002%, n=14). A. EPP amplitudes recorded during low-frequency 0.2 Hz stimulation in control treated preparations (Ctrl) or preparations treated with 2-APB. B. MEPP amplitude and frequency after 30 min treatment with methanol (ctrl) or 2-APB, normalized to their values prior to treatment. C. Paired-pulse facilitation in control treated preparations (white circles) or preparations treated with 2-APB (black circles). D. Normalized EPP amplitudes during low-frequency 0.2 Hz stimulation (time < 0 s), during high-frequency 20 Hz stimulation (indicated by the thick black line) and upon return to low-frequency stimulation (time > 60 s). Black traces, mean values. Gray traces, 95% confidence limits for the mean. The gap in the traces
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was introduced to re-align the recordings from individual preparations to the start of recovery from depression. E. Fast and slow components of depression in the two experimental conditions. In all panels, error bars represent 95% confidence intervals about the mean values.
Fig. 3. Reduction in basal quantal content, decreased paired-pulse facilitation, and increased tetanic depression in nerve-muscle preparations treated with U-73122 (5 µM, n = 20) or vehicle control (DMSO, 0.01%, n = 15). A. EPP amplitudes recorded during low-frequency 0.2 Hz stimulation in control treated preparations (Ctrl) or preparations treated with U-73122. B. MEPP amplitude and frequency after 30 min treatment with DMSO (Ctrl) or U-73122, normalized to their values prior to treatment. C. Paired-pulse facilitation in control treated preparations (white circles) or preparations treated with U-73122 (black circles). D. Normalized EPP amplitudes during low-frequency 0.2 Hz stimulation (time < 0 s), during high-frequency 20 Hz stimulation (indicated by the thick black line) and upon return to low-frequency stimulation (time > 60 s). Black traces, mean values. Gray traces, 95% confidence limits for the mean. The gap in the traces was introduced to re-align the recordings from individual preparations to the start of recovery from depression. E-F. Fast and slow components of depression in the two experimental conditions. G-J, same as for C-F except that the structurally related inactive analog, U-73433, was tested. Error bars represent 95% confidence intervals about the mean values.
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Fig. 1. Reduced paired-pulse facilitation and tetanic depression in nerve-muscle preparations treated with Ryanodine (0.1 or 10 µM) or vehicle control (ethanol, 0.01%). A. EPP amplitudes recorded during lowfrequency 0.2 Hz stimulation in control treated preparations (Ctrl) or preparations treated with ryanodine (Ry, 10 µM). B. MEPP amplitude and frequency after 30 min treatment with ryanodine (Ry, 10 µM), normalized to their values prior to treatment. C. Paired-pulse facilitation in control treated preparations (white circles) or preparations treated with ryanodine (10 µM , black circles). D. Normalized EPP amplitudes during low-frequency 0.2 Hz stimulation (time < 0 s), during high-frequency 20 Hz stimulation (indicated by the thick black line) and upon return to low-frequency stimulation (time > 60 s). Black traces, mean values. Gray traces, 95% confidence limits for the mean. The gap in the traces was introduced to re-align the recordings from individual preparations to the start of recovery from depression. E-F. Fast and slow components of depression in control or ryanodine (10 µM). G-J, same as for C-F except that the concentration of Ryanodine was 0.1 µM. In all panels, error bars represent 95% confidence intervals about the mean values. n=10-25 per group.
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Fig. 2. Increased paired-pulse facilitation and reduced tetanic depression in nerve-muscle preparations treated with 2-APB (10 µM, n=15) or vehicle control (methanol, 0.002%, n=14). A. EPP amplitudes recorded during low-frequency 0.2 Hz stimulation in control treated preparations (Ctrl) or preparations treated with 2APB. B. MEPP amplitude and frequency after 30 min treatment with methanol (ctrl) or 2-APB, normalized to their values prior to treatment. C. Paired-pulse facilitation in control treated preparations (white circles) or preparations treated with 2-APB (black circles). D. Normalized EPP amplitudes during low-frequency 0.2 Hz stimulation (time < 0 s), during high-frequency 20 Hz stimulation (indicated by the thick black line) and upon return to low-frequency stimulation (time > 60 s). Black traces, mean values. Gray traces, 95% confidence limits for the mean. The gap in the traces was introduced to re-align the recordings from individual preparations to the start of recovery from depression. E. Fast and slow components of depression in the two experimental conditions. In all panels, error bars represent 95% confidence intervals about the mean values. 108x217mm (300 x 300 DPI)
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Fig. 3. Reduction in basal quantal content, decreased paired-pulse facilitation, and increased tetanic depression in nerve-muscle preparations treated with U-73122 (5 µM, n = 20) or vehicle control (DMSO, 0.01%, n = 15). A. EPP amplitudes recorded during low-frequency 0.2 Hz stimulation in control treated preparations (Ctrl) or preparations treated with U-73122. B. MEPP amplitude and frequency after 30 min treatment with DMSO (Ctrl) or U-73122, normalized to their values prior to treatment. C. Paired-pulse facilitation in control treated preparations (white circles) or preparations treated with U-73122 (black circles). D. Normalized EPP amplitudes during low-frequency 0.2 Hz stimulation (time < 0 s), during highfrequency 20 Hz stimulation (indicated by the thick black line) and upon return to low-frequency stimulation (time > 60 s). Black traces, mean values. Gray traces, 95% confidence limits for the mean. The gap in the traces was introduced to re-align the recordings from individual preparations to the start of recovery from depression. E-F. Fast and slow components of depression in the two experimental conditions. G-J, same as for C-F except that the structurally related inactive analog, U-73433, was tested. Error bars represent 95% confidence intervals about the mean values.
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