Frequency Of Miniature Endplate Currents Research Articles

Prenatal hyperhomocysteinemia induces oxidative stress and accelerates ‘aging’ of mammalian neuromuscular synapses

Enhanced levels of homocysteine during pregnancy induce oxidative stress and contribute to many age-related diseases. In this study, we analyzed age-dependent synaptic modifications in developing neuromuscular synapses of rats with prenatal hyperhomocysteinemia (hHCY). One of the main findings indicate that the intensity and the timing of transmitter release in synapses of neonatal (P6 and P10) hHCY rats acquired features of matured synaptic transmission of adult rats. The amplitude and frequency of miniature end-plate currents (MEPCs) and evoked transmitter release were higher in neonatal hHCY animals compared to the control group. Analysis of the kinetics of neurotransmitter release demonstrated more synchronized release in neonatal rats with hHCY. At the same time lower release probability was observed in adults with hHCY. Spontaneous transmitter release in neonates with hHCY was inhibited by hydrogen peroxide (H2O2) whereas in controls this oxidant was effective only in adult animals indicating a higher susceptibility of motor nerve terminals to oxidative stress. The morphology and the intensity of endocytosis of synaptic vesicles in motor nerve endings was assessed using the fluorescence dye FM 1-43. Adult-like synapses were found in neonates with hHCY which were characterized by a larger area of presynaptic terminals compared to controls. No difference in the intensity of FM 1-43 fluorescence was observed between two groups of animals. Prenatal hHCY resulted in reduced muscle strength assessed by the Paw Grip Endurance test. Using biochemical assays we found an increased level of H2O2 and lipid peroxidation products in the diaphragm muscles of hHCY rats. This was associated with a lowered activity of superoxide dismutase and glutathione peroxidase. Our data indicate that prenatal hHCY induces oxidative stress and apparent faster functional and morphological "maturation" of motor synapses. Our results uncover synaptic mechanisms of disrupted muscle function observed in hHCY conditions which may contribute to the pathogenesis of motor neuronal diseases associated with enhanced level of homocysteine.

R-spondin 2 promotes acetylcholine receptor clustering at the neuromuscular junction via Lgr5

At the neuromuscular junction (NMJ), acetylcholine receptor (AChR) clustering is mediated by spinal motor neuron (SMN)-derived agrin and its receptors on the muscle, the low-density lipoprotein receptor-related protein 4 (LRP4) and muscle-specific receptor tyrosine kinase (MuSK). Additionally, AChR clustering is mediated by the components of the Wnt pathway. Laser capture microdissection of SMNs revealed that a secreted activator of Wnt signaling, R-spondin 2 (Rspo2), is highly expressed in SMNs. We found that Rspo2 is enriched at the NMJ, and that Rspo2 induces MuSK phosphorylation and AChR clustering. Rspo2 requires Wnt ligands, but not agrin, for promoting AChR clustering in cultured myotubes. Leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5), an Rspo2 receptor, is also accumulated at the NMJ, and is associated with MuSK via LRP4. Lgr5 is required for Rspo2-mediated AChR clustering in myotubes. In Rspo2-knockout mice, the number and density of AChRs at the NMJ are reduced. The Rspo2-knockout diaphragm has an altered ultrastructure with widened synaptic clefts and sparse synaptic vesicles. Frequency of miniature endplate currents is markedly reduced in Rspo2-knockout mice. To conclude, we demonstrate that Rspo2 and its receptor Lgr5 are Wnt-dependent and agrin-independent regulators of AChR clustering at the NMJ.

Effects of 5α-cholestan-3-one on the synaptic vesicle cycle at the mouse neuromuscular junction

We have investigated the effects of 5α-cholesten-3-one (5Ch3, 200 nM) on synaptic transmission in mouse diaphragm. 5Ch3 had no impact on the amplitude or frequency of miniature endplate currents (MEPCs, spontaneous secretion), but decreased the amplitude of EPCs (evoked secretion) triggered by single action potentials. Treatment with 5Ch3 increased the depression of EPC amplitude and slowed the unloading of the dye FM1-43 from synaptic vesicles (exocytosis rate) during high-frequency stimulation. The estimated recycling time of vesicles did not change, suggesting that the decline of synaptic efficiency was due to the reduction in the size of the population of vesicles involved in release. The effects of 5Ch3 on synaptic transmission may be related to changes in the phase properties of the membrane. We have found that 5Ch3 reduces the staining of synaptic regions with the B-subunit of cholera toxin (a marker of lipid rafts) and increases the fluorescence of 22-NBD-cholesterol, indicating a phase change within the membrane. Manipulations of membrane cholesterol (saturation or depletion) strongly reduced the influence of 5Ch3 on both FM1-43 dye unloading and staining with the B-subunit of cholera toxin. Thus, 5Ch3 reduces the number of vesicles which are actively recruited during synaptic transmission and alters membrane properties. These effects of 5Ch3 depend on membrane cholesterol.

Topography and Affinity of Calcium Sensors of Exo- and Endocytosis in Motor Nerve Terminals

Measurements with extracellular microelectrode technique showed that depolarization of motor nerve terminals in frog cutaneous pectoris muscle with high-potassium solution (40 mM K(+)) increased frequency of miniature end-plate currents. Both fast intracellular calcium chelator BAPTA-AM and slow chelator EGTA-AM equally moderated the increase in the frequency of miniature end-plate currents. Intravital fluorescent microscopy with FM 1-43 dye showed that under conditions of stimulation of neurotransmitter exocytosis and secretion with high-potassium solution, internalization of the dye into newly-formed endocytotic synaptic vesicles proceeded both in the control and in the presence of EGTA-AM. In contrast, internalization of the dye was not observed in the presence of BAPTA-AM. It was concluded that asynchronous exocytosis of synaptic vesicles goes on in the active zones enriched with Ca-channels due to activation of high-affinity Ca-site in Ca-macrodomain. Endocytosis of vesicles is probably initiated by Ca-microdomain during activation of low-affinity Ca-site in the immediate proximity to the Ca channel.

Calpain activation impairs neuromuscular transmission in a mouse model of the slow-channel myasthenic syndrome.

The slow-channel myasthenic syndrome (SCS) is a hereditary disorder of the acetylcholine receptor (AChR) of the neuromuscular junction (NMJ) that leads to prolonged AChR channel opening, Ca(2+) overload, and degeneration of the NMJ. We used an SCS transgenic mouse model to investigate the role of the calcium-activated protease calpain in the pathogenesis of synaptic dysfunction in SCS. Cleavage of a fluorogenic calpain substrate was increased at the NMJ of dissociated muscle fibers. Inhibition of calpain using a calpastatin (CS) transgene improved strength and neuromuscular transmission. CS caused a 2-fold increase in the frequency of miniature endplate currents (MEPCs) and an increase in NMJ size, but MEPC amplitudes remained reduced. Persistent degeneration of the NMJ was associated with localized activation of the non-calpain protease caspase-3. This study suggests that calpain may act presynaptically to impair NMJ function in SCS but further reveals a role for other cysteine proteases whose inhibition may be of additional therapeutic benefit in SCS and other excitotoxic disorders.

The role of extracellular calcium in exo- and endocytosis of synaptic vesicles at the frog motor nerve terminals

In the present study we combined FM 1-43 imaging and electrophysiological recording of miniature end-plate currents (MEPCs) to determine the role of extracellular calcium in synaptic vesicle exo- and endocytosis at the frog motor nerve terminals. We replaced extracellular Ca 2+ ions with other bivalent cations (Sr 2+, Ba 2+, Cd 2+, Mg 2+) or used a calcium-free solution and monitored fluorescent staining of the nerve terminals in the presence of caffeine, which promotes the release of Ca 2+ from intracellular stores. Caffeine has induced FM1-43 internalization only in the presence of bivalent cations in the external solution. The exposure of the neuromuscular junction to caffeine in a calcium-free solution caused a reversible failure of FM 1-43 loading and an increase in the nerve terminal width. This effect of a calcium-free solution was not due to a decrease in exocytosis, because caffeine-induced FM1-43 unloading from the previously loaded nerve terminals, as well as a degree of the MEPCs frequency increase, was unchanged. We conclude that the presence of Ca 2+ or other bivalent cations in extracellular space is necessary for endocytosis but not for exocytosis of synaptic vesicles, while transmitter release is promoted by efflux of Ca 2+ from intracellular stores. The effect of extracellular Ca 2+ on endocytosis might be driven by the non-specific interactions with membrane lipids.

Effects of high-potassium solutions and caffeine on synaptic vesicle exoendocytosis processes in the frog neuromuscular junction

Studies on frog skin-pectoris muscle preparations using vital fluorescent microscopy showed that stimulation of transmitter secretion using high-potassium solutions with the endocytosis marker FM 1-43 induced bright spots in all motor nerve terminals, these representing accumulations of vesicles undergoing the exoendocytic cycle in the active zones of nerve endings. Stimulation of transmitter secretion with caffeine evoked bright spots only in some nerve terminals and only in some parts of the terminals. In summer, the number of bright spots on stimulation of transmitter secretion by caffeine increased sharply. Extracellular recording of spontaneous synaptic signals showed that high-potassium solutions, like caffeine, produced dose-dependent increases in the frequency of miniature endplate currents. However, while high-potassium solutions always increased the frequency, this occurred with caffeine in only a proportion of experiments. This leads to the conclusion that exoendocytosis processes can occur both because of the influx of Ca(2+) ions into nerve endings as a result of depolarization (high-potassium solutions) and because of the release of Ca(2+) ions from the endoplasmic reticulum (caffeine). The possible spatial localization of the endoplasmic reticulum in nerve endings is discussed. The endoplasmic reticulum is suggested to have a role in synapse remodeling processes.

Kiss-and-run quantal secretion in frog nerve-muscle synapse.

Electrophysiological and optical methods were used to study exo- and endocytosis of synaptic vesicles underlying secretion of the neurotransmitter from motor nerve terminals in frog sternocutaneous muscle. Increase in extracellular concentration of K+ or sucrose produced similar increase in the frequency of miniature endplate currents recorded by extracellular microelectrode. Fluorescent microscopy revealed bright spots in nerve terminal during stimulation of secretion with high-potassium solutions in the presence of endocytosis marker FM1-43. These spots corresponded to clusters of synaptic vesicles that passed through the cycles of exo- and endocytosis. Subsequent high-potassium stimulation of exocytosis in normal Ringer solution led to disappearance of marker spots, while in hyperosmotic saline the spots were preserved. No spots were seen after stimulation of neurotransmitter secretion with sucrose in the presence of FM1-43. It is concluded that quantal secretion of the neurotransmitter in frog motor nerve endings can be realized via both complete exocytosis of synaptic vesicles with subsequent endocytosis and kiss-and-run mechanism with the formation of a temporary pore.

Transmitter secretion in the frog neuromuscular synapse after prolonged exposure to calcium-free solutions.

Experiments on neuromuscular synapses from frog skin/chest muscle preparations in conditions of extracellular recording addressed changes in the spontaneous and evoked transmitter secretion after long-term (1.5-6 h) maintenance of preparations in calcium-free solution containing EGTA. Use of three microelectrodes for recording of single-quantum postsynaptic signals showed that calcium-free solution altered the characteristic topography of transmitter secretion in nerve terminals, with widening and fusion of groups of transmitter release. These changes persisted after preparations were returned to the initial solution. These data suggest that calcium-free solutions lead to disorganization of the active zones of nerve endings, At initially low extracellular Ca ion concentrations (0.15-0.4 mM), disorganization of active zones induced by prolonged maintenance of preparations in calcium-free solutions led to decreases in the mean amplitude of endplate currents (EPC) because of decreases in their quantum composition, increases in the time course of transmitter secretion, and decreases in the frequency of miniature endplate currents. The relationship between quantum composition of EPC and the extracellular Ca ion concentration showed a sharp displacement towards higher concentrations, without significant changes in the slope of the relationship. At high initial Ca concentrations (1.8 mM), long-term exposure to calcium-free solutions led to a less marked decrease in EPC amplitude. It is suggested that the extra- and intracellular Ca ion concentrations support the maintenance of the characteristic morphofunctional organization of the apparatus responsible for transmitter secretion in frog nerve endings. Disorganization of the active zones leads to disruption of elements involved in transmitter secretion and decreases in the efficiency of secretion.

Thyroid hormones regulate the frequency of miniature end-plate currents in pre- and prometamorphic stages of the tadpole tail.

Thyroid hormones (THs), primarily 3,3',5-triiode-(L)-thyronine (T(3)), have been clearly established as natural inducers of apoptosis during metamorphosis of anuran embryos. We decided to use this phenomenon to test the hypothesis that, prior to genomic activation, T(3) has acute actions in the neuromuscular junction (NMJ) of the tail of amphibian embryos. We detected a dramatic increase in the production of miniature end-plate currents (MEPCs) 2-5 min after continuous application of T(3) (250 nM) using focal recordings under voltage clamp. Furthermore, this increase in the spontaneous release of neurotransmitter, evaluated by the MEPC frequency, was maintained for several hours. Reverse-T(3), the "inhibitory" form of THs, prevented this increase in MEPC frequency, suggesting that this is probably a highly specific action of T(3). In addition, the elevation in MEPC frequency induced by T(3) was unchanged in the presence or absence of extracellular calcium. The T(3)-mediated increase in MEPC frequency was blocked by niflumic acid, a nonsteroidal antinflammatory fenamate used to prevent the apoptotic volume decrease observed in many systems. The present study demonstrated that T(3) induces a remarkable nongenomic action in the NMJ of the tadpole tail at pre- and promatamorphic stages.

Presynaptic function is altered in snake K+-depolarized motor nerve terminals containing compromised mitochondria.

Presynaptic function was investigated at K+-stimulated motor nerve terminals in snake costocutaneous nerve muscle preparations exposed to carbonyl cyanide m-chlorophenylhydrazone (CCCP, 2 M), oligomycin (8 g x ml(-1)) or CCCP and oligomycin together. Miniature endplate currents (MEPCs) were recorded at -150 mV with two-electrode voltage clamp. With all three drug treatments, during stimulation by elevated K+ (35 mM), MEPC frequencies initially increased to values > 350 s(-1), but then declined. The decline occurred more rapidly in preparations treated with CCCP or CCCP and oligomycin together than in those treated with oligomycin alone. Staining with FM1-43 indicated that synaptic vesicle membrane endocytosis occurred at some CCCP- or oligomycin-treated nerve terminals after 120 or 180 min of K+ stimulation, respectively. The addition of glucose to stimulate production of ATP by glycolysis during sustained K+ stimulation attenuated the decline in MEPC frequency and increased the percentage of terminals stained by FM1-43 in preparations exposed to either CCCP or oligomycin. We propose that the decline in K+-stimulated quantal release in preparations treated with CCCP, oligomycin or CCCP and oligomycin together could result from a progressive elevation of intracellular calcium concentration ([Ca2+]i). For oligomycin-treated nerve terminals, a progressive elevation of [Ca2+]i could occur as the cytoplasmic ATP/ADP ratio decreases, causing energy-dependent Ca2+ buffering mechanisms to fail. The decline in MEPC frequency could occur more rapidly in preparations treated with CCCP or CCCP and oligomycin together because mitochondrial Ca2+ buffering and ATP production were both inhibited. Therefore, the proposed sustained elevation of [Ca2+]i could occur more rapidly.

The effects of sodium nitroprusside on mediator release and the functional properties of postsynaptic membranes in the neuromuscular synapse.

Experiments on neuromuscular preparations of frog skin- thoracic muscle and sartorius muscle, using extracellular recording and two-electrode clamping of the muscle fiber membrane potential, were used to study the effects of the nitric oxide donor sodium nitroprusside on endplate currents. At a concentration of 100 microM, sodium nitroprusside sharply decreased the amplitude and quantum composition of the endplate currents, and also decreased the miniature endplate current frequency. The amplitude-time characteristics of miniature endplate currents, the voltage-dependent amplitude, and the decay time constant of miniature endplate currents did not change as compared with controls. However, unlike the situation with other secretion inhibitors, the decrease in endplate current amplitude was not accompanied by increased facilitation in response to rhythmic stimulation or changes in postsynaptic potentiation in conditions of application of pairs of stimuli to muscles. The suppression of acetylcholine secretion was not seen with inactivated sodium nitroprusside solution. These results provide evidence that nitric oxide can be a powerful inhibitor of both spontaneous and evoked transmitter secretion in the neuromuscular synapse, and that this is accompanied by decreases in the efficiency of presynaptic forms of short-term plasticity, while the functional characteristics of the postsynaptic membrane remain unchanged.

Role of mitochondrial dysfunction in the Ca2+-induced decline of transmitter release at K+-depolarized motor neuron terminals.

The present study tested whether a Ca2+-induced disruption of mitochondrial function was responsible for the decline in miniature endplate current (MEPC) frequency that occurs with nerve-muscle preparations maintained in a 35 mM potassium propionate (35 mM KP) solution containing elevated calcium. When the 35 mM KP contained control Ca2+ (1 mM), the MEPC frequency increased and remained elevated for many hours, and the mitochondria within twitch motor neuron terminals were similar in appearance to those in unstimulated terminals. All nerve terminals accumulated FM1-43 when the dye was present for the final 6 min of a 300-min exposure to 35 mM KP with control Ca2+. In contrast, when Ca2+ was increased to 3.6 mM in the 35 mM KP solution, the MEPC frequency initially reached frequencies >350 s-1 but then gradually fell approaching frequencies <50 s-1. A progressive swelling and eventual distortion of mitochondria within the twitch motor neuron terminals occurred during prolonged exposure to 35 mM KP with elevated Ca2+. After approximately 300 min in 35 mM KP with elevated Ca2+, only 58% of the twitch terminals accumulated FM1-43. The decline in MEPC frequency in 35 mM KP with elevated Ca2+ was less when 15 mM glucose was present or when preparations were pretreated with 10 microM oligomycin and then bathed in the 35 mM KP with glucose. When glucose was present, with or without oligomycin pretreatment, a greater percentage of twitch terminals accumulated FM1-43. However, the mitochondria in these preparations were still greatly swollen and distorted. We propose that prolonged depolarization of twitch motor neuron terminals by 35 mM KP with elevated Ca2+ produced a Ca2+-induced decrease in mitochondrial ATP production. Under these conditions, the cytosolic ATP/ADP ratio was decreased thereby compromising both transmitter release and refilling of recycled synaptic vesicles. The addition of glucose stimulated glycolysis which contributed to the maintenance of required ATP levels.

Transmitter release differs at snake twitch and tonic endplates during potassium-induced nerve terminal depolarization.

Twitch and tonic muscle fibers of snake skeletal muscle differ in their synpatic as well as mechanical properties. These experiments were aimed at detemining the basis of the difference in vesicular release properties of nerve terminals at twitch and tonic endplates. Miniature endplate currents (MEPCs) were recorded from voltage-clamped garter snake muscle fibers depolarized by high K+ in either a control Ca2+ or high-Ca2+ solution. MEPC frequency increased at twitch and tonic endplates and remained elevated for 8 h during depolarization in control Ca2+. At twitch endplates depolarized in the presence of high Ca2+, an increase in MEPC frequency was followed by a progressive decline. In contrast, MEPC frequency remained elevated in high Ca2+ at tonic endplates. The observed decrease in MEPC frequency at depolarized twitch endplates in high Ca2+ was not a function of the level of depolarization or initial MEPC frequency, nor was it due to a reduction in MEPC amplitude and loss of MEPCs in baseline noise. An optical assay of presynaptic function in which the activity-dependent dye FM1-43 was used confirmed that quantal releases differs at twitch and tonic endplates. Most twitch nerve terminals were labeled by FM1-43 during prolonged depolarization with control Ca2+ or after brief depolarization with high Ca2+. In contrast, the number of twitch nerve terminals and the degree to which they were stained was greatly reduced after prolonged exposure to high K+ and high Ca2+, whereas depolarized tonic endplates were well stained by FM1-43 during brief and prolonged exposure to high Ca2+. FM1-43 staining also revealed variable levels of quantal release between individual boutons at twitch endplates after prolonged depolarization in high-Ca2+ solution. The observed reduction in presynaptic function at twitch nerve terminals after prolonged depolarization in high-Ca2+ solution was reversible and therefore not due to irreversible damage to terminal boutons. MEPC frequency increased at both twitch and tonic endplates when either Sr2+ or Ba2+ was substituted for high Ca2+ during K(+)-induced depolarization. Over time, in Sr2+ or Ba2+ solutions, MEPC frequency remained elevated at tonic endplates but declined at twitch endplates with a time course similar to that observed in high Ca2+. MEPC amplitudes at both endplates remained constant. We conclude that the regulation of quantal release differs in nerve terminals innervating twitch and tonic endplates and postulate that differential intraterminal accumulation of Ca2+ may underlie the observed difference in presynaptic function.

Effects of pyrocatechol on neuromuscular transmission

Effects of pyrocatechol on neuromuscular transmission were studied both in the frog pectoral-cutaneous muscle and in the mouse phrenic-diaphragmatic preparation by means of extracellular microelectrode recording of synaptic signals. Pyrocatechol applied in a concentration of 0.05 mM increased the frequency of miniature end-plate currents (MEPC) and the amplitude of end-plate current (EPC) by increasing its quantum content. Pyrocatechol also increased the duration of presynaptic response. When voltage-dependent potassium channels had been blocked, pyrocatechol affected neither the EPC quantum content nor the duration of presynaptic response. It is suggested that the pyrocatechol-induced enhancement of transmitter release results from modulatory effects of pyrocatechol on voltage-dependent potassium current in the membrane of a nerve terminal.

Effects of lanthanum at snake twitch and tonic muscle fibre endplates.

1. The effects of 1 mM lanthanum on miniature endplate current (MEPC) frequency, amplitude, and decay time course were studied in voltage-clamped twitch and tonic muscle fibres in the garter snake, Thamnophis. 2. Lanthanum produced a marked increase in MEPC frequency in both fibre types. The maximum frequency in lanthanum was greater at twitch endplates than at tonic endplates although the increase in frequency relative to control levels was as great in tonic fibres as in twitch fibres. 3. In twitch fibres continually exposure to lanthanum, the frequency of MEPCs reached a peak value and then declined progressively until, after approximately 6 h, no MEPCs were recorded. In contrast, at tonic endplates exposed to 1 mM lanthanum, MEPC frequency remained elevated above control levels for periods greater than 20 h. 4. Lanthanum decreased the mean amplitude of MEPCs, skewed the amplitude distribution and increased MEPC duration at both twitch and tonic fibre endplates. 5. Ultrastructural analysis showed that after a 15 min exposure to 1 mM lanthanum, approximately half of the boutons innervating a twitch fibre contained fewer synaptic vesicles than boutons at control endplates, whereas nerve terminals innervating tonic fibre endplates were similar in appearance to those in control preparations. At endplates on both fibres, the postsynaptic membrane was more electron dense than that of control preparations. 6. Following a 6 h exposure to lanthanum, all nerve terminals innervating twitch endplates contained only a few synaptic vesicles and numerous intracellular deposits of electron dense material. The nerve terminals innervating tonic endplates still contained many synaptic vesicles, but the number appeared to be less than that of tonic terminals in untreated preparations. 7. The results demonstrate that lanthanum stimulates spontaneous quantal transmitter release from nerve terminals innervating either twitch or tonic fibres. However, the terminals innervating twitch fibres become depleted of synaptic vesicles, whereas this does not occur as readily in nerve terminals innervating tonic fibres.

Quantal transmitter release at snake twitch and tonic muscle fibres during prolonged nerve terminal depolarization.

1. Miniature endplate currents (MEPCs) were recorded in vitro from voltage-clamped twitch and tonic muscle fibres in costocutaneous muscles of the garter snake, Thamnophis. Recordings were made from fibres in a control sodium-containing solution and then during exposure to an isotonic potassium solution containing either 1.0 mM calcium and 4.2 mM magnesium or 3.6 mM calcium. The experiments were done at two levels of external calcium in order to demonstrate that the change in MEPC frequency was calcium dependent. During the initial exposure to the isotonic potassium solutions, the MEPC frequency was increased manyfold at both twitch and tonic fibres, but it declined progressively with continued exposure. MEPCs were recorded from both fibre types throughout a 20 h exposure to the isotonic potassium solution with 1 mM calcium, but no MEPCs were recorded at most twitch endplates after approximately 6 h in the isotonic potassium solution containing 3.6 mM calcium. In contrast, MEPCs were still present at tonic fibre endplates after 20 h in the isotonic potassium solution containing 3.6 mM calcium. 2. After 30 min in the isotonic potassium solution with 1 mM calcium, the MEPC amplitude recorded from both fibre types was approximately twice that in the control sodium-containing solution. At tonic endplates, the MEPC amplitude was also twofold greater in the isotonic potassium solution with 3.6 mM calcium than in sodium-containing solution. In contrast, after 30 min in the isotonic potassium solution containing 3.6 mM calcium, the MEPC amplitude at twitch endplates was similar to that in control solution. 3. In both fibre types, MEPC amplitude decreased progressively with continued exposure to the isotonic potassium solutions. The progressive decrease in MEPC amplitude was not due to a gradual decrease in postsynaptic sensitivity to acetylcholine. 4. The effects of high potassium were reversible as MEPCs were recorded at twitch fibre endplates in preparations which were returned to the control sodium-containing solution after a 20 h exposure to the isotonic potassium solution containing 3.6 mM calcium. 5. Ultrastructural examination showed that after a 6 h exposure to the isotonic potassium solutions most nerve terminals innervating twitch fibre endplates were devoid of synaptic vesicles whereas at the same time many synaptic vesicles were present in nerve terminals innervating tonic fibre endplates. Surprisingly, numerous synaptic vesicles were present in nerve terminals innervating either fibre type in muscle preparations exposed to the isotonic potassium solutions for 20 h.(ABSTRACT TRUNCATED AT 400 WORDS)

The effects of l-vesamicol, an inhibitor of vesicular acetylcholine uptake, on two populations of miniature endplate currents at the snake neuromuscular junction

The actions of the active l-isomer of vesamicol, an inhibitor of the vesicular storage of acetylcholine, has been studied on spontaneous and evoked acetylcholine release at the snake neuro-muscular junction. Miniature endplate currents and endplate currents were recorded from cut muscle fibres of the garter snake, Thamnophis sirtalis. In controls, prolonged periods of high frequency nerve stimulation produced a bimodal distribution of miniature endplate current amplitudes. The stimulation induced “small-mode” miniature endplate currents had a mean amplitude of around 40–55% of the pre-stimulation miniature endplate current. Relative to the normal-sized post-stimulation miniature endplate current, the proportion and, to a lesser extent, amplitude of the small-mode miniature endplate currents was related to both the frequency and duration of nerve stimulation and to the extracellular calcium ion concentration. In unstimulated preparations, l-vesamicol (2–5 μM) did not affect either endplate current quantal content or miniature endplate current amplitude or frequency. However, at these doses, the mean amplitude of the stimulation-induced, small-mode miniature endplate current was reduced by l-vesamicol in a concentration-dependent manner such that they were not visible at the highest dose. l-Vesamicol had no affect on the mean or coefficient of variance of amplitude of the larger, normal-sized miniature endplate current. Additionally, the stimulation-induced increase in overall miniature endplate current frequency seen in controls was abolished by 5 μM l-vesamicol. After prolonged 10 Hz nerve stimulation endplate current amplitude was markedly reduced in both controls (by 94%) and in the presence of 5 μM l-vesamicol (by 98%). Analysis of endplate current amplitude variance showed that in control the decrease was due to reductions in both quantal content and quantal size while in l-vesamicol the decrease was due entirely to a change in quantal content with no change in quantal size. Thus, we have observed that l-vesamicol selectively reduces the amplitude of a population of stimulation-induced small-mode quanta both as miniature endplate currents and as constituents of endplate currents. We suggest that these quanta are derived from a highly active, readily releasable pool. An action of l-vesamicol on this labile pool is consistent with previous observations on its ability to inhibit the vesicular storage of acetylcholine.

Changes in the synaptic cleft potassium concentration produced by polarizing currents

How much the cleft K + concentration ([K +] c) is changed by a step-like shift of an endplate membrane potential was estimated from the resulting acceleration of frequency of miniature endplate currents and the dependence of miniature endplate currents frequency on bath K + concentration ([K +] 0). The absolute change in [K +] c is generally constant and hence relatively more pronounced the lower the [K +] 0. It is estimated that in physiological solution ([K +] 0 = 2mM), [K +] c is at least tripled (~6 mM) if the muscle membrane is held at the reversal potential ( V r) for the endplate currents. The same step-like shift in muscle membrane potential leads to an almost instantaneous change of endplate current amplitudes or of their time constants of decay. This indicates that cleft K + changes have no effect on the voltage dependence of amplitudes and decay phases of endplate currents (and hence on V r also). The lack of postsynaptic effect of change in [K +] 0 from 0.1 to 10 mM and only small influence of [K +] 0 changes on nerve-evoked quantal release explain these findings.

Effect of axoplasmic transport blockade on end-plate currents in frog muscle fibers

Evoked and spontaneous end-plate currents (EPC) were studied in normal voltage-clamped frog sartorius muscle fibers and 2 weeks after application of colchicine to the nerve innervating the muscle to block axoplasmic transport in its fibers. Application of colchicine was found to reduce the rate of rise and to prolong decay of EPC without affecting the amplitude of the EPC and miniature EPC, the quantum composition of EPC, and the frequency of miniature EPC. The histogram of distribution of the time constant (τ) of EPC decay under normal conditions follows the normal law, but after application of colchicine to the nerve it is shifted to the right, with separation of two modes (τ1 and τ2). Three types of synapses can be distinguished from the character of EPC decay: monoexponential decay with τ1 (44%), biexponential decay with τ1 and τ2 (39%), and monoexponential decay with τ2 (19%). An increase in τ of EPC decay is accompanied by strengthening of the dependence of this process on the clamping voltage. The current-voltage characteristic and reversal potential of EPC are unchanged. It is suggested that the change in character of EPC decay after application of colchicine to the motor nerve is due to the appearance of acetylcholine-activated ionic channels in the muscle membrane with a longer duration of the open state and with potential-dependence of the open state similar to that taking place after muscle denervation.