Decay Of End-plate Currents Research Articles

Methocarbamol blocks muscular Nav 1.4 channels and decreases isometric force of mouse muscles.

The muscle relaxant methocarbamol is widely used for the treatment of muscle spasms and pain syndromes. To elucidate molecular mechanisms of its action, we studied its influence on neuromuscular transmission, on isometric muscle force, and on voltage-gated Na+ channels. Neuromuscular transmission was investigated in murine diaphragm-phrenic nerve preparations and muscle force studied on mouse soleus muscles. Nav 1.4 channels and Nav 1.7 channels were functionally expressed in eukaryotic cell lines. Methocarbamol, at 2 mM, decreased the decay of endplate currents, slowed the decay of endplate potentials and reduced tetanic force of soleus muscles. The drug reversibly inhibited current flow through muscular Nav 1.4 channels, while neuronal Nav 1.7 channels were unaffected. The study provides evidence for peripheral actions of methocarbamol on skeletal muscle. Muscular Na+ channels are a molecular target of methocarbamol. Since Nav 1.7 currents were unaffected, methocarbamol is unlikely to exert its analgesic effect by directly blocking Nav 1.7 channels.

Differential Effects of Mefloquine Enantiomers on a Cholinergic Synapse

Racemic mefloquine activates spontaneous vesicle release (SVR) from motor nerve endings and inhibits endplate acetylcholinesterase (AChE). Such cholinergic effects may reduce mefloquine's safety as an antimalarial drug. We tested the hypothesis that (+) and (‐) mefloquine differentially affect the neuromuscular junction (NMJ). Intracellular recordings at NMJs of mouse Triangularis sterni muscles suggested that 30 min exposure to 10 μM (+) or (‐) enantiomer alters the frequency of miniature endplate potentials (mEPPs). Quantitative comparisons were not possible since (+) reduced mEPP amplitude. Therefore, we recorded miniature endplate currents (mEPCs). (+) and (‐) significantly (p<0.01) increased mEPC frequency to 59.7 ± 20.05 Sec‐1 (mean ± SEM, N=21 NMJs) and 17.2 ± 1.98 Sec‐1 (N=14), respectively, from the control value of 5.6 ± 0.76 Sec‐1 (N=25). (+) also reduced amplitude (p<0.01) to 2.9 ± 0.18 nAmps (N=21) from the control value of 3.4 ± 0.18 nAmps (N=25); (‐) did not affect mEPC amplitude. (+), but not (‐), significantly (p<0.01) prolonged the endplate current decay time constant to 2.5 ± 0.16 mSec (N=12) from the control value of 1.1 ± 0.05 mSec (N=16). Thus, (+) may reduce conductance and prolong open time of the endplate acetylcholine receptor (AChR) as well as activate SVR. Neither (+) nor (‐) altered stimulus‐evoked quantal release. Assay of whole muscle homogenates revealed that while both enantiomers (1 to 100 μM) inhibit AChE, (‐) is more potent. The data suggest that (‐) is a less potent activator of SVR but a more potent inhibitor of AChE than (+) mefloquine. Supported by the Kirby Foundation.

Altered active zones, vesicle pools, nerve terminal conductivity, and morphology during experimental MuSK myasthenia gravis.

Recent studies demonstrate reduced motor-nerve function during autoimmune muscle-specific tyrosine kinase (MuSK) myasthenia gravis (MG). To further understand the basis of motor-nerve dysfunction during MuSK-MG, we immunized female C57/B6 mice with purified rat MuSK ectodomain. Nerve-muscle preparations were dissected and neuromuscular junctions (NMJs) studied electrophysiologically, morphologically, and biochemically. While all mice produced antibodies to MuSK, only 40% developed respiratory muscle weakness. In vitro study of respiratory nerve-muscle preparations isolated from these affected mice revealed that 78% of NMJs produced endplate currents (EPCs) with significantly reduced quantal content, although potentiation and depression at 50 Hz remained qualitatively normal. EPC and mEPC amplitude variability indicated significantly reduced number of vesicle-release sites (active zones) and reduced probability of vesicle release. The readily releasable vesicle pool size and the frequency of large amplitude mEPCs also declined. The remaining NMJs had intermittent (4%) or complete (18%) failure of neurotransmitter release in response to 50 Hz nerve stimulation, presumably due to blocked action potential entry into the nerve terminal, which may arise from nerve terminal swelling and thinning. Since MuSK-MG-affected muscles do not express the AChR γ subunit, the observed prolongation of EPC decay time was not due to inactivity-induced expression of embryonic acetylcholine receptor, but rather to reduced catalytic activity of acetylcholinesterase. Muscle protein levels of MuSK did not change. These findings provide novel insight into the pathophysiology of autoimmune MuSK-MG.

Kinetics of neurotransmitter release in neuromuscular synapses of newborn and adult rats

The kinetics of the phasic synchronous and delayed asynchronous release of acetylcholine quanta was studied at the neuromuscular junctions of aging rats from infant to mature animals at various frequencies of rhythmic stimulation of the motor nerve. We found that in infants 6 (P6) and 10 (P10) days after birth a strongly asynchronous phase of quantal release was observed, along with a reduced number of quanta compared to the synapses of adults. The rise time and decay of uni-quantal end-plate currents were significantly longer in infant synapses. The presynaptic immunostaining revealed that the area of the synapses in infants was significantly (up to six times) smaller than in mature junctions. The intensity of delayed asynchronous release in infants increased with the frequency of stimulation more than in adults. A blockade of the ryanodine receptors, which can contribute to the formation of delayed asynchronous release, had no effect on the kinetics of delayed secretion in the infants unlike synapses of adults. Therefore, high degree of asynchrony of quantal release in infants is not associated with the activity of ryanodine receptors and with the liberation of calcium ions from intracellular calcium stores.

Prolongation of Evoked and Spontaneous Synaptic Currents at the Neuromuscular Junction after Activity Blockade Is Caused by the Upregulation of Fetal Acetylcholine Receptors

It has been shown previously in a number of systems that after an extended block of activity, synaptic strength is increased. We found that an extended block of synaptic activity at the mouse neuromuscular junction, using a tetrodotoxin cuff in vivo, increased synaptic strength by prolonging the evoked endplate current (EPC) decay. Prolongation of EPC decay was accompanied by only modest prolongation of spontaneous miniature EPC (MEPC) decay. Prolongation of EPC decay was reversed when quantal content was lowered by reducing extracellular calcium. These findings suggested that the cause of EPC prolongation was presynaptic in origin. However, when we acutely inhibited fetal-type acetylcholine receptors (AChRs) using a novel peptide toxin (alphaA-conotoxin OIVA[K15N]), prolongation of both EPC and MEPC decay were reversed. We also blocked synaptic activity in a mutant strain of mice in which persistent muscle activity prevents upregulation of fetal-type AChRs. In these mice, there was no prolongation of EPC decay. We conclude that upregulation of fetal-type AChRs after blocking synaptic activity causes modest prolongation of MEPC decay that is accompanied by much greater prolongation of EPC decay. This might occur if acetylcholine escapes from endplates and binds to extrajunctional fetal-type AChRs only during large, evoked EPCs. Our study is the first to demonstrate a functional role for upregulation of extrajunctional AChRs.

Mechanisms of the Effect of Dimephosphone on Synaptic Transmission in the Frog Neuromuscular Junction

We studied the influence of dimephosphone, an organophosphorus drug with a broad spectrum of therapeutic effects on the peripheral and central nervous systems, on postsynaptic end-plate currents (EPC) in the frog neuromuscular junction. Dimephosphone was demonstrated to decrease in a voltage-independent manner the EPC amplitude and to prolong the EPC decay. These effects are not related to inhibition of acetylcholinesterase. We propose a theoretical interpretation of the observed phenomena based on the model of blockade of an open ion channel of the acetylcholine receptor and conclude that postsynaptic receptors are one of the most probable targets for the action of dimephosphone.

The dissociation of acetylcholine from open nicotinic receptor channels

Ligand-gated ion channels bind agonists with higher affinity in the open than in the closed state. The kinetic basis of this increased affinity has remained unknown, because even though the rate constants of agonist association to and dissociation from closed receptors can be estimated with reasonable certainty, the kinetics of the binding steps in open receptors have proven to be elusive. To be able to measure the agonist-dissociation rate constant from open muscle nicotinic receptors, we increased the probability of ligand unbinding from the open state by engineering a number of mutations that speed up opening and slow down closing but leave the ligand-binding properties unchanged. Single-channel patch-clamp recordings from the wild-type and mutant constructs were performed at very low concentrations of acetylcholine (ACh). The durations of individual channel activations were analyzed assuming that "bursts" of fully liganded (diliganded) receptor openings can be terminated by ligand dissociation from the closed or open state (followed by fast closure) or by desensitization. This analysis revealed that ACh dissociates from diliganded open receptors at approximately 24 s(-1), that is, approximately 2,500 times more slowly than from diliganded closed receptors. This change alone without a concomitant change in the association rate constant to the open state quantitatively accounts for the increased equilibrium affinity of the open channel for ACh. Also, the results predict that both desensitization and ACh dissociation from the open state frequently terminate bursts of openings in naturally occurring gain-of-function mutants (which cause slow-channel congenital myasthenia) and therefore would contribute significantly to the time course of the endplate current decay in these disease conditions.

Acetylcholine receptor δ subunit mutations underlie a fast-channel myasthenic syndrome and arthrogryposis multiplex congenita

Limitation of movement during fetal development may lead to multiple joint contractures in the neonate, termed arthrogryposis multiplex congenita. Neuromuscular disorders are among the many different causes of reduced fetal movement. Many congenital myasthenic syndromes (CMSs) are due to mutations of the adult-specific ε subunit of the acetylcholine receptor (AChR), and, thus, functional deficits do not arise until late in gestation. However, an earlier effect on the fetus might be predicted with some defects of other AChR subunits. We studied a child who presented at birth with joint contractures and was subsequently found to have a CMS. Mutational screening revealed heteroallelic mutation within the AChR δ subunit gene, δ756ins2 and δE59K. Expression studies demonstrate that δ756ins2 is a null mutation. By contrast, both fetal and adult AChR containing δE59K have shorter than normal channel activations that predict fast decay of endplate currents. Thus, δE59K causes dysfunction of fetal as well as the adult AChR and would explain the presence of joint contractures on the basis of reduced fetal movement. This is the first report of the association of AChR gene mutations with arthrogryposis multiplex congenita. It is probable that mutations that severely disrupt function of fetal AChR will underlie additional cases.

Linalool modifies the nicotinic receptor–ion channel kinetics at the mouse neuromuscular junction

Linalool is a monoterpene compound reported to be a major component of essential oils in various aromatic species. Several linalool-producing species are used in traditional medical systems. Among these is Aeolanthus suaveolens G. Dom (Labiatae) which is used as an anticonvulsant in the Brazilian Amazon. Psychopharmacologicalin vivo evaluation of linalool showed that this compound has dose-dependent marked sedative effects at the central nervous system (CNS), including hypnotic, anticonvulsant and hypothermic properties. It has been suggested that these neurochemical effects might be ascribed to the local anaesthetic activity of linalool. The present study reports an inhibitory effect of linalool on the acetylcholine (ACh) release and on the channel open time in the mouse neuromuscular junction. These findings could provide a rational basis to confirm the traditional medical use of linalool-producing plant species. Indeed, our data demonstrate some interactions in the modulation of the ACh release at the mouse neuromuscular junction, which are well correlated with the suggested molecular mechanisms. Linalool induced a reduction of the ACh-evoked release. The possibility that this effect could be ascribed to some interaction with pre-synaptic function is noteworthy. Moreover, the inhibitory effect induced on the kinetics of the miniature end-plate current decay demonstrates a local anaesthetic action, either on the voltage or on the receptor-activated channels.

Electrophysiological and Biochemical Effects of Single and Multiple Doses of the Organophosphate Diazinon in the Mouse

Diazinon is an organophosphorus compound (OP) widely used in pesticides. The relationship between dose of diazinon, inhibition of acetylcholinesterase, and effect on neuromuscular transmission has been studied in a mouse model. Inhibition of acetylcholinesterase activity occurred within 1 h, was maximal by 3 h and remained inhibited for at least 24 h. Blood, brain, diaphragm, and soleus acetylcholinesterase activities were differentially affected by diazinon. Brain and soleus activities were not affected by low doses. Multiple daily dosing of diazinon caused a cumulative decrease in acetylcholinesterase activity, although to a lesser extent in brain and soleus. Diazinon had no effect on the activity of neuropathy target esterase. Plasma and brain levels of diazinon peaked at 15 min after dosing and declined with a half-life of 2.5 h. Metabolic products of diazinon were cleared from the urine within 24 h. Increased miniature end-plate current half decay times occurred in a dose-dependent manner. Single doses of diazinon caused an increase in the jitter (variability of latencies) of evoked action potentials recorded in the diaphragm but did not affect end-plate potential (EPP) jitter. Multiple lower doses of diazinon caused an increase in EPP jitter after 28 days. This effect on nerve function was delayed and occurred when acetylcholinesterase activity had returned to control levels. The results indicate that diazinon produces long-term electrophysiological changes in neurotransmission following repeated dosing in the mouse. This has implications for the current use of diazinon; however, there is a need to further define the mechanism of this effect.

Hexamethonium- and methyllycaconitine-induced changes in acetylcholine release from rat motor nerve terminals.

1. The neuronal nicotinic receptor antagonists hexamethonium and methyllycaconitine (MLA) have been used to study the putative prejunctional nicotinic ACh receptors (AChRs) mediating a negative-feedback control of ACh release from motor nerve terminals in voltage-clamped rat phrenic nerve/ hemidiaphragm preparations. 2. Hexamethonium (200 microM), but not MLA (0.4-2.0 microM), decreased the time constant of decay of both endplate currents (e.p.cs) and miniature endplate currents (m.e.p.cs), indicating endplate ion channel block with hexamethonium. However, driving function analysis and reconvolution of e.p.cs and m.e.p.cs indicated that this ion channel block did not compromise the analysis of e.p.c. quantal content. 3. At low frequencies of stimulation (0.5-2 Hz), hexamethonium (200 microM) and MLA (2.0 microM) increased e.p.c. quantal content by 30-40%. At high frequencies (50-150 Hz) neither compound affected e.p.c. quantal content. All effects on quantal content were paralleled by changes in the size of the pool of quanta available for release. 4. The low frequency augmentation of e.p.c. quantal content by hexamethonium was absent when extracellular [Ca2+] was lowered from 2.0 to 0.5 mM. 5. At the concentrations studied, MLA and hexamethonium produced a small (10-20%) decrease in the peak amplitude of m.e.p.cs. 6. Neither apamin (100 nM) nor charybdotoxin (80 nM) had effects on spontaneous or nerve evoked current amplitudes at any frequency of stimulation. Thus the ability of nicotinic antagonists to augment e.p.c. quantal content is not due to inhibition of Ca(2+)-activated K(+)-channels. 7. We suggest that hexamethonium and MLA increase evoked ACh release by blocking prejunctional nicotinic AChRs. These receptors exert a negative feedback control over evoked ACh release and are probably of the alpha-bungarotoxin-insensitive neuronal type.

Desensitization shortens the high-quantal-content endplate current time course in frog muscle with intact cholinesterase.

1. The desensitization induced by bath-applied carbachol or acetylcholine (ACh) and potentiated by proadifen (SKF 525A) was studied in the frog sartorius with intact synaptic acetylcholinesterase (AChE). 2. The reduction in the density and number of postsynaptic receptors produced by desensitization lowered the amplitude of the endplate currents (EPCs) and shortened the EPC decay when the quantal content (m) of the EPC was about 170 and when multiple release of quanta at single active zones was highly probably. The shortening of high-quantal-content EPCs persisted for at least 15 min after the wash-out of agonists, at a time when the amplitude had recovered fully. 3. The decay times of the low-quantal-content EPCs recorded from preparations pretreated with 5 mM Mg2+ (m approximately 70) and single-quantum miniature endplate currents (MEPCs) were not affected by carbachol, ACh or proadifen. 4. The desensitization of ACh receptors potentiated by proadifen, prevented completely the 6- to 8-fold prolongation of EPC which was induced by neostigmine inhibition of synaptic AChE. 5. It is assumed that high-quantal-content EPCs increase the incidence of multiple quanta release at single active zones and the probability of repetitive binding of ACh molecules which leads to EPC prolongation. The shortening which persists after complete recovery of the amplitude during wash-out of the exogenous agonist is probably due to 'trapping' of ACh molecules onto rapidly desensitized receptors and the reduced density of functional AChRs during the quantum action.

Monoethylcholine as a flase transmitter precursor at the fog and mouse neuromuscullar junctions

Monoethylcholine (MECH0 enters motor nerve terminals where it is made into acetylmonoethylcholine (AMECH). AMECH opens endpate channels for about half of the average duration observed when they are opened by acetylcholine (ACH). Therefore when AMECH is present in a quatum the endplate currents decay more rapidly. MECH has been used to measure quantal turnover in motor nerve terminals. We find that the incorporation of AMECH into quanta is blocked by vesamicol, and inhibitor of ACH transport into synaptic vesicles. AMECH is incorporated more rapidly when acetylcholinesterase is inhibited, when the choline uptake inhitor, hemicholinium-3, is present or when extracellular Na + (required for active CH uptake) is replaced with methylamine. This suggests that in the absence of these inhibitors CH obtained from released ACH is recycled. Therefore, experiments on the rate of incorporation of MECH are misleading unless CH recycling is prevented. Previous work also suggested that MECH is incorporated at a faster rate into those quanta which are released by stimulation than into those released spontaneously. We concluded that quanta released spontaneously and following nerve stimulation proabably come from the same pool. The distribution of t 1 2 's during the incorporation of MECH can be accounted for in the framework of recent studies of the recyclingt of synaptic vesicles. We concluded that false transmitter is a valuable tool for studying the loading of quanta, but that there are several complications to be considered when trying to use it to measure the turnover of the population of quanta.

Neuromuscular blocking profile of the vecuronium analogue, Org-9487, in the rat isolated hemidiaphragm preparation.

1. The neuromuscular effects of the short-acting aminosteroid muscle relaxant Org-9487 have been studied in the in vitro rat phrenic nerve/hemidiaphragm preparation by use of twitch tension and electrophysiological recording techniques. 2. Org-9487 (5-100 microM) produced a concentration-dependent decrease in the amplitude of twitches (0.1 Hz) and tetanic contractions (50 Hz) evoked by motor nerve stimulation. The compound produced fade of force during both 50 Hz stimulation and train-of-four stimulation at 2 Hz, indicating a prejunctional component of action. 3. Anticholinesterases only partially reversed the effect of Org-9487 on twitch responses. This was possibly because, at the concentrations required to block twitches in the rat, Org-9487 itself was found to possess significant anticholinesterase activity. 4. Org-9487 (3 microM) increased the rundown of endplate current amplitudes during a 2 s train of 50 Hz nerve stimulation. This was because Org-9487 increased the quantal content of the first endplate current in the train without affecting acetylcholine release towards the latter part of the train. 5. Org-9487 (10 microM) produced a voltage-dependent decrease in the time constant of decay of endplate currents at 32 degrees C and 0.5 Hz, indicative of a block of endplate ion channels. The blocking rate constant increased with membrane hyperpolarization.

Physiological role of desensitized cholinoceptors in skeletal muscle

The effect of desensitized cholinoceptors on the time course of end-plate currents was evaluated in frog skeletal muscle at a high (physiological) level of acetylcholine secretion in the presence of active acetylcholinesterase, with two-electrode recording of the membrane potential. When the number of cholinoceptors was small so that they did not appreciably affect the amplitude of end-plate currents or the parameters of onequantum responses (miniature currents), the decay of multiquantum currents was significantly accelerated. Moreover, the presence of cholinoceptors drastically reduced the ability of acetylcholinesterase inhibitors to prolong the decay of end-plate currents. It is suggested that desensitized cholinoceptors in a synapse with a physiological level of acetylcholine secretion and active acetylcholinesterase may bind free acetylcholine with high affinity and thus supplement the well-known physiological role of acetylcholinesterase in limiting the reactivation of postsynaptic membrane cholinoceptors

Modelling endplate currents: dependence on quantum secretion probability and decay of miniature current.

Quantification of the time course and amplitude of endplate currents (EPC) was made with respect to dispersion of quanta secretion and to changes in the exponential decay of miniature endplate currents (tau mepc). The relationship between RPC amplitude and tau mepc follows a double-exponential curve with tau1 = 0.3 ms and tau2 = 6 ms. If the amplitude of fully synchronised EPC is taken as 100%, then the loss of EPC amplitude is already 42% with "physiological" parameters of dispersion (the half-rise and decay constant of distribution of secretion probability = 0.5 ms, taumepc = 1 ms). This loss is even more substantial if secretion is more dispersed or miniature endplate currents decay faster.

A correlation between quantal content and decay time of endplate currents in frog muscles with intact cholinesterase.

1. The relationship between quantal content and prolongation of endplate currents (EPC) was studied in the frog sartorius with intact synaptic acetylcholinesterase. 2. The prolongation of EPC was more pronounced in endplates with a higher quantal content both before and after potentiation of quantal release by 4-aminopyridine (4-AP). When the quantal content of EPC was lowered, either by high Mg2+ or repetitive stimulation, the EPC decay constant was reduced. 3. A certain critical value of about 120 quanta per nerve impulse was found, at which point the decay of EPC remained constant even through the quantal content was reduced further. 4. The reduction in both density and number of postsynaptic receptors, produced by alpha-bungarotoxin and (+)-tubocurarine led to a profound reduction in EPC decay during the progressive fall in EPC amplitude in both 4-AP-treated and -untreated endplates. Both drugs are known to produce a shortening of EPC in anti-cholinesterase (anti-ChE)-treated muscles, due to a decrease in receptor density and less frequent repetitive binding of ACh. 5. It is assumed that the prolongation of multiquantal EPC is caused by an increased ACh concentration near the receptors, which may provide the opportunity for repetitive binding even with full cholinesterase activity. The critical quantum content of about 120 might be the number of quanta at which the probability of multiple release at single active zones is increased above zero.

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.

Effects of "non-quantal" acetylcholine on postsynaptic membrane senstitivity: Effects of ouabain, and mimicry by exogenous acetylcholine

The development of postsynaptic potentiation (PSP) and desensitization due to "non-quantal" acetylcholine that occurs when acetylcholinesterase (AChE) is inhibited was studied using the Na,K-ATPase inhibitor, ouabain, to alter (initially increasing, then decreasing) the level of non-quantal acetylcholine secretion, and exogenous acetylcholine. When ouabain increased non-quantal secretion the time constant (τ) of the miniature end-plate current (MEPC) decay increased, i.e., PSP developed. The later the application of ouabain relative to inhibition of AChE, the greater the degree of PSP. During the next phase when non-quantal secretion was inhibited the MEPC time course shortened more rapidly than in the controls, i.e., desensitization occurred. If ouabain abolished non-quantal secretion before AChE had been inhibited τ did not change, and neither PSP nor desensitization developed. When AChE was not inhibited ouabain had no effect on τ. When ACh was continuously applied at 20 nmol·liter−1, similar to the nonquantal concentration, the shortening of τ slowed down, and the signal amplitude declined more rapidly than in controls. Addition of exogenous ACh (50 nmol·liter−1) after acceleration of MEPC decay had developed caused τ to increase to its initial value. The combined appearance of PSP and desensitization during the action of non-quantal ACh, and the sustained desensitization after removal of released ACh from the synaptic cleft are discussed.

Characteristics of synaptic transmission in reinnervating rat skeletal muscle.

Synaptic transmission, and its sensitivity to the effects of 3,4-diaminopyridine (3,4-DAP) and the phosphatase 2,3-butanedione monoxime (BDM), was examined for "crushed fiber" preparations of rat extensor digitorum longus muscle undergoing reinnervation after nerve crush. While mean quantal content (m) of endplate potentials (EPPs) was low early during reinnervation (10-24 days after nerve crush), elevation of temperature or extracellular calcium concentration restored m toward normal. However, m achieved control values for reinnervating preparations exposed to 3,4-DAP. 3,4-DAP also activated quiescent motor nerve terminals: after exposure to this drug, synaptic transmission was detected as early as 8 days after nerve crush. BDM too activated quiescent regenerating motor nerve terminals and increased m to normal. It also prolonged EPP and endplate current decay, suggesting a pre-synaptic effect on the synchrony of transmitter release and/or a post-synaptic effect on the open time of acetylcholine-gated endplate channels. While the effects of temperature, extracellular calcium, 3,4-DAP, and BDM suggest that regenerating nerve terminals can mobilise a reserve of quanta, this reserve is abnormally low, since hemicholinium-3 caused rapid rundown of EPP amplitude at repetitively stimulated regenerating endplates.