Miniature Endplate Currents Research Articles

Activation of protein kinase C increases acetylcholine release from frog motor nerves by a direct action on L-type Ca(2+) channels and apparently not by depolarisation of the terminal.

The effects of the dihydropyridine Ca(2+) channel antagonist nimodipine and the protein kinase C inhibitors staurosporine and calphostin C on the changes in the electrophysiological indices of quantal acetylcholine release induced by a 4-beta-phorbol ester were studied at the frog neuromuscular junction. 4-beta-Phorbol 12-myristate 13-acetate (200 nM) caused an increase in the frequency of miniature endplate potentials and miniature endplate currents and in the quantal content of endplate potentials and endplate currents. These effects were not replicated by 4-alpha-phorbol 12,13-didecanoate (200 nM). Nimodipine (1 microM) itself had no effect on the frequency of miniature endplate potentials and miniature endplate currents and it had no effect on the quantal content. Nimodipine inhibited by 83-98% the increase in these parameters induced by 4-beta-phorbol 12-myristate 13-acetate. The increase in the frequency of miniature endplate potentials and currents caused by KCl (12 mM) matched the increase caused by 20 min exposure to 4-beta-phorbol 12-myristate 13-acetate. Nimodipine did not reduce the increase in frequency caused by KCl. Unlike 4-beta-phorbol 12-myristate 13-acetate, KCl (12 mM) prevented neuromuscular transmission. The effects of prior exposure of muscles to staurosporine (5 microM) on 4-beta-phorbol 12-myristate 13-acetate-induced increases in quantal acetylcholine release were inconsistent. In some pretreated fibres, 4-beta-phorbol 12-myristate 13-acetate caused increases in miniature endplate potential frequency and quantal content which were as great as the largest values encountered in fibres that had not been pretreated. In others, 4-beta-phorbol 12-myristate 13-acetate did not have a marked effect; the frequency of the spontaneous potentials and the quantal content of endplate potentials recorded in the presence of 4-beta-phorbol 12-myristate 13-acetate were sometimes less than their respective control values. Pretreatment with calphostin C (500 nM) was more consistent; it prevented by 93-100% the 4-beta-phorbol 12-myristate 13-acetate-induced increases in the frequency of miniature endplate potentials and quantal content.Overall, from these results we suggest that activation of protein kinase C increases quantal acetylcholine release by opening quiescent L-type Ca(2+) channels in motor nerve terminals at resting potential and apparently not by depolarisation.

Temperature compensation of postsynaptic currents from the extraocular muscle of temperate teleost fishes.

Miniature end plate currents were recorded from white inferior oblique extraocular muscle fibres of one temperate marine teleost (Aldrichetta forsteri, Family Mugilidae) and two temperate freshwater teleosts (Galaxias fasciatus, Family Galaxiidae and Oncorhynchus mykiss, Family Salmonidae). Miniature end plate currents were digitised and averaged over a temperature range of 5-25 degrees C. For each species, decay of miniature end plate currents was exponential and exhibited a strong temperature dependence. Lower temperatures resulted in prolonged decay phases, which decreased exponentially as a function of absolute temperature. Although values of the exponential time constant tau (tau) obtained for each species at 5 degrees C, 15 degrees C and 25 degrees C were significantly different, at any given temperature, there were no significant differences between tau values for the three species, despite differences in phylogeny (different families) and habitat (marine versus freshwater). At their normal temperature of 15 degrees C, mean values of tau for the three species ranged from 840 micros to 940 micros, and apparent activation energies ranged from -41 kJ mol(-1) deg(-1) to 50 kJ mol(-1) deg(-1). These observations confirm earlier reports that teleost miniature end plate currents are consistently shorter than those of other vertebrates.

Repetitive nerve stimulation decreases the acetylcholine content of quanta at the frog neuromuscular junction.

We investigated how elevated quantal release produced by motor nerve stimulation affects the size of the quanta. The motor nerve was stimulated at 10 Hz in preparations in which excitation-contraction coupling was disrupted. Two hundred stimuli reduced the size of the time integrals of the miniature endplate currents ([integral]MEPCs), measured at the same junction immediately after stimulation, by 16 %. Three thousand stimuli reduced size by 23 %. When the solution contained 10 microM neostigmine (NEO) 3000 stimuli reduced [integral]MEPCs by 60 %, because with acetylcholinesterase (AChE) inhibited, [integral]MEPC size is more sensitive to changes in acetylcholine (ACh) content. Similar decreases in miniature endplate potential size ([integral]MEPP) followed repetitive stimulation of contracting preparations. The depolarization produced by iontophoretic pulses of ACh was scarcely changed by 3000 nerve stimuli at 10 Hz, suggesting that the decreases in miniature sizes are largely due to less ACh released per quantum. Following 3000 stimuli at 10 Hz the sizes of the [integral]MEPCs increased back to pre-stimulus values with a half-time of 8-10 min. Recovery was blocked by (-)-vesamicol (VES), by hemicholinium-3 (HC3) and by nicotinic cholinergic agonists - all of which inhibit ACh loading into synaptic vesicles. The number of quanta in the total store was estimated by releasing them with carbonyl cyanide m-chlorophenylhydrazone (CCCP). CCCP releases fewer quanta after stimulation than from unstimulated controls. After resting for hours following stimulation, the releasable number increased, even when ACh loading inhibitors were present. We conclude that the inhibitors do not block a significant fraction of the ACh loading into reformed reserve vesicles and propose that ACh can be loaded in a series of steps.

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 role of desensitisation in decay time of miniature endplate currents in frogs Rana ridibunda and Rana temporaria.

A new comparative characteristic of endplate microphysiology has been introduced. It is the feasibility of receptors to become desensitised as demonstrated on two frog species, Rana temporaria and Rana ridibunda: the decay times (tau(MEPC)) of single quantum miniature endplate currents (MEPCs) in the sartorius muscles of both species were about 1 ms and were not affected by the desensitisation-promoting agent proadifen when AChE was active. However, when the desensitisation was induced by anticholinesterase neostigmine and promoted by proadifen, the prolongation of tau(MEPC) from 1 ms was almost twice as great in Rana temporaria (tau(MEPC) = 4.4 ms) than in Rana ridibunda (tau(MEPC) = 3.1). This indicates that desensitisation reduces the number of available receptors and lowers the number of available ACh molecules for repetitive binding by trapping them by desensitised, high-affinity receptors significantly more in Rana ridibunda than in Rana temporaria. The application of proadifen, a promoter of desensitisation, decreased the prolongation of MEPCs in both species, but this shortening was more rapid in Rana ridibunda than in Rana temporaria. It is concluded that the desensitisation-induced reduction in the density, and the number of postsynaptic receptors is significantly higher at Rana ridibunda than in Rana temporaria endplates.

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.

Factors influencing the low-frequency associated nicotinic ACh autoreceptor-mediated depression of ACh release from rat motor nerve terminals.

1. We have studied the inhibitory autoreceptor control of acetylcholine (ACh) release from rat motor nerve endings using an electrophysiological technique to quantify evoked ACh release in isolated hemidiaphragm muscles. Quantal ACh release (m) was estimated from the ratio of amplitudes of nerve evoked endplate currents and spontaneously occurring miniature endplate currents. 2. The nicotinic ACh receptor agonist cytisine (1 microM) decreased m at 0.5 Hz by around 20% but had no effect on m at 50 Hz. Changing the extracellular Ca(2+) concentration from 1.8 mM to either 0.45 or 3.6 mM abolished the effect of cytisine on m at 0.5 Hz. The nicotinic ACh receptor antagonist hexamethonium (200 microM) increased m at 0.5 Hz by 15 - 20%. 3. The effects of cytisine and hexamethonium on m at 0.5 Hz were blocked by 10 microM verapamil, which itself significantly increased m. However, the effects of cytisine and hexamethonium on m at 0.5 Hz were not sensitive to 10 microM of the calmodulin antagonist, W-7. This concentration of W-7 attenuates effects on ACh release mediated by facilitatory prejunctional nicotinic ACh autoreceptors. 4. Our present observations are suggestive of actions of cytisine and hexamethonium to activate and inhibit respectively negative-feedback prejunctional nicotinic ACh autoreceptors. Further, they strengthen the case for the existence of two separate and independent autoregulatory mechanisms for the control of ACh release from motor nerve terminals and give a preliminary insight into the cellular mechanism involved in the autoinhibition of ACh release.

Recovery from Open Channel Block by Acetylcholine during Neuromuscular Transmission in Zebrafish

At larval zebrafish neuromuscular junctions (NMJs), miniature end plate currents (mEPCs) recorded in vivo have an unusually fast time course. We used fast-flow application of acetylcholine (ACh) onto outside-out patches to mimic the effect of synaptic release onto small numbers of ACh receptor channels (AChRs). Positively charged ACh acted at hyperpolarized potentials and at millimolar concentrations as a fast ("flickering") open channel blocker of AChRs. Because of filtering, the open channel block resulted in reduced amplitude of single channel currents. Immediately after brief (1 msec) application (without significant desensitization) of millimolar ACh at hyperpolarized potentials, a slower, transient current appeared because of delayed reversal of the block. This rebound current depended on the ACh concentration and resembled in time course the mEPC. A simple kinetic model of the AChR that includes an open channel-blocking step accounted for our single channel results, as well as the experimentally observed slowing of the time course of mEPCs recorded at a hyperpolarized compared with a depolarized potential. Recovery from AChR block is a novel mechanism of synaptic transmission that may contribute in part at all NMJs.

The Temperature Sensitivity of Miniature Endplate Currents Is Mostly Governed by Channel Gating: Evidence from Optimized Recordings and Monte Carlo Simulations

The temperature dependence of miniature endplate current (MEPC) amplitude ( A c), 20–80% rise time ( t r), and 90–33% fall-time ( t f) was determined for lizard ( Anolis carolinensis) intercostal muscle using broadband extracellular (EC) and voltage clamp (VC) recordings. Voltage clamp methods were optimized for the fast MEPC rising phase using custom electronics. From 0–43°C, A c increased by ∼4.2-fold, while t r and t f decreased by ∼3.6- and ∼9.5-fold, respectively. Arrhenius plots were smoothly curved, with small apparent Q 10 ( A c) or (Q 10) −1 ( t r and t f) values mostly well below 2.0. Nearly identical extracellular and voltage clamp results ruled out measurement artifacts, even for the shortest t r values (<60 μs). Monte Carlo simulation of MEPCs showed that a single underlying rate cannot determine the observed temperature dependence. To quantitatively reproduce the experimental t f results, a minimal model required activation energies of 46.0 (Q 10 ∼ 2.0) and 63.6 (Q 10 ∼ 2.5) kJ mol −1 for channel opening and closing, respectively, and accounted for most of the observed changes in A c and t r as well. Thus, relatively large but offsetting temperature sensitivities of channel gating mostly govern and minimize the temperature dependence of MEPCs, preserving the safety factor for neuromuscular transmission. Additional temperature-sensitive parameters that could fine-tune the minimal model are discussed.

Localization effects of acetylcholine release from a synaptic vesicle at the neuromuscular junction

A two-dimensional compartment model devised for the appropriate representation of the transient process of the spontaneous generation of miniature endplate current (MEPC) at the neuromuscular junction is applied for clarifying the biochemical significance of the quantal release mechanism of acetylcholine (ACh), a typical neurotransmitter, in the synaptic chemical transmission process. The simulation analysis with the model demonstrates that the localization of the ACh release due to the fusion of a synaptic vesicle with the presynaptic membrane has significant effects on the amplitude of MEPC and that the stronger effects are caused with the smaller diffusion coefficients of ACh in the cleft. The sharpest and highest response of MEPC is achieved when the release area is about 4 times to the natural release through the narrow pore. On the other hand, the actual localization corresponding to the natural release of ACh makes the amplitude of MEPC higher by a factor about 2.5 compared with that in the most extended release of ACh examined, implying that the natural release mechanism works as an amplifier of the MEPC with the fixed amount of ACh available.

Empty synaptic vesicles recycle and undergo exocytosis at vesamicol-treated motor nerve terminals.

We investigated whether recycled cholinergic synaptic vesicles, which were not refilled with ACh, would join other synaptic vesicles in the readily releasable store near active zones, dock, and continue to undergo exocytosis during prolonged stimulation. Snake nerve-muscle preparations were treated with 5 microM vesamicol to inhibit the vesicular ACh transporter and then were exposed to an elevated potassium solution, 35 mM potassium propionate (35 KP), to release all preformed quanta of ACh. At vesamicol-treated endplates, miniature endplate current (MEPC) frequency increased initially from 0.4 to >300 s-1 in 35 KP but then declined to <1 s-1 by 90 min. The decrease in frequency was not accompanied by a decrease in MEPC average amplitude. Nerve terminals accumulated the activity-dependent dye FM1-43 when exposed to the dye for the final 6 min of a 120-min exposure to 35 KP. Thus synaptic membrane endocytosis continued at a high rate, although MEPCs occurred infrequently. After a 120-min exposure in 35 KP, nerve terminals accumulated FM1-43 and then destained, confirming that exocytosis also still occurred at a high rate. These results demonstrate that recycled cholinergic synaptic vesicles that were not refilled with ACh continued to dock and undergo exocytosis after membrane retrieval. Thus transport of ACh into recycled cholinergic vesicles is not a requirement for repeated cycles of exocytosis and retrieval of synaptic vesicle membrane during prolonged stimulation of motor nerve terminals.

Maturation of neuromuscular transmission during early development in zebrafish.

We have examined the rapid development of synaptic transmission at the neuromuscular junction (NMJ) in zebrafish embryos and larvae by patch-clamp recording of spontaneous miniature endplate currents (mEPCs) and single acetylcholine receptor (AChR) channels. Embryonic (24-36 h) mEPCs recorded in vivo were small in amplitude (<50 pA). The rate of mEPCs increased in larvae (3.5-fold increase measured by 6 days), and these mEPCs were mostly of larger amplitude (10-fold on average) with (</=5-fold) faster kinetics. Intracellular labeling with Lucifer yellow indicated extensive coupling between muscle cells in both embryos and larvae (</=10 days). Blocking acetylcholinesterase (AChE) with eserine had no effect on mEPC kinetics in embryos at 1 day and only partially slowed (by approximately 1/2) the decay rate in larvae at 6 days. In acutely dissociated muscle cells, we observed the same two types of AChR with conductances of 45 and 60 pS and with similar, brief (<0.5 ms) mean open times in both embryos and larvae. We conclude that AChR properties are set early during development at these early stages; functional maturation of the NMJ is only partly shaped by expression of AChE and may also depend on postsynaptic AChR clustering and presynaptic maturation.

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.

Evaluation of characteristic parameters for the neurotransmitter release mechanisms at the neuromuscular junction

The process of neurotransmitter release at the neuromuscular junction needs to be represented appropriately in modeling of the synaptic chemical transmission as a reaction-diffusion system. The release mechanisms of the expanding pore and the acceleration are analyzed by the computer simulation with respect to the effects of the characteristic parameters in the mechanisms on spontaneous generation of the miniature endplate current (MEPC), leading to the following evaluation. In the expanding pore mechanism the expanding rate of the pore more than 10 nm ms −1 and the diffusion coefficient of acetylcholine in the synaptic cleft ( D c) of about 1.0×10 −6 cm 2 s −1 yield the maximum amplitude, the rise time and the decay time constant of the MEPC in agreement with the empirical data. In the active release mechanism the 10-fold acceleration of the natural diffusion and a similar value of D c are required to suit for the empirical MEPC.

Effects of Some Phosphorylacetic Acid Hydrazides on Periferal Cholinegric Neurotransmission

Cholinergic deficit has received considerable attention with respect to the development of therapeutic agents designed to ameliorate the cognitive symptomatology of the dementias. The influence of (2-chloroethoxy) (p-N,N- dimethylaminophenyl) phosphorylacetic acid hydrazide (CAPAH) and diphenylphosphorylacetic acid hydrazide (PHOSENAZID) on the processes of the synaptic transmission in the neuromuscular synapses of frog and rat was studied. Experiments revealed that the effectiveness of CAPAH was concentration- dependent: in low concentrations (10 - 100 nM) neuromuscular junction miniature end-plate currents (MEPC) were attenuated by 15 - 20 % (sensitising effect); at higher concentrations (0.1 mM) h4EPC amplitude was reduced. This effect was similar to that seen with blockers of the sodium channel component of the nicotinic receptor The HCI-salt of CAPAH also had a sensitising effect in low concentrations (1 - 10 nh4). By contrast, PHOSENAZID was not effective at low concentrations. These differences in the effects of CAPAH and PHOSENAZID on cholinergic neurotransmission appear dependent in changes in the phosphyl fragment of the molecule.

Modulation of Synaptic Efficacy and Synaptic Depression by Glial Cells at the Frog Neuromuscular Junction

The ability of perisynaptic glial cells to modulate transmitter release and synaptic depression was studied at the frog neuromuscular junction (nmj). Injection of GTPγS in perisynaptic Schwann cells (PSCs), glial cells at this synapse, induced a reduction in the amplitude of nerve-evoked synaptic responses but had no effect on the frequency, the amplitude, or the duration of the miniature endplate currents (MEPCs). Also, paired pulse facilitation was not affected. The reduction in transmitter release was mediated by pertussis toxin– (PTX) sensitive and insensitive G proteins. Blockade of G proteins in PSCs with GDPβS reduced synaptic depression induced by high frequency trains of stimuli, whereas activation of G proteins occluded it. Hence, the activation by endogenous neurotransmitters of G proteins in PSCs induced a profound depression in neurotransmitter release.

Analysis of Synaptic Transmission in the Neuromuscular Junction Using a Continuum Finite Element Model

There is a steadily growing body of experimental data describing the diffusion of acetylcholine in the neuromuscular junction and the subsequent miniature endplate currents produced at the postsynaptic membrane. To gain further insights into the structural features governing synaptic transmission, we have performed calculations using a simplified finite element model of the neuromuscular junction. The diffusing acetylcholine molecules are modeled as a continuum, whose spatial and temporal distribution is governed by the force-free diffusion equation. The finite element method was adopted because of its flexibility in modeling irregular geometries and complex boundary conditions. The resulting simulations are shown to be in accord with experiment and other simulations.

Constitutive Secretion of Exogenous Neurotransmitter by Nonneuronal Cells: Implications for Neuronal Secretion

Fibroblasts in cell culture were loaded with exogenous neurotransmitter acetylcholine (ACh). ACh secretion from loaded cells was detected by whole-cell patch clamp recordings from Xenopus myocytes manipulated into contact with ACh-loaded cells. Two different approaches were used for ACh loading. In the first approach, fibroblasts were incubated in the culture medium containing ACh. Recordings from myocytes revealed fast inward currents that resemble miniature endplate currents found at neuromuscular synapses. The currents observed in recordings from myocytes were due to exocytosis of ACh-containing vesicles. Although exogenous ACh penetrated through the plasma membrane of fibroblasts during incubation and was present in the cytoplasm at detectable levels, cytoplasmic ACh did not contribute to the quantal ACh secretion. In the second approach, exogenous ACh was loaded into the cytoplasm of fibroblasts by microinjection. Under these experimental conditions, fibroblasts also exhibited spontaneous quantal ACh secretion. Analysis of the exocytotic events in fibroblasts following two different protocols of ACh loading revealed that the vesicular compartments responsible for uptake of exogenous ACh are associated with the endocytic recycling pathway. Extrapolation of our results to neuronal cells suggest that in cholinergic neurons, in addition to genuine synaptic vesicles, ACh can be secreted by the vesicles participating in endosomal membrane recycling.

Fluoroquinolone antibiotics block neuromuscular transmission.

Fluorinated 4-quinolones are widely used antibiotics. Several case reports describe the exacerbation of muscle weakness in myasthenia gravis patients treated with fluoroquinolones. We studied the effects of norfloxacin, ofloxacin, and pefloxacin on miniature endplate potentials (MEPPs) and currents. These antibiotics progressively decreased the amplitude of the MEPPs as drug concentrations were increased from 12.5 to 100 mg/L. Fluoroquinolones should be used only with great caution in disorders that compromise the safety margin of neuromuscular transmission.

Making quantal analysis easier and more accurate

Problems inherent in applying quantal analysis to synapses are discussed. These include the dispersion in time of the evoked quantal releases and the skew to the right in the distribution of the sizes of the spontaneous miniature potentials. To circumvent some of the problems, quantal analysis was performed at the neuromuscular junction by dividing each individual endplate current (EPC) by the mean miniature endplate current (MEPC) or each individual time integral of the endplate current (∫ EPC) by the mean ∫ MEPC. The results were evaluated by comparing the resulting distributions to the predictions of Poisson's law, based on the number of stimuli not followed by a response. The quantal distributions obtained from the ∫ MEPCs by this `point-by-point' were in excellent agreement with these predictions. This method may make quantal analysis easier and more reliable.