Frequency Of Miniature End-plate Potentials Research Articles

Adaptation of quantal content to decreased postsynaptic sensitivity at single endplates in alpha-bungarotoxin-treated rats.

1. Rats were injected once every 48 h with alpha-bungarotoxin (alpha BTX) for periods up to 6 weeks. Injections caused weakness of facial muscles which lasted about 8 h. Hemidiaphragms were dissected for biochemical and electrophysiological measurements. 2. In muscles from animals treated for 2-3 weeks with toxin, the binding of 125I-alpha BTX was reduced to 58%, and the ACh content to 81% of control values. Choline acetyltransferase activity was unchanged. ACh release evoked by 3 Hz nerve stimulation was increased to 175% of control values. 3. The use of mu-conotoxin, which specifically blocks muscle action potentials, enabled the recording of full-sized endplate potentials (EPPs) and miniature endplate potentials (MEPPs) at normal muscle membrane potentials (-70 to -80 mV). The amplitude of MEPPs was decreased to 57% in muscles from animals treated for 3 weeks with alpha BTX. The mean of the quantal contents, calculated from the ratio of the corrected EPPs and the MEPPs, was increased to 154%. 4. Within individual muscles of both alpha BTX-treated and control rats, there was an inverse relationship between the quantal content of an endplate and its MEPP amplitude. 5. The MEPP frequency of endplates from control muscles was positively correlated with the quantal content. However, this correlation was not found in alpha BTX-affected muscles. 6. Three hours after a single injection of alpha BTX the amplitude of the MEPPs was reduced to about 60% of control values but no increase of the quantal content was found. During the first few days of alpha BTX treatment the quantal content gradually increased; it reached a plateau between 20 and 30 days. 7. The results suggest the existence of an adaptive mechanism, operating at individual endplates, in which retrograde signals at the motor nerve terminals modulate ACh release when neuromuscular transmission is endangered by block of acetylcholine receptors.

The relation between transmitter release and ca 2+ entry at the mouse motor nerve terminal: Role of stochastic factors causing heterogeneity

The relation between quantal transmitter release and presynaptic Ca 2+ /Ba 2+ entry at the mouse neuromuscular junction was studied, making use of the finding that in the presence of Ba 2+ trains of nerve stimuli or brief nerve terminal depolarizations elicit “tails” of raised miniature end-plate potential frequency ( f m) that reflect entry of Ba 2+ per pulse, and hence effectiveness of pulses in opening Ca 2+ /Ba 2+ channels; at the same time these pulses elicit end-plate potentials. With nerve stimulation in the presence of Ba 2+ and Ca 2+ and modulation of release by raised Mg 2+ or bekanamycin, slopes of log quantal content ( m) vs log apparent Ba 2+ entry per pulse were close to 4, which is the same as the Hill coefficient for Ba 2+ cooperativity derived from other data. With depolarizing pulses of varied intensity, however, similar plots gave slopes close to 2, with Ba 2+ alone or in a mixture of Ca 2+ and Ba 2+. Thus, the relation between transmitter release and Ca 2+ (or Ba 2+) entry apparently depends upon how entry is varied; varying the numbers of channels opened is not the same as varying ion entry per channel. A mathematical model was developed to examine the consequences of heterogeneity of local Ca 2+ (or Ba 2+) between release sites, arising because of stochastic variation of number and time course of Ca 2+ channels opened per site; the experimental results were consistent with this model. It was therefore concluded that release is normally governed by intracellular Ca 2+ close to points of Ca 2+ entry through channels; stochastic factors give rise to more release than if Ca 2+ were homogeneously distributed. If Ca 2+ channels are uniformly close to release sites the average number of channels opened per site per action potential may be as low as 4.

Nature of increase in quantal release by the thallous ion at frog end plates with and without nerve stimulation.

The monovalent thallous ion (Tl) was evaluated at the frog end plate in vitro with intracellular microelectrodes. Recordings included end plate potentials (EPPs), and miniature end plate potentials (MEPPs). Replacement of extracellular potassium (K) by 2.5 mM Tl (a) caused increases in MEPP and EPP amplitudes, MEPP frequency, and quantal content, and (b) caused complete recovery of the EPP facilitation index at BAPTA-loaded nerve terminals. Tl's effects were reversible and concentration dependent, and persisted for > 3 h. The increase in MEPP frequency and its rate of decline due to Tl washout were more pronounced at 0 calcium (Ca)-2 mM EGTA than at 0.3 mM EGTA, suggesting that Tl's effects were not due to elevation of internal Ca. Unlike heavy metal ions reportedly capable of substituting for Ca, 0.2 mM Tl did not block, but further enhanced, elevated MEPP frequencies, occurring after nerve stimulation or in high K, to greater levels with barium (Ba) than with Ca. 200 nM omega-conotoxin (omega-CTX) blocked Tl's effect, indicating that Tl primarily entered the nerve terminal via Ca channels. A 50% reduction in sodium (Na) did not modify Tl's effect, although removal of K in the presence of 20 microM ouabain and 2.5 mM Tl caused an exaggerated increase in MEPP frequency, which decreased with a 50% reduction in Na. Based on the analysis, Tl neither substituted for Ca nor elevated internal Ca and Na, nor were its effects antagonized by ouabain; Tl increased quantal secretion, possibly by a fusogenic mechanism, after its entry into the nerve terminal.

Calcium currents at motor nerve endings: absence of effects of adenosine receptor agonists in the frog.

1. The effects of adenosine (50 microM) and 2-chloroadenosine (1-25 microM) were studied on Ca2+ currents in frog motor nerve endings. 2. Ca2+ currents associated with the synchronous, neurally evoked release of acetylcholine (ACh) were measured using either perineural or patch recording methods. Tetraethylammonium and/or 3,4-diaminopyridine were employed to block K+ currents. 3. Ca2+ currents were depressed by omega-conotoxin (1.5-2.5 microM), Cd2+ (100 microM-2 mM), Co2+ (500 microM-5 mM) or by a reduction of the extracellular calcium concentration. Such currents were also observed when Sr2+ was substituted for Ca2+. Both ACh release and Ca2+ currents at motor nerve endings have been reported to be insensitive to 1,4-dihydropyridine antagonists in this species. 4. Adenosine receptor agonists did not affect Ca2+ currents at concentrations that produced maximal inhibition of ACh release. 5. The effects of adenosine receptor agonists were examined on asynchronous K(+)-dependent ACh release under conditions in which the Ca2+ concentration gradient is likely to be reversed (Ca(2+)-free Ringer solution containing 1 mM EGTA). ACh release was measured by monitoring the frequency of occurrence of miniature endplate potentials (MEPPs). In Ca(2+)-free solutions containing 1 mM EGTA, high K+ depolarization caused a decrease in MEPP frequency, presumably because it elicits the efflux of Ca2+ from the nerve ending via membrane Ca2+ channels in a reverse Ca2+ gradient. 6. The Ca2+ channel blocker Co2+, which blocks the exit of Ca2+ from the nerve ending, increased the frequency of MEPPs in a concentration-dependent manner in a reverse Ca2+ gradient. 7. Adenosine or 2-chloroadenosine inhibited ACh release in a reverse Ca2+ gradient. 8. The results suggest that blockade of Ca2+ entry is not responsible for the inhibitory effects of adenosine at frog motor nerve endings.

Effects of calcium on the dynamic process of transmitter release which generates either skew- or bell-MEPPs

Miniature endplate potential (MEPP) amplitudes, MEPP frequencies and ratios of skew: bell-MEPPs were determined as well as synaptic vesicle diameters and densities at the mouse diaphragm neuromuscular endplate during exposure to elevated calcium concentrations. Additions of external Ca 2+ had variable effects on MEPP frequencies and percentages of skew-MEPPs, regardless of concentrations used (1–25 mM). Nevertheless, changes in MEPP amplitudes were most sensitive (4-fold decrease) to low value increases of Ca 2+. Changes in MEPP frequencies produced by an increase in Ca 2+ were very sensitive to initial frequencies as well as the initial calcium concentration. An increase in Ca 2+ usually increased MEPP frequency (providing skew-MEPPs were measured). Changes in the percentage of skew-MEPPs were extremely variable (4–90%) and these changes depended on initial frequencies, initial skew- to bell-MEPP ratios and age of the mouse. With a change in Ca 2+ concentration, synaptic vesicle diameters and densities remained constant during changes in MEPP frequencies and large changes in the skew:bell-MEPP ratios; and vesicle numbers were sometimes slightly increased. Because of the wide range in MEPP frequencies and amplitudes, this study demonstrates that the effect of various treatments should be evaluated on identified endplates and that analyses of randomly selected endplates must consider the large variability between endplates. These results show that the skew-MEPP class must not be ignored in studies of spontaneous MEPP release, and that initial frequencies and age of the mouse are also important in evaluating changes in skew-MEPP to bell-MEPP ratios. The rapid changes in skew- to bell-MEPP classes indicate that MEPP class and size are determined at the moment of release by the state of the release process as proposed by Kriebel et al. (1990). Because changes in calcium concentration can immediately alter the ratio of skew- to bell-MEPPs we conclude that the release process has two states to generate the two classes of MEPPs, and that the release process is very sensitive to conditions so that states are easily changed. We propose that the release process meters transmitter in subunit amounts to form both classes of MEPPs and that the calcium ions modulate the process.

Ca(2+)‐dependent and ‐independent components of transmitter release at the frog neuromuscular junction.

1. When a Ca2+ chelator, bis (O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), was loaded into the presynaptic nerve terminal of the frog neuromuscular junction (NMJ), facilitation, measured as an increase in endplate potential (EPP) amplitudes during a train of ten stimulations at 100 Hz, was greatly decreased within 20 min of BAPTA-AM (the acetoxymethyl ester of BAPTA) perfusion, and remained at a constant low level thereafter, suggesting that [Ca2+]i at the presynaptic nerve terminal was buffered by BAPTA. 2. Detailed examination of the two components of facilitation of EPP amplitude in the BAPTA-loaded NMJs showed that the fast component was lost almost completely, while the slow component was unaffected by loaded BAPTA. Augmentation and potentiation were also unaffected by BAPTA. 3. Under external Ca(2+)-free conditions (with 1 mM-EGTA), both augmentation and potentiation of miniature endplate potential (MEPP) frequency were clearly observed after tetanic stimulation in the normal NMJ, and were also unaffected by loaded BAPTA. 4. The above findings strongly support the residual Ca2+ hypothesis for the fast component of facilitation, and suggest that the three slower processes (the slow component of facilitation, augmentation and potentiation) occur independently of [Ca2+]i. This Ca2+ independence was supported by the fact that facilitation and potentiation have multiplicative effects on the amount of release. 5. The quantal content of the first EPP in the train remained unchanged throughout the time course of BAPTA loading for most NMJs. This suggests that [Ca2+]i immediately adjacent to Ca2+ channels at the active zone triggers transmitter release and is little affected by loaded BAPTA. 6. MEPP frequency was almost unchanged during BAPTA loading, suggesting that the basal [Ca2+]i remained unchanged close to the dissociation constant of BAPTA for Ca2+ (108 nM). 7. The slow component of facilitation had a multiplicative relationship with augmentation and potentiation, suggesting that the underlying mechanism for the slow component of facilitation differs from that for augmentation and potentiation.

Multiplicative and additive Ca(2+)-dependent components of facilitation at mouse endplates.

1. Facilitation of endplate potentials (EPPs) and frequency of miniature endplate potentials (MEPPs) were studied, in the presence of low Ca2+/raised Mg2+, in isolated mouse hemidiaphragm, using pseudo-random sequences of nerve stimulation and automated (computer) counting of MEPPs and quantal components of EPPs. 2. The facilitation in quantal content of EPPs (m) produced by one or more antecedent stimuli was accompanied by facilitation of MEPP frequency (fm) that was similar in magnitude and substantially less than expected if facilitation reflects persistent (residual) intraterminal Ca2+. The time course of 'phasic' quantal release, associated with the EPP, was little if at all altered with facilitation. 3. The magnitude and time course of facilitation was consistent with two distinct presynaptic processes, each manifest both in m and fm, (i) an effect to multiply transmitter release, and (ii) residual Ca2+ which adds to Ca2+ brought in by nerve impulses. These have distinct time courses. 4. After loading nerve terminals with bis (O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), facilitation of m and fm became very small. 5. At sufficiently low Ca2+/raised Mg2+ facilitation of m and fm became very small although latency histograms showed clear EPPs. However, the multiplicative component of facilitation became maximal at Ca2+/Mg2+ concentrations giving an average m value less than 0.1, corresponding to about 5% of normal Ca2+ entry per pulse. At lower Ca2+, facilitation was restored when EPPs were made larger using 4-aminopyridine. 6. With EPPs elicited by brief 'direct' nerve terminal depolarizations, facilitation was graded with pulse intensity (and m) and could be much less than with EPPs with similar m evoked by nerve stimuli at lower Ca2+ and/or higher Mg2+. 7. It was concluded that fast facilitation is primarily multiplicative and reflects activity within the nerve terminal of a Ca(2+)-sensitive process distinct from that generating Ca(2+)-dependent release.

Reversal by cysteine of the cadmium-induced block of skeletal neuromuscular transmission in vitro.

1. Neuromuscular transmission in isolated nerve-muscle preparations was blocked by exposure to Cd2+ for less than 30 min or more than 2 h. The abilities of cysteine, Ca2+ or 3,4-diaminopyridine (3,4-DAP) to reverse the blockade induced by Cd2+ were studied. 2. On the mouse hemidiaphragm preparation, exposure to Cd2+ (10 microM) for 10 to 20 min induced a blockade which was easily reversed by increasing the extracellular Ca2+ concentration (5-10 mM) or by 3,4-DAP (100 microM). Exposure to Cd2+ (3-10 microM) for over 2 h led to a blockade which was not reversed by Ca2+ (5-15 mM) or 3,4-DAP (100 microM). Cysteine (1 mM) was able to reverse completely the blockade induced by both brief and prolonged exposures to Cd2+. 3. In chick biventer cervicis preparations, Cd2+ (100 microM) decreased the twitch height of indirectly stimulated preparations without affecting responses to exogenously applied acetylcholine, carbachol or KCl. Cysteine (1-3 mM) had no appreciable effect on twitch responses to indirect stimulation or to exogenously applied agonists but fully reversed the blockade induced by Cd2+ (100 microM). 4. In mouse triangularis sterni preparations, Cd2+ (1-30 microM) depressed the evoked quantal release of acetylcholine. Concentrations of Cd2+ which completely blocked endplate potentials (e.p.ps) were without significant effect on miniature endplate potential (m.e.p.p.) amplitude and frequency or time constant of decay. Cysteine (1-10 mM) alone had no effect on e.p.ps or m.e.p.ps, but completely reversed the blockade induced by Cd2+.6. In addition to the competitive blocking action of Cd2+ at the prejunctional Ca2+ channels, long exposure to Cd2+ leads to a blockade that is not competitive. This probably involves binding of Cd2+" at an extracellular thiol site on, or close to, voltage-operated Ca2+' channels.

Early postnatal changes in presynaptic potassium sensitivity

Amplitude histograms of miniature endplate potentials (MEPPs) and the overall frequency of skew-MEPPs and bell-MEPPs were examined in 5 and 15 mM potassium solutions at postnatal day (PD) 3, PD 10 and PD 27 neuromuscular junctions. Temporal non-uniformities in spontaneous release produced clusters of bell-MEPPs at PD 0-PD 3 junctions. PD 3 nerve terminals that preferentially release skew-MEPPs (5 mM potassium) were significantly ( P < 0.01) less sensitive to elevations in potassium than more mature (PD 10) junctions that preferentially released bell-MEPPs. Increases in the potassium concentration at PD 3 junctions increased the frequency of bell-MEPPs and altered the MEPP amplitude distribution profile by significantly ( P < 0.01) reducing the percentage of skew-MEPPs. Although the potassium sensitivity of PD 10 and PD 27 preparations were as expected for adult preparations, there was an increase in overall MEPP frequency in 5 mM potassium between PD 10 and PD 27. These results suggest that early postnatal increases in the number of presynaptic calcium channels establish adult levels of depolarization sensitivity and promote the preferential spontaneous release of bell-MEPPs. Since these changes occur during an early period of synapse elimination, they may play a critical role in synapse stabilization.

Magnetic field influence on acetylcholine release at the neuromuscular junction.

The effects of a static magnetic field on the frequency of miniature end-plate potentials (MEPPs), recorded from the murine phrenic nerve-diaphragm preparation, were studied. In the presence of a 1,200-G field, statistically significant changes in MEPP frequency were observed. There was a modest increase in frequency at temperatures at and below 34 degrees C and a prominent decrease in frequency at temperatures above 35 degrees C. This temperature-dependent phenomenon was not seen in the absence of calcium in the perfusate. These results suggest that, at its phase transition temperature, the diamagnetic anisotropy of the presynaptic membrane is sufficient to influence neurotransmitter release by altering the function of the transmembrane calcium transfer mechanism.

Effects of lectins on quantal release at the frog neuromuscular junction

This work was initiated because pretreatment with concanavalin A was reported to abolish the increase in spontaneous quantal release produced by hypertonic solutions [Gorio A. and Mauro A. (1979) J. gen. Physiol. 73, 245–263]. This suggested that lectins might be valuable tools for investigating the role of glycoproteins in the response to tonicity. We compared muscles soaked for 2 h in hypertonic solution containing concanavalin A with paired muscles soaked in hypertonic solution without lectin. The lectin treatment decreased miniature end-plate potential frequencies in Ringer and in hypertonic solutions compared with the controls. Even after lectin treatment hypertonic solutions and elevated K + solutions increased miniature end-plate potential frequencies, and the proportional increases were the same as in controls. The lectin treatment lowered baseline frequency, but the preparation still responded to hypertonic solutions. Concanavalin A effects appeared after treatment for more than 1 h and required concentrations of 10 μg/ml or higher. Higher concentrations did not produce more effect. Similar results were obtained with four other lectins with different sugar specificities. Treatment in hypertonic solution without lectin produces a similar, but smaller, decrease in baseline frequency. Concanavalin A pretreatment had no detectable effects on evoked release or facilitation. We conclude that the effects of lectins on quantal release are not mediated by binding to a single sugar group. The lectins do not produce a unique effect; they exaggerate the changes produced by hypertonic pretreatment. All of the effects could be accounted for by a reduction in baseline [Ca 2+] in the nerve terminal. Such reductions are produced by lectins in many cell types.

Diversity among mouse motor nerve terminals with respect to release transmitter quanta

1. 1. The aim of this work was to reexamine whether a positive correlation exists between the frequency ( F, sec −1) of miniature endplate potentials (m.e.p.ps) and the quantal content ( m) of endplate potentials (e.p.ps) or between quantal content, frequency and twin-pulse facilitation of transmitter release at a large number of neuromuscular junctions in the mouse. 2. 2. The values of F and m were both measured intracellulary at endplates of mouse diaphragm in a high Mg 2+/low Ca 2+ bathing solution. 3. 3. Values of both F and m varied from junction to junction. Smaller values of F were correlated with smaller values of m, and vice versa, resulting in a linear relationships. Histograms of F and m were skewed towards smaller values. 4. 4. E.p.ps evoked by twin pulses gave the quantal contents of the first ( m1) and second ( m2) responses. 5. 5. The ratio of m 2 to m1 varied from junction to junction. A histogram of this ratio was skewed towards smaller values. 6. 6. The ratio of m2 to m1 showed larger fluctuations at junctions with smaller values of F or m1 but was focused around 1 at junctions with larger values of F or m1. 7. 7. The skewed parts of the histograms of F, m and m2/ m1 accounted for the major population of junctions. 8. 8. These results support the hypothesis that an intrinsic ability to release transmitter plays a role in regulation of the evoked output of transmitter at neuromuscular junctions in the mouse. 9. 9. Such an ability is not correlated with the twin-pulse facilitation of transmitter release.

Effects of activators and inhibitors of protein kinase A on increases in quantal size at the frog neuromuscular junction.

Adrenaline, permeable cyclic adenosine monophosphate (cAMP) derivatives and insulin are known to elicit an increase in quantal size at the frog neuromuscular junction, primarily by increasing the amount of acetylcholine (ACh) per quantum. The quantal size increases produced by adrenaline or cAMP were antagonized by the protein kinase inhibitor H8 N-[2-(methylamino)ethyl]-5-isoquinolonesulfonamide. The increase in quantal size produced by insulin was not prevented by H8. Quantal size is also increased by pretreatment in hypertonic solution; this increase was also antagonized by H8. The H8 did not alter the increase in miniature endplate potential (MEPP) frequency produced by the hypertonic solution. A permeable cGMP derivative had no effect on quantal size. The diastereomer (Sp)-cAMPS (cyclic 3',5'-phosphothoate) activates protein kinase A(PKA). It elicited an increase in quantal size. The (Rp)-cAMPS isomer is known to inhibit PKA; it had no effect on quantal size. The increase in quantal size produced by hypertonic solution was antagonized by (Rp)-cAMPS but not by (Sp)-cAMPS. Brief exposure to a hypertonic solution containing a phosphodiesterase inhibitor followed by incubation in the inhibitor leads to an increase in quantal size. We conclude that one pathway for signaling for an increase in quantal size involves activation of PKA and that hypertonic pretreatment acts via this pathway.

Focal, extracellular recording of slow miniature junctional potentials at the mouse neuromuscular junction.

Miniature endplate potentials (MEPPs) with slow rising phase can be attributed either to burst of transmitter releases or to distortion of conduction from remote releasing sites. The spontaneous activity of neuromuscular junctions recorded extracellularly at mouse diaphragms using sharp electrodes was analyzed to test these two hypotheses. The miniature junctional potentials (MEJPs) frequencies observed intracellularly as compared to MEPP frequency measured intracellularly in controls indicate that most events recorded extracellularly are induced by the presence of the electrode. All types of MEPPs (bell-MEPPs, skew-MEPPs, slow-, and giant MEPPs) previously described with intracellular recording methods (Vautrin and Kriebel, Neuroscience 41:71-88, 1991) were observed extracellularly and showed similar characteristics. This means that the presynaptic and postsynaptic zones that generate these synaptic events are restricted within areas of a few micrometers squared of synaptic contact. Long rise times of extracellularly recorded synaptic spontaneous events may be explained by multiple transmitter releases at intervals shorter than the rise time of individual events, which postsynaptic responses fuse into a single peak.

Miniature endplate potentials induced by ammonium chloride, hypertonic shock, and botulinum toxin.

Intracellular recordings were made at the neuromuscular junction (NMJ) of the mouse diaphragm to study alteration of miniature endplate potential (MEPP) amplitude and rise time after different treatments. Following either hyperosmotic shock or 3 to 5 min of incubation in 10 to 50 mM ammonium chloride (NH4Cl) (replacing NaCl, a treatment which is known to raise intracellular pH) MEPP frequencies increased and the amplitudes of MEPPs decreased. These treatments as well as type A botulinum toxin (BoTx) gradually prolonged the rising phase of some MEPPs, which increased their time-to-peak (slow-MEPPs; Vautrin and Kriebel: Neuroscience 41:71-88, 1991) and increased eventually their amplitude. Fasciculation after hyperosmotic shock or during NH4Cl challenge was blocked by D-tubocurarine and was due to large slow-MEPPs that reached threshold for the muscle fiber action potential. The development of fasciculation provided the time course for the development of giant-MEPPs. Increased frequency of giant MEPP is accompanied by a block of the nerve-evoked muscle contraction. Effects of BoTx on spontaneous release were functionally antagonized either by NH4Cl or hyperosmotic shock. NH4Cl delayed BoTx blockage of bell-MEPPs. Data suggest that BoTx alters the formation of transmitter packets gradually but similarly to other treatments which increase incidence of skew-MEPPs.

The effect of 3,3,-dipyridylmethyl-1-phenyl-2-indolinone on the neuromuscular transmission in the rodent skeletal muscles

The effects of a cognition enhancer, 3,3-dipyridylmethyl-1-phenyl-2-indolinone (DPMPI) (21.5–645 μM), on neuromuscular transmission were studied electrophysiologically on diaphragms of mouse and rat and the soleus muscle of rat. The drug DPMPI (21.5–645μM) increased both direct and indirect twitch tension of mouse diaphragm. It also increased (a) the frequency of miniature endplate potentials and (b) the quantal content of endplate potential. However, DPMPI (64.5 μM) affected neither the amplitude of the directly elicited action potential of soleus muscle in the rat nor the magnitude of the resting membrane potential of mouse diaphragm, although DPMPI (215 μM) decreased the amplitude of the compound action potential of phrenic nerve. Based on these results, it is concluded that DPMPI had several effects on neuromuscular transmission, i.e. it (a) facilitated the transmitter releasing process of the motor nerve terminal, (b) decreased the conduction in the phrenic nerve and (c) increased the directly elicited twitch tension.

Effect of muscle stretching on the activity of neuromuscular transmission

The purpose of this study was to investigate how muscle stretching affects the activity of neuromuscular transmission. The magnitude of the post-tetanic potentiation (PTP) of miniature end-plate potential (m.e.p.p.) frequency was measured in the rat soleus muscle at resting length and stretched length. The parameters of the magnitude of PTP can be indicators of the kinetics of Ca2+ metabolism in the nerve terminal. One of the parameters, normalized initial post-tetanic frequency (f), was significantly increased by stretching muscle 10% (P less than 0.05) and 20% (P less than 0.01) of its resting length. Another parameter, the time constant of augmentation (tau a), was not significantly changed by muscle stretching. The time constant of potentiation (tau p) was significantly increased by 20% muscle stretching (P less than 0.05). These results indicate that the Ca2+ conductance, especially the voltage-dependent Ca2+ influx, of the nerve terminal could be increased by muscle stretching. Greater Ca2+ conductance of the nerve terminal would increase intracellular free Ca2+. Consequently, the probability of transmitter release would be increased, because Ca2+ is closely related to the activity of the transmitter release mechanism.

Subcellular mechanism and site of action of ionic lanthanum at the motor nerve terminal.

The mechanism by which ionic lanthanum (La3+) increases and subsequently decreases spontaneous transmitter release was investigated by recording miniature endplate potentials (MEPPs) at frog neuromuscular junctions. Addition of tetrodotoxin and Co2+ delayed the onset of MEPP frequency increase but did not otherwise prevent the response. Dinitrophenol substantially reduced but did not eliminate the increase, whereas 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester (TMB-8) completely abolished it. Thus, La3+ does not act by depolarizing the terminal or by substituting for Ca2+ at transmitter release sites. Instead, it appears to enter the terminal through Na+ channels and promote Ca2+ release from intracellular organelles. The profound depletion of transmitter with time may be due to the high turnover of transmitter coupled with the inhibition of metabolic processes by La3+.

Depression of miniature endplate potential frequency by acetylcholine and its analogues in frog.

1. Acetylcholine (ACh), 7.5 x 10(-5) M, and carbachol, 5 x 10(-6) M (CCh) depressed the frequency of miniature endplate potentials (m.e.p.ps) in the frog (Rana temporaria) sartorius neuromuscular junction with active acetylcholinesterase to about 50-55% of the controls. 2. A similar depression was produced by the nicotinic agonists, nicotine, suberyldicholine and tetramethylammonium. 3. The muscarinic agonists, oxotremorine, methylfurmethide and methacholine were without effect on m.e.p.p. frequency. The muscarinic antagonist, atropine and the nicotinic antagonist, (+)-tubocurarine, had no effect on the depression of m.e.p.p. frequency evoked by CCh. 4. The ganglionic blockers, benzhexonium and IEM-1119, were also without effect on the CCh-evoked depression of m.e.p.p. frequency. 5. Pretreatment of muscles with anticholinesterases did not prevent the CCh-induced drop in m.e.p.p. frequency. 6. The effect of CCh was proportionally the same as in the controls in preparations where the m.e.p.p. frequency was changed by elevation of K+ and in the presence of theophylline, noradrenaline, dibutyryl adenosine 3':5'-cyclic monophosphate (db cyclic AMP) and db cyclic GMP. 7. An inhibitor of Na+,K(+)-ATPase, ouabain, 5 x 10(-5) mol l-1, prevented or reversed the depression of m.e.p.p. frequency by CCh. However, the depression was present in a nominally K(+)-free medium. Insulin and adrenaline, which are considered to be Na+,K(+)-ATPase activators, were without effect on depression of m.e.p.p. frequency. 8. The depression of m.e.p.p. frequency by 5 x 10(-6) M CCh was the same at temperatures between 5 and 30 degrees C with a Q10 near to 1.0. When threshold amounts of CCh were used (6 x 10-7 and 3 x 10-7 M), the depression was less at higher temperatures.9. The receptive structures responsible for the CCh (or ACh)-evoked depression of m.e.p.p. frequency differ pharmacologically from muscarinic, nicotinic ganglionic and neuromuscular junction ACh-receptors as well as from the synaptic cholinesterase, in contrast to previous reports (Duncan & Publicover, 1979).The low temperature-dependence points to the possibility that physical rather than biochemical processes are limiting in this presynaptic effect of cholinomimetics.

Structure and function of the neuromuscular junction in young adultmdx mice

Dystrophin, the protein product of the gene responsible for X-linked muscular dystrophies, shares structural features with the cytoskeletal proteins spectrin and alpha-actinin. Like spectrin, it is localized at the cytoplasmic surface of the sarcolemma and is particularly concentrated in the subsynaptic region of the neuromuscular junction. Mdx mice have a profound deficiency of dystrophin and develop a necrotizing myopathy in the first weeks of life. Abnormalities of the neuromuscular junction, including a redistribution of postsynaptic molecules and reduction in synaptic folding, are also observed. We have studied these mice to see whether the lack of dystrophin has a specific effect on the structure and function of their neuromuscular junctions. Using a fore-limb muscle from 8 week old mdx mice we confirm the previously described postsynaptic structural changes and in addition show that many nerve terminals are abnormally complex. We demonstrate that these structural abnormalities are found exclusively at neuromuscular junctions on regenerated muscle fibres. Despite these structural abnormalities, miniature endplate potential frequency, the quantal content of endplate potentials, the amplitude and time course of miniature endplate currents and the number of acetylcholine receptors at the postsynaptic membrane are normal in mdx mice of this age. We conclude that in the mdx mouse the absence of dystrophin from the postsynaptic membrane has little direct effect on the function of the neuromuscular junction but that degeneration and regeneration of muscle fibres leads to remodelling of both its pre- and postsynaptic components.