Amplitude Of Miniature Endplate Potentials Research Articles

Inositol derivatives modulate spontaneous transmitter release at the frog neuromuscular junction

One of the consequences of G-protein-coupled receptor activation is stimulation of phosphoinositol metabolism, leading to the generation of IP 3 and its metabolites 1,3,4,5-tetrakisphosphate (IP 4) and inositol 1,2,3,4,5,6-hexakisphosphate (IP 6). Previous reports indicate that high inositol polyphosphates (IP 4 and IP 6) are involved in clathrin-coated vesicular recycling. In this study, we examined the effects of IP 4 and IP 6 on spontaneous transmitter release in the form of miniature endplate potentials (MEPP) and on enhanced vesicular recycling by high K + at frog motor nerve endings. In resting conditions, IP 4 and IP 6 delivered intracellularly via liposomes, caused concentration-dependent increases in MEPP frequency and amplitude. Pretreatment with the protein kinase A (PKA) inhibitor H-89 or KT 5720 reduced the IP 4-mediated MEPP frequency increase by 60% and abolished the IP 6-mediated MEPP frequency increases as well as the enhancement in MEPP amplitude. Pretreatment with antibodies against phosphatidylinositol 3-kinase (PI 3-K), enzyme also associated with clathrin-coated vesicular recycling, did not alter the IP 4 and IP 6-mediated MEPP frequency increases, but reduced the MEPP amplitude increase by 50%. In our previous reports, IP 3, but not other second messengers releasing Ca 2+ from internal Ca 2+ stores, is able to enhance the MEPP amplitude. In order to dissociate the effect of Ca 2+ release vs. metabolism to IP 4 and IP 6, we evaluated the effects of 3-deoxy-3-fluoro-inositol 1,4,5-trisphosphate (3F-IP 3), which is not converted to IP 4 or IP 6. 3F-IP 3 produced an increase then decrease in MEPP frequency and a decrease in MEPP amplitude. In elevated vesicle recycling induced by high K +-Ringer solution, IP 4 and IP 6 have similar effects, except decreasing MEPP frequency at a higher concentration (10 −4 M). We conclude that (1) high inositol polyphosphates may represent a link between IP 3 and cAMP pathways; (2) the IP 3-induced increase of MEPP amplitude is likely to be due to its high inositol metabolites; (3) PI 3-K is not involved in the IP 4 and IP 6-mediated MEPP frequency increases, but may be involved in MEPP size.

Extra- and intracellular sphingosylphosphorylcholine promote spontaneous transmitter release from frog motor nerve endings.

Similar to phosphatidylinositol bisphosphate, sphingomyelin breakdown generates several lipids, including sphingosylphosphorylcholine (SPC), that are putative signaling molecules. The present study was undertaken to evaluate the involvement of SPC in transmitter release process. Intracellular recordings were made from isolated frog sciatic-sartorius nerve-muscle preparations, and the effects of SPC on neurosecretion in the form of miniature endplate potentials (MEPPs) were assessed. Extracellular application of SPC mixture (D,L-SPC) at 1, 10, and 25 microM increased the MEPP frequency by 68, 96, and 127%, respectively. D-erythro-SPC (dissolved in dimethyl sulfoxide but not coupled to bovine serum albumin), but not L-threo-SPC, was active extracellular; the former (at 10 microM) increased the MEPP frequency by 143%. D-erythro-SPC treatment did not significantly change the median amplitude or frequency-distribution of MEPPs. Intracellular delivery via liposomes, in which 10, 100, or 1000 microM SPC mixture was entrapped in liposomal aqueous phase, induced a concentration-dependent increase in MEPP frequency of 45, 91, and 100%, respectively. D-erythro-SPC and L-threo-SPC at the concentration of 100 microM increased the MEPP frequency by 117 and 67%, respectively, or 91 and 61%, respectively, when coupled to bovine serum albumin. Pretreatment with thapsigargin significantly reduced but did not abolish the effects of extracellular D-erythro-SPC (10 microM) or liposomes containing 100 microM D-erythro-SPC. Liposomes filled with 100 microM D-myo-inositol 1,4,5-trisphosphate (IP3) enhanced the MEPP frequency to the same magnitude as 100 microM D-erythro-SPC entrapped in liposomes. However, administration of 100 microM D-erythro-SPC and IP3 entrapped in the same liposomes enhanced the MEPP frequency by 70%, which was less than that produced by these two compounds alone. The result provides the first electrophysiological evidence that SPC can modulate transmitter release by an extra- or intracellular action at the frog motor nerve ending.

Neurotoxic and myotoxic actions of Naja naja kaouthia venom on skeletal muscle in vitro

The neuromuscular and skeletal muscle actions of Naja naja kaouthia snake venom were studied in mammalian (rat left hemidiaphragm) and avian (chick biventer cervicis) nerve–muscle preparations. The venom (5 and 10 μg/ml) produced neuromuscular blockade (85% in 36.8±2.0 min, mean±SEM, n=5, and 18±0.6 min, n=3, p<0.01, respectively) in the rat preparation. That the phospholipase A 2 (PLA 2) activity of the venom is involved in this effect was evaluated by inhibiting this enzyme with p-bromophenacyl bromide. This resulted in significantly ( p<0.01) increasing the time required for 85% blockade with 5 and 10 μg/ml to 54±4.6 min ( n=3) and 29±0.6 min ( n=3), respectively. In chick preparations, the venom (5 μg/ml) produced neuromuscular blockade in 14.0±1.8 min ( n=5). The contractures to exogenous acetylcholine were completely inhibited by the venom, whereas those to 134 μM KCl were partially blocked in chick preparations ( n=4, n=3, respectively). The venom (5 μg/ml) produced a progressive decrease in the amplitude of miniature end-plate potentials (m.e.p.ps) in the rat hemidiaphragm, but did not alter the resting membrane potential at 5 μg/ml. Neostigmine (5.8 μM) immediate and partially reversed the 85% blockade produced by venom (61%, n=3) in rat preparations, as did 4-aminopyridine (53 μM) (∼59%, n=3). The 4-aminopyridine and neostigmine also restored the m.e.p.ps to pre-venom (control) values. In rat preparations, the venom damaged 47%±11% and 62.7±3.6% of the muscle fibers at concentrations of 5 and 10 μg/ml, respectively. For venom in which PLA 2 activity was inhibited, the corresponding values were 38±11.8% (5 μg/ml) and 67±9.6% (10 μg/ml). These findings suggest a post-synaptic neurotoxic action for N. n. kaouthia venom, and that inhibiting phospholipase activity of the venom reduces significantly the neuromuscular block but not the direct myotoxicity.

The vasoactive peptide urotensin II stimulates spontaneous release from frog motor nerve terminals.

1. The effect of urotensin II (U-II) on spontaneous transmitter release was examined in the frog to see if the biological activity of this vasoactive peptide extended to neural tissues. 2. In normal Ringer solution, frog and human U-II (fU-II and hU-II, respectively) caused concentration-dependent, reversible increases in miniature endplate potential (MEPP) frequency, with hU-II about 22 times more potent than fU-II. hU-II caused a dose-dependent increase in MEPP amplitude, whereas fU-II caused an increase, followed by a decrease with higher concentrations. 3. Increasing extracellular Ca(2+) three-fold had no effect on the MEPP frequency increase to 25 microM hU-II. Pretreatment with thapsigargin to deplete endoplasmic reticulum Ca(2+) caused a 61% reduction in the MEPP frequency increase to 25 microM hU-II. 4. Pretreatment with the phospholipase C inhibitor U-73122 caused a 93% reduction in the MEPP frequency increase to 25 microM hU-II and a 15% reduction in the increase in MEPP amplitude. Pretreating with antibodies against the inositol 1,4,5-trisphosphate (IP(3)) type 1 receptor using liposomal techniques reduced the MEPP frequency increase by 83% but had no effect on MEPP amplitude. 5. Pretreating with protein kinase C inhibitors (bisindolylmaleimide I and III) had no effect on the response to 25 microM hU-II, but pretreating with protein kinase A inhibitors (H-89 and KT5720) reduced the MEPP frequency increase by 88% and completely abolished the increase in MEPP amplitude. 6. Our results show that hU-II is a potent stimulator of spontaneous transmitter release in the frog and that the effect is mediated by IP(3) and cyclic AMP/protein kinase A.

Sphingosine 1-phosphate enhances spontaneous transmitter release at the frog neuromuscular junction.

Intracellular recordings were made from isolated frog sciatic-sartorius nerve-muscle preparations, and the effects of sphingosine 1-phosphate (S1-P) on miniature endplate potentials (MEPPs) were studied. Extracellular application of S1-P (1 and 30 micro M) had no significant effects on the frequency and amplitude of MEPPs. Delivery into nerve terminals by liposomes containing 10(-5), 10(-4) or 10(-3) M S1-P was associated with a concentration-dependent increase in MEPP frequency of 37, 63 and 86%. The per cent of median MEPP amplitude was not significantly changed, but there was an increase in the number of 'giant' MEPPs. Pre-exposure of the preparations to S1-P 10(-5) but not 10(-8) M entrapped in liposomes for 15 min blocked the effects of subsequent superfusion of S1-P (10(-4) M)-filled liposomes on MEPP frequency. Thus, intracellular S1-P receptors seem to undergo 'desensitization' to higher concentrations of S1-P. The result provides the first evidence that S1-P acting intracellularly but not extracellularly enhances spontaneous transmitter release at the frog neuromuscular junction.

New evidence for a presynaptic action of prednisolone at neuromuscular junctions.

The action of prednisolone at the neuromuscular junction was studied in mouse isolated phrenic nerve-diaphragm and rat external popliteal/sciatic nerve-tibialis anterior muscle preparations. Prednisolone (0.03 mM and 0.3 mM) did not alter the twitch-tension in phrenic nerve-diaphragm preparations after 120 min, but increased the frequency (170 +/- 4%) and amplitude (200 +/- 13%) of miniature end-plate potentials. Quantal content was not influenced by the glucocorticoid treatment. Prednisolone (400 microg/kg) did not change the twitch-tension in rat external popliteal/sciatic nerve-tibialis anterior muscle preparations. However, this steroid (0.3 mM) prevented the neuromuscular blockade by d-tubocurarine (1.45 microM) in mouse preparations by 70 +/- 10% (P < 0.05). A similar effect (82 +/- 6% protection, P < 0.05) occurred in rats treated with prednisolone (400 microg/kg) before d-tubocurarine (225 microg/kg). In phrenic nerve-diaphragm preparations, prednisolone (0.3 mM) increased (13 +/- 4%, p < 0.05) the twitch-tension in the presence of beta-bungarotoxin (1 microM), and prevented the blockade produced by this toxin (0.15 microM) in its third phase of action. This presynaptic facilitatory effect may contribute to the usefulness of prednisolone in myasthenia gravis.

Neuromuscular action of Bothrops lanceolatus (Fer de lance) venom and a caseinolytic fraction

A protein capable of inducing neuromuscular blockade in avian preparations and of depolarizing mouse diaphragm muscle was isolated from Bothrops lanceolatus venom using gel filtration and ion-exchange chromatography. The purified protein was a single chain polypeptide with an estimated molecular mass of 27.5 kDa by SDS–PAGE and had caseinolytic activity (13.3 units/mg), but no phospholipase A 2. B. lanceolatus venom (50 μg/ml) and the caseinolytic protein (20 μg/ml) produced contracture and progressive irreversible blockade (50% in 25±5 min (SEM) and 45±15 min, respectively), in indirectly stimulated chick biventer cervicis preparations. The contractile responses to acetylcholine (ACh; 37 and 74 μM, n=6) were inhibited by venom and the caseinolytic protein, whereas those to potassium (13.4 mM, n=6) were not. Membrane resting potential measurements in mouse hemidiaphragm preparations showed that B. lanceolatus venom and the purified protein caused depolarization which was prevented by d-tubocurarine (14.6 mM). The venom produced a slight increase in the amplitude and frequency of miniature end-plate potentials, but this effect was not seen with the purified fraction. These results suggest that the purified protein acts exclusively post-synaptically.

Possible involvement of neurokinin receptors in CNS and neuromuscular synapse in the realization of the effects of CAPAH and substance P.

Nootropic non-anticholinesterase organophosphorus preparation CAPAH and substance P produced similar and dose-dependent effects on the amplitude and temporal parameters of miniature endplate potentials in mammalian neuromuscular synapse. Neurokinin receptor antagonist Win-51.708 abolished the effects of these agents. In behavioral experiments substance P moderated the mnemotropic and antidepressant effects of CAPAH. It was assumed that neurokinin receptors are the targets of CAPAH and substance P in CNS and neuromuscular synapse.

Action of Micrurus dumerilii carinicauda coral snake venom on the mammalian neuromuscular junction

The venoms of coral snakes (mainly Micrurus species) have pre- and/or postsynaptic actions, but only a few of these have been studied in detail. We have investigated the effects of Micrurus dumerilii carinicauda coral snake venom on neurotransmission in rat isolated phrenic nerve-diaphragm muscle and chick biventer cervicis preparations stimulated directly or indirectly. M. d. carinicauda venom (5 or 10 μg/ml) produced neuromuscular blockade in rat (85–90% in 291.8±7.3 min and 108.3±13.8, respectively; n=5) and avian (95.0±2.0 min; 5 μg/ml, n=5) preparations. Neostigmine (5.8 μM) and 3,4-diaminopyridine (230 μM) partially reversed the venom-induced neuromuscular blockade in rat nerve-muscle preparations. In neither preparation did the venom depress the twitch response elicited by direct muscle stimulation. The contractures induced by acetylcholine in chick preparations were inhibited by the venom (95–100%; n=4; p<0.05). In rat preparations, the venom produced a progressive decrease in the amplitude of miniature end-plate potentials (m.e.p.ps control frequency=69.3±5.0/min and control amplitude=0.4±0.2 mV) until these were abolished. Neostigmine (5.8 μM) and 3,4-diaminopyridine (230 μM) partially antagonized this blockade of m.e.p.ps. The resting membrane potential was not altered with the venom (10 μg/ml). M. d. carinicauda venom produced dose-dependent morphological changes in indirectly stimulated mammal preparations. Twenty-five per cent of muscle fibers were affected by a venom concentration of 5 μg/ml, whilst 60.7% were damaged by 10 μg of venom/ml. In biventer cervicis preparations, the morphological changes were slower in onset and were generally characterized by undulating fibers and, to a lesser extent, by zones of disintegrating myofibrils. A venom concentration of 5 μg/ml damaged 52.2% of the fibers. These findings indicate that M. d. carinicauda venom has neurotoxic and myotoxic effects and that the neuromuscular blockade involves mainly a postsynaptic action.

IgM monoclonal antibody against terminal moiety of GM2, GalNAc-GD1a and GalNAc-GM1b from a pure motor chronic demyelinating polyneuropathy patient: effects on neurotransmitter release

We describe a patient with a pure motor chronic demyelinating polyneuropathy with an IgM monoclonal component showing anti-GM2, GalNAc-GD1a and GalNAc-GM1b reactivity whose common epitope appears to be -[GalNAcβ1-4Gal(3-2αNeuAc)β1]. We used intracellular recording to study how IgM from this patient affected neurotransmitter release in the mouse diaphragm in vitro. Adding serum (and specifically, the purified monoclonal IgM component) blocked the nerve-evoked response in both quantal content and evoked endplate potential (EPP) amplitude in a complement-independent and reversible manner. The IgM increased the frequency of spontaneous miniature endplate potentials (MEPPs) in a complement-dependent and reversible manner but had no effect on MEPP amplitude.

GABA Enhances Transmission at an Excitatory Glutamatergic Synapse

GABA mediates both presynaptic and postsynaptic inhibition at many synapses. In contrast, we show that GABA enhances transmission at excitatory synapses between the lateral gastric and medial gastric motor neurons and the gastric mill 6a and 9 (gm6a, gm9) muscles and between the lateral pyloric motor neuron and pyloric 1 (p1) muscles in the stomach of the lobster Homarus americanus. Two-electrode current-clamp or voltage-clamp techniques were used to record from muscle fibers. The innervating nerves were stimulated to evoke excitatory junctional potentials (EJPs) or excitatory junctional currents. Bath application of GABA first decreased the amplitude of evoked EJPs in gm6a and gm9 muscles, but not the p1 muscle, by activating a postjunctional conductance increase that was blocked by picrotoxin. After longer GABA applications (5-15 min), the amplitudes of evoked EJPs increased in all three muscles. This increase persisted in the presence of picrotoxin. beta-(Aminomethyl)-4-chlorobenzenepropanoic acid (baclofen) was an effective agonist for the GABA-evoked enhancement but did not increase the postjunctional conductance. Muscimol activated a rapid postsynaptic conductance but did not enhance the amplitude of the nerve-evoked EJPs. GABA had no effect on iontophoretic responses to glutamate and decreased the coefficient of variation of nerve-evoked EJPs. In the presence or absence of tetrodotoxin, GABA increased the frequency but not the amplitude of miniature endplate potentials. These data suggest that GABA acts presynaptically via a GABA(B)-like receptor to increase the release of neurotransmitter.

Adult dystrophic (mdx) endplates exhibit reduced quantal size and enhanced quantal variation.

Examination of miniature endplate potential (MEPP) distributions indicated that the average quantal size is significantly reduced by approximately 44% in young adult mdx mice (5-7 weeks). The average quantum declined further to approximately 37% of non-dystrophic levels in mature (6- to 24-month) mdx mice. Young adult non-dystrophic and mdx endplates and mature non-dystrophic endplates exhibited a linear relationship between the mean and variance of uniquantal MEPP amplitude distributions. Mature mdx endplates, however, exhibited a distinctly nonlinear relationship characterized by large increases in variance at larger mean MEPP amplitudes. These results indicate a reduced average density of functional acetylcholine (ACh) receptors (AChRs) at mdx endplates that, in mature preparations, is associated with greater temporal or spatial variability in the density of functional AChRs apposed to individual release sites.

Habitual exercise enhances neuromuscular transmission efficacy of rat soleus muscle in situ.

Rat motor nerve terminals and the endplates they interact with exhibit changes to varying patterns of use, as when exposed to increased activation in the form of endurance exercise training. The extent to which these changes affect neuromuscular transmission efficacy is uncertain. In this study, the effects of habitual exercise on the electrophysiological properties of neuromuscular transmission in rat soleus muscle were investigated using a novel in situ approach. Consistent with previous reports, miniature endplate potential frequency was enhanced by habitual exercise. Other passive properties, such as resting membrane potential, miniature endplate potential amplitude, and "giant" miniature endplate potential characteristics were unaltered by the training program. Full-size endplate potentials were obtained by blocking soleus muscle action potentials with mu-conotoxin GIIIb. Quantal content values were 91.5 and 119.9 for control and active groups, respectively (P < 0.01). We also measured the rate and extent of endplate potential amplitude rundown during 3-s trains of continuous stimulation at 25, 50, and 75 Hz; at 50 and 75 Hz, we found both the rate and extent of rundown to be significantly attenuated (10--20%) in a specific population of cells from active rats (P < 0.05). The results establish the degree of activity-dependent plasticity as it pertains to neuromuscular transmission in a mammalian slow-twitch muscle.

Effect of tetraalkylammonium derivative of 6-methyluracil on amplitude and temporal parameters of miniature endplate potentials in frog neuromuscular junction.

The effect of C-547, a tetraalkylammonium derivative of 6-methyluracil, a novel highly selective acetylcholinesterase inhibitor, on frog neuromuscular junction was studied. In concentrations 10(-9)-10(-7)M the preparation increased the amplitude and temporal parameters of miniature endplate potentials. In contrast to the effect of C-574 on purified acetylcholinesterase from mammals, the effect of this agent on frog neuromuscular junction was reversible. In a concentration of 10(-6)M the preparation apart from anticholinesterase activity produced a parasympatholytic effect manifested in a decrease in amplitude and decay time constant of miniature endplate potentials accompanied by a decrease in spontaneous transmitter secretion. After washout, the parasympatholytic effect recovered more slowly, but disappeared more rapidly compared to anticholinesterase activity. These findings suggest that parasympatholytic effect of C-547 results from direct action on receptor-channel complexes in the endplate membrane.

PTX-sensitive and -insensitive synaptic modulation at the frog neuromuscular junction.

Pharmacological manipulations were used to examine the role of G proteins in modulating synaptic transmission at the frog neuromuscular junction. Pertussis toxin (PTX, a G protein antagonist) increased end-plate potential (epp) amplitude but had no effect on the amplitude or frequency of miniature end-plate potentials. Mastoparan (a G protein agonist) decreased epp amplitude, while suramin (an antagonist) increased epp amplitude. The results suggest that PTX-sensitive G proteins tonically modulate synaptic transmission by reducing the amount of transmitter released in response to presynaptic action potentials. We also showed that endogenous ATP decreased transmitter release via P2 receptor in a PTX-insensitive manner. Thus, at least two distinct mechanisms regulate neuromuscular transmission; one is coupled to PTX-sensitive G proteins and the other is not.

Disruption of actin impedes transmitter release in snake motor terminals.

To investigate the role of actin in vertebrate nerve terminals, nerve-muscle preparations from garter snake (Thamnophis sirtalis) were treated with the actin-depolymerizing agent latrunculin A. Immunostaining revealed that actin filaments within presynaptic motor terminal boutons were disrupted by the drug. In preparations loaded with the optical probe FM1-43, destaining was reduced by latrunculin treatment, suggesting that transmitter release was partially blocked. Latrunculin treatment did not influence the amplitude or time course of spontaneous miniature endplate potentials (MEPPs). Similarly, endplate potentials (EPPs) evoked at low frequency were comparable in control and latrunculin-treated curarized preparations. Brief tetanic stimulation of the muscle nerve (25 Hz, 90 s) depressed EPP amplitudes in both control and latrunculin-treated preparations. After tetanus, EPPs elicited at 0. 2 Hz in control preparations recovered rapidly (0-5 min) and completely (usually potentiating to above pre-tetanus levels; 130 +/- 11 %, mean +/- s.e.m.). In contrast, EPPs evoked in latrunculin-treated preparations recovered slowly (8-10 min) and incompletely (84 +/- 8 %). The influence of latrunculin on post-tetanic EPPs depended on its concentration in the bath (KD = 3. 1 microM) and on time of incubation. These observations argue that actin filaments facilitate transmitter release rather than impede it. Specifically, actin may facilitate mobilization of vesicles towards the releasable pools.

The modulation of skeletal muscle contraction by FMRFamide-related peptides of the locust

The external ventral protractor muscle of the VIIth abdominal segment, M234, is a skeletal muscle that possesses receptors that recognize a range of FMRFamide-related peptides and application of these peptides results in an increase in the amplitude of neurally evoked contractions with little or no effect on basal tonus. FLRFamide itself has the same biologic activity as the extended peptides, whereas truncation to LRFamide or RFamide results in a peptide with no biologic activity. The receptors recognizing these extended FLRFamides, which include SchistoFLRFamide, seem to be different from the SchistoFLRFamide receptors found on locust oviduct visceral muscle. SchistoFLRFamide and the non-peptide mimetic, benzethonium chloride (Bztc), increase the frequency and amplitude of miniature endplate potentials, increase the amplitude of neurally evoked excitatory junction potentials, and result in a hyperpolarisation of resting membrane potential. Bztc, however, also abolishes the active membrane response that may explain its ability to decrease neurally evoked contractions.

Transmitter quantal size in Torpedo electrocytes is determined by frequency of release

Miniature end-plate potentials (MEPPs) were focally recorded from the cytoplasmic surface of electrocytes in isolated columns of the Torpedo electric organ. Double electrode studies showed that the junctional area was restricted to 12 μm 2. MEPP frequencies ranging from 1/min to 400/s were controlled with electrode advancement against the cytoplasmic surface. Stable membrane potentials and noise levels indicated constant intracellular, focal recording conditions. Focal MEPPs are only 1–3 mV and MEPP amplitudes smoothly decreased with an increase in MEPP frequency which demonstrates a process that meters quantal size at moment of release. Thus, release if not from a prepackaged store. MEPP interval analyses showed that events are weakly interactive at low frequencies and periodic at higher frequencies. The interdependency of MEPP amplitudes and intervals indicates that the mechanism of release controls both rate and quantal size. We propose that the amplitude and frequency dependencies of MEPPs at the Torpedo nerve–electrocyte junction are best described by a membrane channel (e.g., mediatophore, Israël and Dunant, Neurochem. Int. 28 (1996) 1–9) that meters transmitter from a presynaptic store.

Fractal dimensions in the occurrence of miniature end-plate potential in a vertebrate neuromuscular junction

1. 1. Occurrence of miniature endplate potentials (MEPP) in the sartorius muscle of Rana catesbiana in high Mg 2+ Ringer solution were observed in standard intracellular recording. Intervals and amplitudes of sequentially occurring MEPP were registered and analyzed. 2. 2. Interval histograms of a time series of MEPP showed exponential-like pattern as reported in the classical study by Fatt and Katz (1952). The cumulative distribution of the intervals plotted in logarithmic axes showed two distinct phases. In shorter intervals (<ls), curve along exponential decay was observed, and in longer intervals (≥ls) linear decay can be seen. The latter power-law relation gave dimensions of 4.111 ± 0.812 (mean and S.D.). Self-similarity in longer range implies a time-scale invariant nature and may suggest fractal nature in restoration process of synaptic vesicles, while exponential decay in the short time interval range implies random release of transmitter packet from the readily releasable pool. 3. 3. Fluctuation of amplitudes in sequentially occurred MEPP were analyzed according to Higuchi's cumulative route-length analysis. The estimates for sequential amplitude curve showed the power-law relation in a logarithmic plot whose inclination (=D) estimated with linear regression analysis was 1.996±0.007 (mean and S.D.). Thia results indicate that fluctuation in the amplitude of MEPP shows possible maximum complexity as a graphic curve in 2-D plane. Similar result was obtained for fluctuation of intervals of successively occurring MEPP.

Pathological changes induced by PhTx1 from Phoneutria nigriventer spider venom in mouse skeletal muscle in vitro

The `armed' spider Phoneutria nigriventer is responsible for most human accidents involving spiders in Brazil. The effects of fraction Tx1 (PhTx1) from the venom of this spider were investigated by physiological and morphological methods using the mouse phrenic nerve-diaphragm preparation. PhTx1 (1 and 5 μg) did not affect the twitch tension of muscle fibers under indirect electrical stimulation. At this same concentration, PhTx1 also did not alter the miniature end-plate potential (mepp) frequency and amplitude, nor did it change the resting membrane potential 60 min after addition to the preparation. Light microscopy (LM) revealed that in muscles incubated with PhTx1 a number of fibers were morphologically altered, as evidenced by microvacuolization and myofibril hypercontraction and loss within 15 min after toxin administration. Transmission electron microscopy (TEM) showed sarcoplasmic reticulum swelling, disorganization of the sarcomeres and mitochondrial damage, and occasionally, sarcolemmal discontinuities with a persisting basal membrane. The intra-muscular fascicles of the phrenic nerve showed myelinated axons with vacuolated myelin sheaths as well as peri- and intra-axoplasmic vacuoles. The neuromuscular junction changes were variable, but were rarely severe. Thus, although PhTx1 did not depolarize or hyperpolarize the neuromuscular junction, it was nevertheless toxic to a restricted number of muscle fibers and nerve structures. The site of action of PhTx1 may involve the sarcolemma and axolemma as suggested by the morphological abnormalities which could reflect hydroelectrolytic disturbances.