Frequency Of Miniature End-plate Potentials Research Articles

A new conotoxin isolated from Conus consors venom acting selectively on axons and motor nerve terminals through a Na+-dependent mechanism.

A novel conotoxin was isolated and characterized from the venom of the fish-hunting marine snail Conus consors. The peptide was identified by screening chromatography fractions of the crude venom that produced a marked contraction and extension of the caudal and dorsal fins in fish, and noticeable spontaneous contractions of isolated frog neuromuscular preparations. The peptide, named CcTX, had 30 amino acids and the following scaffold: X11CCX7CX2CXCX3C. At the frog neuromuscular junction, CcTx at nanomolar concentrations selectively increased nerve terminal excitability so that a single nerve stimulation triggered trains of repetitive or spontaneous synaptic potentials and action potentials. In contrast, CcTx had no noticeable effect on muscle excitability even at concentrations 100 x higher than those that affected motor nerve terminals, as revealed by direct muscle stimulation. In addition, CcTx increased miniature endplate potential (MEPP) frequency in a Ca2+-free medium supplemented with ethylene glycol-bis-(beta-aminoethyl ether)-N,N,N', N'-tetraacetic acid (EGTA). Blockade of voltage-dependent sodium channels with tetrodotoxin (TTX) either prevented or suppressed the increase of MEPP frequency induced by the toxin. CcTx also produced a TTX-sensitive depolarization of the nodal membrane in single myelinated axons giving rise, in some cases, to repetitive and/or spontaneous action potential discharges. In addition, CcTx increased the nodal volume of myelinated axons, as determined using confocal laser scanning microscopy. This increase was reversed by external hyperosmolar solutions and was prevented by pretreatment of axons with TTX. It is suggested that CcTx, by specifically activating neuronal voltage-gated sodium channels at the resting membrane potential, produced Na+ entry into nerve terminals and axons without directly affecting skeletal muscle fibres. CcTx belongs to a novel family of conotoxins that targets neuronal voltage-gated sodium channels.

The neuromuscular action of Ancylometes sp. spider venom in the rat phrenic nerve–diaphragm preparation

The effects of Ancylometes sp. venom on muscle contraction and bioelectrical potentials were investigated in the rat phrenic nerve–diaphragm muscle preparation. The venom (50 μg/ml) depolarized the diaphragm muscle fiber membranes. This effect was abolished by tetrodotoxin and by reduction of the sodium concentration of the Tyrode solution. The increase in the frequency of miniature end plate potentials induced by the venom was also suppressed by tetrodotoxin (3 μM). These results indicate that the venom may activate voltage-dependent sodium channels in cell membranes. All of the effects of Ancylometes sp. venom on this nerve–muscle preparation (i.e. increase in twitch tension, spontaneous small phasic contractions and increase in the frequency of miniature end-plate potentials) may be explained in terms of its action on sodium channels.

Long-term use-dependent enhancement of impulse-induced exocytosis by adrenaline at frog motor nerve terminals.

Adrenaline (5-20 microM) use-dependently increased end-plate potentials (EPPs) in normal Ringer solution (containing d-tubocurarine to partially block acetylcholine receptors) and a low Ca2+, high Mg2+ solution for more than several hours and decreased the coefficient of variation of EPP amplitude in the latter solution in frog neuromuscular junctions. The amplitude and frequency of miniature EPPs and impulse-induced increases in intraterminal Ca2+ concentration were unaffected. Adrenaline thus causes sustained enhancement of impulse-induced exocytosis by acting at a mechanism of exocytosis downstream to Ca2+ entry.

Calcium channels coupled to neurotransmitter release at neonatal rat neuromuscular junctions.

1. The effects of different calcium channel blockers (omega-agatoxin IVA (omega-Aga IVA), omega-conotoxin GVIA (omega-CgTx GVIA) and dihydropyridines) were tested on spontaneous and evoked transmitter release at embryonic and newborn rat neuromuscular junctions (NMJs). 2. The nerve-evoked transmitter release quantal content (m) was strongly reduced by the P/Q-type voltage-dependent calcium channel (VDCC) blocker omega-Aga IVA (100 nM) at newly formed endplates of embryos and 0- to 11-day-old rats, in agreement with the effect of this blocker on transmitter release at mature and reinnervating muscles. 3. omega-CgTx GVIA (1-5 microM), the N-type VDCC blocker, also caused a significant reduction in m at newly formed NMJs early in development (embryos and 0- to 4-day-old rats), while it was ineffective in more mature animals (5- to 11-day-old rats). 4. L-type channel blockers, nitrendipine (1 microM) and nifedipine (1 microM), did not significantly affect neurally evoked release at developing NMJs. However, nifedipine (10 microM) was able to increase m significantly at 0- to 4-day-old rat NMJs. 5. At developing NMJs, K+-evoked transmitter release was dependent on Ca2+ entry through VDCCs of the P/Q-type family (100 nM omega-Aga IVA reduced 70 % of the K+-evoked miniature endplate potential frequency). N- and L-type VDCC blockers did not affect this type of release. 6. We conclude that at rat neuromuscular junctions the presynaptic calcium channel types involved in transmitter release undergo developmental changes during the early postnatal period.

A Ca2+-induced Ca2+ release mechanism involved in asynchronous exocytosis at frog motor nerve terminals.

The extent to which Ca2+-induced Ca2+ release (CICR) affects transmitter release is unknown. Continuous nerve stimulation (20-50 Hz) caused slow transient increases in miniature end-plate potential (MEPP) frequency (MEPP-hump) and intracellular free Ca2+ ([Ca2+]i) in presynaptic terminals (Ca2+-hump) in frog skeletal muscles over a period of minutes in a low Ca2+, high Mg2+ solution. Mn2+ quenched Indo-1 and Fura-2 fluorescence, thus indicating that stimulation was accompanied by opening of voltage-dependent Ca2+ channels. MEPP-hump depended on extracellular Ca2+ (0.05-0.2 mM) and stimulation frequency. Both the Ca2+- and MEPP-humps were blocked by 8-(N, N-diethylamino)octyl3,4,5-trimethoxybenzoate hydrochloride (TMB-8), ryanodine, and thapsigargin, but enhanced by CN-. Thus, Ca2+-hump is generated by the activation of CICR via ryanodine receptors by Ca2+ entry, producing MEPP-hump. A short interruption of tetanus (<1 min) during MEPP-hump quickly reduced MEPP frequency to a level attained under the effect of TMB-8 or thapsigargin, while resuming tetanus swiftly raised MEPP frequency to the previous or higher level. Thus, the steady/equilibrium condition balancing CICR and Ca2+ clearance occurs in nerve terminals with slow changes toward a greater activation of CICR (priming) during the rising phase of MEPP-hump and toward a smaller activation during the decay phase. A short pause applied after the end of MEPP- or Ca2+-hump affected little MEPP frequency or [Ca2+]i, but caused a quick increase (faster than MEPP- or Ca2+-hump) after the pause, whose magnitude increased with an increase in pause duration (<1 min), suggesting that Ca2+ entry-dependent inactivation, but not depriming process, explains the decay of the humps. The depriming process was seen by giving a much longer pause (>1 min). Thus, ryanodine receptors in frog motor nerve terminals are endowed with Ca2+ entry-dependent slow priming and fast inactivation mechanisms, as well as Ca2+ entry-dependent activation, and involved in asynchronous exocytosis. Physiological significance of CICR in presynaptic terminals was discussed.

Enhancement of spontaneous transmitter release at neonatal mouse neuromuscular junctions by the glial cell line-derived neurotrophic factor (GDNF).

1. The acute effects of neurotrophic factors on the frequency of spontaneous transmitter release (miniature endplate potentials (MEPPs)) from motor nerve terminals has been examined in skeletal muscles of neonatal mice aged between 9 and 20 days. The following factors were tested at a concentration of 50 ng ml-1: brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), ciliary neuronotrophic factor (CNTF), leukaemia inhibitory factor (LIF), insulin-like growth factors 1 and 2 (IGF-1 and IGF-2), and glial cell line-derived neurotrophic factor (GDNF). In some experiments, the responses to 2 microM LaCl3 and 10 mM K+, or to 2-5 nM purified alpha-latrotoxin (alpha-LTX) were also measured. 2. Neither BDNF, NT-3, NT-4, LIF, IGF-1 or IGF-2 - singly or in combination - caused any significant change in MEPP frequency. GDNF, however, produced a highly significant, 2-fold increase in neurotransmitter release that was reproduced in fourteen muscles. 3. Potentiation of MEPP frequency in GDNF was of the same order as that induced by tetanic stimulation or substitution of the bathing medium with hypertonic saline; but substantially less than that induced either by lanthanum ions or alpha-latrotoxin. 4. The data suggest that concentrations of GDNF that produce maximal enhancement of motoneurone survival in vitro and in vivo also produce acute, non-saturating enhancement in transmitter release at immature mammalian neuromuscular synapses. Taken together with other reports, these findings suggest that GDNF may mediate both functional and structural plasticity of neonatal neuromuscular junctions.

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.

Calcitonin gene-related peptide acts presynaptically to increase quantal size and output at frog neuromuscular junctions.

1. Calcitonin gene-related peptide (CGRP) is found in dense-cored vesicles in the motor nerve terminal. 2. Exogenous CGRP increased the size of the quanta. The increase in size reached a maximum after about 40 min. The lowest effective concentration of human CGRP (hCGRP) was 0.8 nM. The action of hCGRP was antagonized by (-)-vesamicol, a drug that blocks active acetylcholine (ACh) uptake into synaptic vesicles, so it appears that hCGRP increases size by adding more ACh to the quanta. The action of hCGRP was antagonized by drugs that block the activation of protein kinase A (PKA). (In other preparations CGRP also activates PKA.) 3. The hCGRP effect was not blocked by fragment 8-37, an antagonist of one class of CGRP receptor. 4. hCGRP increases evoked quantal output and miniature endplate potential (MEPP) frequency, again by activating PKA. 5. CGRP release was measured by radioimmunoassay. Release was increased by depolarization with elevated K+, but the amounts released appear to be below those needed to affect quantal size or output. Moreover, although elevated K+ can increase quantal size it acts by a pathway that does not involve PKA. We suggest that the most likely target of endogenously released CGRP is the regulation of circulation of the muscle.

Short term plasticity caused by Ca 2+ induced Ca 2+ release in the frog motor nerve terminals

The actions of ephedrine were studied using intracellular recording techniques in guinea pig intercostal muscle fiber. After application of ephedrine (10−5 −10−4 M), miniature end-plate potential (MEPP) amplitude was decreased in a concentration-dependent manner. A greater depression in amplitude of end-plate potentials (EPP) was observed in preparations immobilized with d-tuboeurarine. In addition, ephedrine depressed the transient depolarization produced by iontophoretically applied acetylcholine (ACh).In normal Krebs solution and high Mg2+ solution, 10−4 M ephedrine increased MEPP frequency but did not alter the quantal content of EPPs in high Mg2+ blocked muscle. In these preparations, EPP amplitude did not change significantly. These results suggested that in the absence of a postsynaptic receptor blocking agent, a slight presynaptic action of ephedrine may offset the decreased responsiveness of the postsynaptic membrane. In addition, no significant alterations of resting membrane potential or the electrical characteristics of the membrane were recorded.Pretreatment of a mouse phrenic nerve-diaphragm preparation with ephedrine did not protect against the irreversible blockade by α-bungarotoxin. These results suggested that ephedrine may depress ACh potentials, MEPPs, and EPPs in dTc-blocked muscle by acting postsynaptically at a site on or associated with the ACh receptor other than the ACh recognition site.

Spontaneous acetylcholine release in mammalian neuromuscular junctions.

Spontaneous secretion of the neurotransmitter acetylcholine in mammalian neuromuscular synapsis depends on the Ca2+ content of nerve terminals. The Ca2+ electrochemical gradient favors the entry of this cation. We investigated the possible involvement of three voltage-dependent Ca2+ channels (VDCC) (L-, N-, and P/Q-types) on spontaneous transmitter, release at the rat neuromuscular junction. Miniature end-plate potential (MEPP) frequency was clearly reduced by 5 microM nifedipine, a blocker of the L-type VDCC, and to a lesser extent by the N-type VDCC blocker, omega-conotoxin GVIA (omega-CgTx, 5 microM). On the other hand, nifedipine and omega-CgTx had no effect on K(+)-induced transmitter secretion. omega-Agatoxin IVA (100 nM), a P/Q-type VDCC blocker, prevents acetylcholine release induced by K+ depolarization but failed to affect MEPP frequency in basal conditions. These results suggest that in the mammalian neuromuscular junction Ca2+ enters nerve terminals through at least three different channels, two of them (L- and N-types) mainly related to spontaneous acetylcholine release and the other (P/Q-type) mostly involved in depolarization-induced neurotransmitter release. Ca(2+)-binding molecule-related spontaneous release apparently binds Ca2+ very rapidly and would probably be located very close to Ca2+ channels, since the fast Ca2+ chelator (BAPTA-AM) significantly reduced MEPP frequency, whereas EGTA-AM, exhibiting slower kinetics, had a lower effect. The increase in MEPP frequency induced by exposing the preparation to hypertonic solutions was affected by neither external Ca2+ concentration nor L-, N-, and P/Q-type VDCC blockers, indicating that extracellular Ca2+ is not necessary to produce hyperosmotic neurosecretion. On the other hand, MEPP frequency was diminished by BAPTA-AM and EGTA-AM to the same extent, supporting the view that hypertonic response is promoted by "bulk" intracellular Ca2+ concentration increases.

Cholinergic agonists decrease quantal output at the frog neuromuscular junction by targeting a calcium channel blocked by omega-conotoxin.

Nicotinic cholinergic agonists are known to decrease synchronous evoked quantal output at the frog neuromuscular junction [Van der Kloot 1993, J Physiol (Lond) 468:567-589]. Here we also show that carbachol decreases the frequency of miniature endplate potentials (FMEPP) in solutions containing elevated levels of K+ and Ca2+. Carbachol did not decrease FMEPP in hypertonic solutions or in solutions containing the Ca2+ ionophore ionomycin and Ca2+. We conclude that the nicotinic agonists decrease Ca2+ influx through voltage-gated Ca2+ channels. Carbachol did not alter two-pulse facilitation. A blocker of N-type Ca2+ channels, omega-conotoxin GVIA, antagonized the nicotinic agonist-induced decrease in evoked quantal output. The effect of carbachol was not altered by omega-conotoxin MVIIC, a blocker of P-type and certain other Ca2+channels. The Ca2+ channel targeted by the nicotinic agonists appears to be of the N-type.

Endurance exercise modulates neuromuscular junction of C57BL/6NNia aging mice.

The effect of age and endurance exercise on the physiology and morphology of neuromuscular junctions (NMJ) of gluteus maximus muscle was studied in C57BL/6NNia mice. Mice were exercised, starting at 7 or 25 mo of age, at 28 m/min for 60 min/day, 5 days/wk for 12 wk, on a rodent treadmill. Intracellular recordings of spontaneous miniature endplate potentials (MEPP) and the quantal content of endplate potentials (EPP) were recorded from NMJ of 10- and 28-mo-old control and exercised mice. Endurance exercise resulted in significant increases in MEPP amplitudes (23%), quantal content, and safety margin, and a significant decrease in MEPP frequency of young mice, with no change in resting membrane potential or membrane capacitance. Three months of endurance exercise resulted in an increase in MEPP frequency (41%) and decreases in MEPP amplitudes (15%), quantal content, and safety margin of old mice. Endurance exercise resulted in significantly larger nerve terminals (24%) in young animals, suggesting functional adaptation. Nerve terminals in exercised 28-mo-old mice were smaller than in the corresponding control mice, an indication that exercise minimized age-related nerve terminal elaboration. It is concluded that the different physiological responses of young and old gluteus maximus muscles to endurance exercise parallel their morphological responses. This suggests that the mouse NMJ undergoes a process of physiological and morphological remodeling during aging, and such plasticity could be modulated differently by endurance exercise.

Overexpression of adhesion molecule L1 in NG108-15 neuroblastoma X glioma hybrid cells enhances dibutyryl cyclic AMP-induced neurite outgrowth and functional synapse formation with myotubes.

The role of adhesion molecule L1 in synapse formation was examined by transient transfection of L1 cDNA in neuroblastoma x glioma hybrid NG108-15 cells. L1 overexpression was found in approximately 50% of the transfected NG108-15 cell population. Neurite outgrowth induced by 0.25 mM dibutyryl cyclic AMP (cAMP) was much greater in L1-transfected NG108-15 cells than that in nontransfected and mock-transfected cells. The proportion of cells with neurites and the number of neurites per cells were increased in L1-transfected cells after 2 days of dibutyryl cAMP treatment. The proportion of cells with branched neurites and the average length of neurites were higher at day 4. A significantly higher rate of synapse formation with myotubes was apparent in the late phase of coculture (days 4-7) in L1-transfected cells than in control cells. The miniature end-plate potential frequency in myotubes was the same for the three types of NG108-15 cells. These results show that overexpression of L1 in NG108-15 cells facilitates synaptic connections by enhancing branching and elongation of neurites induced with dibutyryl cAMP, rather than by increasing probability of acetylcholine release.

Novel effects of guanidine on the neuromuscular junction

1. 1. The effects of guanidine on the isolated mouse phrenic nerve diaphragm (MPND) and chick biventer cervicis (CBC) neuromuscular preparations were determined by myographic and electrophysiological methods. 2. 2. Guanidine at concentrations of 5–10 mM induced an initial facilitation followed by neuromuscular blockade in both preparations. In the isolated MPND such blockade was associated with the abolition of miniature end-plate potentials (MEPPs), but in the CBC the acetylcholine-induced contracture remained unimpaired. After guanidine removal, a heretofore undescribed pronounced facilitation of neuromuscular transmission associated with an increase in MEPP frequency was observed. Simultaneously, the muscular contractions exhibited delayed relaxation and aftercontractions. 3. 3. The K + channel opener, cromakalim (100–200 μM) inhibited both the well-described initial and the novel postremoval facilitatory effects of guanidine in a concentration-dependent manner. These findings are consistent with the proposal that guanidine blocks K + channels in motor nerve endings. 4. 4. The guanidine-induced NMB was reverted by increasing the Ca 2+ concentration (1.8–5 mM) in the nutritive solution. 5. 5. Tetrodotoxin (TTX, 1.56 μM) did not influence the increase in MEPPS frequency induced by guanidine (10 mM) but did reduce the rise in MEPPS frequency observed after guanidine removal. 6. 6. The present findings indicate that the effects of guanidine on the neuromuscular junction are more complex than currently described because they include a neuromuscular blockade and a postremoval facilitation previously unreported in the literature.

Dynamic responses of presynaptic terminal membrane pools following KCl and sucrose stimulation

The cholinergic presynaptic terminals of Torpedo electric organ have been examined morphometrically following stimulation by KCl and sucrose. The objective was to confirm correlations predicted by the vesicle hypothesis between miniature end-plate potentials (MEPPs) and morphometric changes in terminal ultrastructure. Both secretegogues generated high frequencies of MEPPs and also distinctive though differing ultrastructural changes. The synaptic vesicles show classes of 68 and 90 nm diameters and both store acetylcholine (ACh). KCl stimulation depleted the 90 nm class first whereas sucrose reversed the order of depletion. Very few instances of actual vesicle fusion were seen. Dose-response correlations between vesicle density and secretegogue strength (mM) and duration were higher with sucrose. Both secretegogues produced declines in vesicle numbers and densities and yielded multimodal distributions of large vesicles with an average 160 nm mean diameter. No meaningful correlations were detected between numbers of MEPPs and vesicles and little evidence was found to indicate that vesicles were fusing to terminal plasma membrane in numbers approximating MEPP release. Linear regression analysis was used to quantitatively examine relationships between the vesicle membrane pool and other pools of the putative exo/endocytotic pathway. Correlation coefficients between vesicle and terminal plasma membrane pools were non-significant and of positive sign, indicating independent, similar responses. Non-significant, negative coefficients were obtained when vacuole and 160 nm vesicle membrane values were included. These tests further argue against claims that vesicles are actively fusing with the plasma membrane. These conflicting findings for both secretegogues preclude meaningful correlations between vesicle changes and numbers of MEPPs generated and again emphasize the difficulty of validating the vesicle hypothesis by ultrastructural means. On the other hand, the study shows that vesicular, vacuolar and terminal membrane pools are dynamically changing during transmitter release, presumably interacting with cytosolic membrane constituents. A dynamical release process therefore has been proposed to account for the two classes of MEPPs, the rapid changes in class ratio and the mutable characteristics of the bell-MEPP that presently challenge the quantal-vesicular claims of prepackaged, immutable, exocytotically released packets of transmitter. This model features a state for each MEPP class with class and size determined at moment of release. For example, a single flicker of a channel would generate the sub-MEPP (defined subunit of an MEPP) and 7–20 flickering channels would generate the bell-MEPP.

Sodium-dependent increase in quantal secretion induced by brevetoxin-3 in Ca2+-free medium is associated with depletion of synaptic vesicles and swelling of motor nerve terminals in situ

Brevetoxin-3 at nanomolar concentrations markedly enhanced spontaneous quantal transmitter release from neuromuscular junctions equilibrated in a Ca2+-free EGTA medium. After about 3h, the sustained increase in miniature endplate potential frequency led to an exhaustion of transmitter release. This increase still occurred after loading the nerve terminals with the Ca2+ chelator bis-(aminophenoxy)ethanetetra-acetate or after pretreatment with various pharmacological agents known to prevent Ca2+ release from intracellular pools, but was completely prevented by the Na+ channel blocker tetrodotoxin. Brevetoxin-3 also increased miniature endplate potential frequency from junctions treated with botulinum type-A toxin, but to a smaller extent than at normal junctions. At normal junctions, brevetoxin-3 exposure for 2h increased the three-dimensional projected area of living motor nerve terminals in situ by about 74% while at botulinum type-A poisoned junctions a similar toxin exposure caused only a 29% increase. Tetrodotoxin prevented such effects, indicating that they are related to both Na+ entry into the terminals and increased quantal transmitter release. Ultrastructural examination of nerve terminals from junctions exposed for 3h to brevetoxin-3 revealed profound depletions of clear and large dense core synaptic vesicles and an increase in coated vesicles and axolemma infoldings. These results indicate that brevetoxin-3 impairs the recycling of clear synaptic vesicles and are consistent with our immunofluorescent observations showing that synaptophysin epitopes can be revealed without nerve terminal permeabilization. In contrast, no such changes were detected in nerve terminals poisoned with botulinum type-A toxin which, after 3h exposure to brevetoxin-3, retained their synaptic vesicles and had a normal appearance.We conclude that tetrodotoxin-sensitive Na+ entry into motor nerve terminals induced by brevetoxin-3 triggers external Ca2+-independent asynchronous quantal transmitter release, blocks synaptic vesicle recycling and induces swelling of the terminals. We suggest that an excess of cytoplasmic Na+per se can activate the asynchronous neurotransmitter release process.

Pharmacologic inhibition of twin-pulse facilitation of release of transmitter quanta at the mouse neuromuscular junction

1. 1. The frequency (F, s −1) of miniature endplate potentials and the quantal content (m) of endplate potentials were simultaneously measured intracellularly at mouse diaphragm endplates in a bath solution that contained 0.6 mM Ca 2+ ions and 5 mM Mg 2+ ions. 2. 2. Twin pulses at 4-ms intervals gave the quantal contents of the first ( m1) and second ( m2) responses. The ratio of m2/ml was taken as an indicator of the temporal facilitation of the release of transmitter. 3. 3. Lead ions (Pb 2+; 10 μM), bis( o-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid (BAPTA; loaded for 60 min at 200 μM), and chlortetracycline (CTC; loaded for 30 min at 80 μM) reduced the values of F and m2/ml. Pb 2+ ions and CTC reduced the value of m, whereas BAPTA did not. ω-Agatoxin (ωAGT; 10 ng/ml) reduced the value of m without affecting F or m2/ m1. 4. 4. These results suggest that synaptic facilitation is modifiable by agents that can affect systems which buffer intracellular levels of Ca 2+ ions.

Calcium channels involved in synaptic transmission at the mature and regenerating mouse neuromuscular junction.

1. The involvement of the different types of voltage-dependent calcium channels (VDCCs) in synaptic transmission at the mature and newly formed mammalian neuromuscular junction was studied by evaluating the effects of L-, P/Q- and N-type VDCC antagonists on transmitter release in normal and reinnervating levator auris preparations of adult mice. 2. Nerve-evoked transmitter release was blocked by omega-agatoxin IVA (omega-AgaIVA), a P/Q-type VDCC blocker, both in normal and reinnervating endplates (100 nM omega-AgaIVA caused > 90% inhibition). The N-type VDCC antagonist omega-conotoxin GVIA (omega-CgTX; 1 and 5 microM), as occurs in normal preparations, did not significantly affect this type of release during reinnervation. Nitrendipine (1-10 microM), an L-type VDCC blocker, strongly antagonized release in reinnervating muscles (approximately 40-69% blockade) and lacked any effect in normal preparations. 3. In reinnervating muscles, spontaneous release was not dependent on Ca2+ entry through either P- or L-type VDCCs. Neither 100 nM omega-AgaIVA nor 10 microM nitrendipine affected the miniature endplate potential (MEPP) frequency or amplitude. 4. At the newly formed endplates, K(+)-evoked release was dependent on Ca2+ entry through VDCCs of the P-type family (100 nM omega-AgaIVA reduced approximately 70% of the K(+)-evoked MEPP frequency). L-type VDCCs were found not to participate in this type of release (10 microM nitrendipine lacked any effect). 5. In reinnervating muscles, the L-type VDCC blocker, nitrendipine (10 microM), provoked a significant increase (approximately 25%) in the latency of the evoked endplate potential (EPP). This drug also caused an increase (approximately 0.3 ms) in the latency of the presynaptic currents. The P/Q- and Ny-type VDCC blockers did not affect the latency of the EPP. 6. These results show that at newly formed mouse neuromuscular junctions, as occurs in mature preparations, VDCCs of the P-type family play a prominent role in evoked transmitter release whereas N-type channels are not involved in this process. In addition, signal conduction and transmitter release become highly sensitive to nitrendipine during reinnervation. This suggests that L-type VDCCs may play a role in synaptic transmission at the immature mammalian neuromuscular junction.

Potentiation of spontaneous acetylcholine release from motor nerve terminals by glutamate in Xenopus tadpoles

Extracellular application of glutamate (100 μM) increased the spontaneous secretion of acetylcholine, as well as the amplitude and decay time of miniature endplate potentials at developing neuromuscular synapses in Xenopus tadpoles. Kainate, quisqualate and N-methyl- d-aspartate (100 μM each) increased miniature endplate potential frequency by 26-, 13- and four-fold, respectively. The rank order of efficacy at 100 μM was kainate > quisqualate > N-methyl- d-aspartate > glutamate. The effect of kainate on miniature endplate potential frequency was inhibited by 6-cyano-2,3-dihydroxy-7-nitroquinoxaline (20 μM), but not by (±)-2-amino-5-phosphonovalerate (20 μM). Treatment with the voltage-dependent Ca 2+ channel blockers verapamil (10 μM), Cd 2+ (100 μM) or ω-conotoxin (1 μM) inhibited the potentiating action of kainate on miniature endplate potential frequency. On the other hand, 1 S,3 R-1-aminocyclopentane-1,3-dicarboxylate (300 μM), a glutamate metabotropic receptor agonist, inhibited the spontaneous acetylcholine release, which was antagonized by the application of 2-amino-3-phosphonopropionate (500 μM). The potentiating effect of glutamate receptor agonists on the miniature endplate potential frequency declined or disappeared in older Xenopus tadpoles. Quisqualate (100 μM) and N-methyl- d-aspartate (100 μM) but not kainate (30 μM) increased the amplitude and decay time of miniature endplate potential, whereas 1 S,3 R-1-aminocyclopentane-1,3-dicarboxylate (300 μM) only increased the decay time of miniature endplate potentials. These results suggest that there are kainate/quisqualate and N-methyl- d-aspartate receptors existing in the motor nerve terminals of younger Xenopus tadpoles and the activation of these receptors potentiates spontaneous acetylcholine release through increasing Ca 2+ influx. Our data suggest that the presynaptic glutamate receptors on cholinergic terminals may be involved in feedback regulation of acetylcholine secretion at earlier embryonic stages.

Presynaptic effects of a trinitrobenzene analogue at the frog neuromuscular junction.

1. Application of 0.15 mM 1-(hydroxyethylamino)-2,4,6-trinitrobenzene (HEATNB) to the frog neuromuscular junction induced a marked increase (4.0-fold) in the amplitude of nerve-evoked end-plate potentials (EPPs) obtained from intracellular and extracellular records, but only a slight increase (1.9-fold) in the frequency of miniature EPPs (MEPPs) obtained from intracellular records. The effects of HEATNB on EPP amplitude and MEPP frequency showed a similar time course, reaching a plateau level approximately 40 min after the start of application and returning to the control level after wash. The difference in the effects of HEATNB on EPP and MEPP frequency suggests that it specifically enhances synchronous transmitter release. 2. Comparing the effects and structure of HEATNB with those of 2,4,6-trinitrobenzene-1-sulfonic acid, we conclude that the observed increase in transmitter release is due to the effects of the trinitrobenzene moiety of those reagents. 3. The distribution of MEPP amplitude was unchanged by HEATNB treatment, indicating that its effects are presynaptic. 4. Among four components of short-term synaptic plasticity, HEATNB greatly decreased (approximately 70%) augmentation and increased (approximately 50%) potentiation, but had little effect on fast and slow facilitations. These results suggest that each of the short-term plasticities has a different mechanism and that HEATNB affects the same mechanisms as those of augmentation. 5. Even when a calcium chelator, bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid, was loaded into the presynaptic nerve terminal, the effects of HEATNB were not changed in nature, suggesting that effects of HEATNB persist independently of intracellular Ca2+ concentration. 6. These observations suggest that HEATNB may affect specific protein(s) involved primarily in synchronous transmitter release and not asynchronous release.