Transmitter release from insect excitatory motor nerve terminals

1. Intracellular and extracellular electrodes were used to study spontaneous and impulse-linked release of transmitter at locust retractor unguis nerve-muscle synapses.2. At most extracellular recording sites the amplitude distributions of the excitatory post-synaptic potentials (e.p.s.p.s) were apparently non-Poisson. However, interpretation of these amplitude distributions was complicated by the effect on the extracellular recordings of the complex structural organization of the retractor unguis nerve terminal with its spatially distinct transmitter release sites extending over distances of 15-30 mum.3. The spontaneous miniature excitatory post-synaptic potentials (min e.p.s.p.s) did not occur at random intervals, bursts of min e.p.s.p.s being frequently recorded. As a result the spontaneous release of transmitter rarely approximated a Poisson process.4. For a period of at least 390 msec following a conditioning nerve impulse a test e.p.s.p. was facilitated and the probability of spontaneous transmitter release was enhanced. A large primary phase of facilitation of impulse-linked and spontaneous release was invariably followed by one or more secondary phases of smaller magnitude.     

The interaction of presynaptic polarization with calcium and magnesium in modifying spontaneous transmitter release from mammalian motor nerve terminals

1. The relationship between motor terminal polarization and miniature end-plate potential (m.e.p.p.) frequency was examined in the presence of various Ca, Mg and K concentrations ([Ca], [Mg] and [K]) and also at modified bathing osmolarity levels. The polarization changes were obtained with ;electrotonic' and ;focal' polarizing currents and with rapid changes in bathing [K].2. M.e.p.p. frequency increased exponentially with electrotonic depolarizing currents, but failed to decrease similarly with hyperpolarizing currents. An increase in bathing [K] to 15 mM increased the sensitivity of the terminals to presynaptic hyperpolarization.3. The slope, on semilogarithmic coordinates, of the function relating m.e.p.p. frequency to electrotonic polarizing currents (the release-current function) was unchanged when bathing [Ca] was raised from 2 to 8 mM. When [Ca] was reduced to 0.5 mM the slope of this function was reduced initially but eventually approached the same slope as in control [Ca]. A similar effect was also found in the presence of 15 mM-KCl.4. The relationship between m.e.p.p. frequency and log [K], at various [Ca], resembled the relationships between m.e.p.p. frequency and presynaptic polarizing currents.5. An increase in bathing [Mg] or osmolarity had a similar effect to a reduction of [Ca].6. Tetrodotoxin (TTX) at a concentration of 10(-6) g/ml. was found to reduce m.e.p.p. frequency, at various [K], by a constant fraction of about 30%.7. In some of the junctions ;anodic break-down' was observed. An examination of this phenomenon with focal polarizing currents disclosed an unusual type of ;anodic break-down', with rapid ;on' and ;off' responses. This phenomenon may indicate that release depends on the influx of positively charged particles into the nerve terminals.8. It is concluded that nerve terminal depolarization accelerates exponentially the activity of a membrane component bearing three Ca molecules, the rate of acceleration being independent of bathing [Ca].     

Species specific morphology of mammalian motor nerve terminals

Summary Motor nerve terminals in the diaphragm, extensor digitorum longus and soleus muscles of young and adult rat, hamster and guinea pig were studied with the light microscope after staining with methylene blue. In adult animals the nerve terminals are smaller in the diaphragm than in the two other muscles. In the rat and hamster the extensor digitorum longus and soleus terminals are of similar area, but the terminals in extensor digitorum longus are shorter. In the guinea pig the terminals are smaller and shorter in soleus than in extensor digitorum longus. The density of nerve terminal varicosities is lowest in the diaphragm in all three species. In the rat and hamster the density is higher in extensor digitorum longus than in soleus. In the guinea pig the converse is found. In all three muscles the density of varicosities is higher in the rat than in the hamster and guinea pig. The nerve terminal branches in the diaphragm are mostly organized in one group. In the rat and hamster the soleus terminal branches are more separated in groups than the extensor digitorum longus terminal branches. In the guinea pig the number of groups is almost the same in the two muscles. These muscle and species-specific differences appear already in very young animals.     

Membrane events related to transmitter release in mouse motor nerve terminals captured by ultrarapid cryofixation

Summary The sequence of structural changes occurring in the presynaptic membrane during transmitter release was studied at the mouse neuromuscular junction using the combined quick-freezing and cryosubstitution techniques. The mouse levator auris longus (LAL) muscle was stimulated by two means: either, chemically, by soaking 5 min before freezing in a physiological solution containing 25mm potassium chloride or, electrically, by applying, 10 ms before freezing, a single supramaximal stimulus to the nerve-muscle preparation treated with 50 M 3,4-diaminopyridine (3,4-DAP) and 100 M (+)tubocurarine. In both cases, the preparations were maintained at approximately 5 °C, 5 min prior to freezing, in order to prolong nerve membrane changes. In most experiments, tannic acid (0.1%) was added to the substitution medium for better preservation of membranes. The different steps of warming in the substitution medium were strictly controlled from –90 °C to 4 °C. When fixed under chemical stimulation, the presynaptic membrane appeared very sinuous and synaptic vesicles were seen apposed to specialized sites facing subjunctional folds. When submitted to a single electrical stimulus, after treatment with 3,4-diaminopyridine, features of synaptic vesicle fusion were observed at these specialized sites which appear similar by their morphology, their macromolecular organization (already described) and their functional changes to active zones of the frog neuromuscular junction. Other images suggested that with 3,4-diaminopyridine which causes a pronounced and long-lasting release of transmitter, some vesicles collapse after exocytosis instead of being locally reformed by endocytosis.     

Transmitter release triggered by a local depolarization in motor nerve terminals of the frog: role of calcium entry and of depolarization

Quantal synaptic currents (EPSCs) were elicited at neuromuscular junctions of frogs, applying current pulses through the recording current-clamp electrode. Facilitation of a test-EPSC by a preceding EPSC of variable amplitude was determined. This facilitation after a prepulse and release during the prepulse were found to have different dependences on depolarizing current amplitude. In addition, if intraterminal calcium [( Ca]i) was raised by a train of EPSCs, small depolarizations with little additional Ca entry elicited release which depended strongly on pulse amplitude. It is concluded that, in addition to [Ca]i, depolarization of the terminal directly controls quantal release. This depolarization dependence of release is shown to be the probable cause for termination of release after a large Ca entry.     

On the mechanism by which calcium and magnesium affect the spontaneous release of transmitter from mammalian motor nerve terminals

1. The frequency of miniature end-plate potentials (m.e.p.p.s) was recorded from neuromuscular junctions in rat diaphragm phrenic nerve preparations in vitro after preparations had soaked in solutions containing Ca in concentrations between 10(-10) and 10(-2)M and a similar range of [Mg].2. Ethylenediamine tetra-acetate (EDTA) and ethyleneglycol bis (beta-aminoethyl ether) tetra-acetate (EGTA) buffers were added to prepare solutions with [Ca] and [Mg] below 10(-4)M. A computer program was used to estimate the free [Ca(2+)] in these solutions, and it was shown that the effects of Ca could be attributed to the free [Ca(2+)] in the bathing solution.3. M.e.p.p.s could still be detected without difficulty after soaking preparations for 6-8 hr in solutions containing EDTA or EGTA buffers and no added Ca. The basal frequency was unchanged upon exhibition of Ca in concentrations up to 10(-5)M and/or Mg in concentrations up to 10(-3)M.4. Ca in concentrations of and above 10(-4)M accelerated m.e.p.p. frequency from the basal level. This effect reached a maximum in [Ca] of 10 mM and raising the [Ca] above this level did not further change frequency. These effects were explained by the combination of Ca molecules with a nerve terminal receptor site. It was postulated that this combination allosterically activated the spontaneous release mechanism.5. Mg could accelerate m.e.p.p. frequency in the absence of added Ca. The interactions of Ca and Mg upon m.e.p.p. frequency indicated that Ca and Mg competed for the same sites.6. Raising the [H(+)] of the bathing medium accelerated m.e.p.p. frequency. This effect was thought to be exerted partly by combination with the same receptor sites as Ca and Mg and partly by variation of the ionization of the CaCl(2) of the bathing solution.     

Transmitter release from nerve terminals evoked by depolarization pulses contains a short phase of repression

The time course of quantal transmitter release after a depolarization pulse was measured at frog and crayfish motor nerve terminals. Test pulses were arranged to elicit low release, and the delay of first releases and the median of distributions of release times were defined for large (>2000 stimuli) samples. Small, subthreshold depolarizing postpulses were added directly after the test pulses. Such postpulses of 1 to 4 ms duration prolonged the delay of first releases and shifted the median of the time course of release by up to 3 ms (at 0°C) depending on the duration and on the amplitude of the post-pulses. These latency shifts, which have been observed after prolonged depolarizations by other authors, were statistically highly significant. The results of post-pulses were very similar at neuromuscular junctions of frog and crayfish. It is concluded that depolarization in addition to the promotion of release has a short repressing action on release which is partly responsible for synaptic delay.Supported by the Deutsche Forschungsgemeinschaft Preliminary reports of part of the present results have appeared Dudel 1985 a, b).     

An examination of the effects of osmotic pressure changes upon transmitter release from mammalian motor nerve terminals

1. When the frequency of miniature end-plate potentials (m.e.p.p.s) was measured at neuromuscular junctions in rat diaphragm nerve preparations in vitro bathed in solutions having osmolarities between 200 and 700 m-osmoles/l. it was found that m.e.p.p. frequency was transiently increased by exposure to osmotic gradients exceeding 75 m-osmoles/l., and then declined, within 1 hr, to a steady level slightly higher than the control level of frequency. Smaller osmotic gradients caused a maintained increase in m.e.p.p. frequency. E.p.p. quantal content was initially increased and later profoundly decreased upon exposure of preparations to solutions with an osmotic pressure of 500 or 600 m-osmoles/l. but was unaffected by less hypertonic solutions.2. Variation of the Ca or Mg content of the bathing solutions did not alter these effects of osmotic pressure on the early transient increase in m.e.p.p. frequency or e.p.p. quantal content but affected the late steady increase in m.e.p.p. frequency.3. The value of the transient increase in m.e.p.p. frequency was exponentially related to the osmotic gradient in the range 0-300 m-osmoles/l. with a Q(10) of 1.95 (range 11-34 degrees C). Greater osmotic gradients did not further increase m.e.p.p. frequency. Variation of the ionic strength of the bathing medium did not influence osmotic effects upon frequency.4. The discrepancy between the effects of osmotic gradients upon spontaneous and nerve-impulse induced transmitter release was explained by an occlusion of the osmotic effects by depolarization of nerve terminals. Time-course studies showed that in the presence of 20 mM-KCl the m.e.p.p. frequency increase in response to an increase in osmotic pressure was small and was followed by a reduction in frequency to below control levels while osmotic pressure changes had no immediate effect upon m.e.p.p. frequency in solutions containing 30 mM-KCl.5. It was concluded that increased osmotic gradients could release transmitter by a mechanism independent of Ca and of nerve terminal depolarization.6. It is suggested that the initial transient effects of changes of osmotic gradient upon transmitter release are related to flow of water through the nerve terminal membrane, while the later effects are related to nerve terminal volume changes.     

Control of quantal transmitter release at frog's motor nerve terminals

Motor terminals on the cutaneous pectoris muscle of the frog were depolarized by current pulses through the recording macro-pathch-clamp electrode and the resulting quantal release was measured (excitation blocked with TTX). Above a threshold release increased very steeply with depolarization until saturation was approached. The dependence of release on duration of depolarization was even steeper: doubling pulse duration often produced more than 100-fold release (early facilitation) Distributions of delays of quantal release after the depolarization pulse were determined for wide ranges of depolarizations and pulse durations. The shape of these distributions was little affected by large changes in average release; increasing the temperature from 0°C to 10°C about halved the time scale of the distributions. Lengthening the depolarization from 0.5 to 6 ms produced a latency shift: the distributions of delays were shifted by almost the increase in pulse duration. At 5–6 ms pulse duration a few releases occurred during the final millisecond of the pulse. It is suggested that the time course of the phasic release is not controlled by the time course of changes in intracellular calcium concentration, but by an activator which is produced about proportional to supra-threshold pulse amplitude and duration, and that this activator effects release with a cooperativity of 6–7. An additional depolarization produced repressor is responsible for the minimum delay.Supported by the Deutsche Forschungsgemeinschaft     

Control of quantal transmitter release at frog's motor nerve terminals

Quanta of transmitter were released from motor nerve terminals of the frog by a depolarizing 'releasing pulse'. 'Modulating pulses' were subthreshold for release; pre-pulses were added directly before and post-pulses directly after the releasing pulse. Modulating depolarization pulses enhanced release up to 20-fold, and such hyperpolarizations suppressed release up to 10-fold. Pre- and post-pulses were about equally effective. In a wide range these modulations did not affect the facilitation of a test-EPSC by the preceding releasing pulse; modulation thus is not mediated by changes in Ca-inflow. It is suggested that phasic release is largely controlled by an 'activator' which is generated by depolarization, and that modulating pulses increase this activator when depolarizing, and decrease this activator below its resting level if hyperpolarizing. If an interval was interposed between pre- and releasing pulse, the modulating effect decreased very steeply with increasing interval for the first 2 ms, and much slower for longer intervals. Distributions of delays of quantal releases showed a time course of decay very similar to the decay of modulation with increasing interval. Both decays may reflect the exponential decay of activator. Depolarizing post-pulses increased the minimal synaptic delay and the delay of maximal release, and hyperpolarizing ones had the opposite effects. They are interpreted to modulate the generation and decay of a 'repressor', which is produced by depolarization and is responsible for the minimal synaptic delay and the delayed maxima of release. A speculative scheme of interactions of [Ca]i, activator and repressor is discussed.     

The origin of the post-tetanic hyperpolarization of mammalian motor nerve terminals

1. Motor nerve terminals in magnesium-poisoned rat hemidiaphragm-phrenic nerve preparations in vitro were stimulated with short depolarizing pulses of approximately threshold strength and the evoked antidromic responses recorded from the phrenic nerve. The percentage of these 1/sec or 0.5/sec stimuli to which there was no antidromic response was used as a quantitative measure of the terminal excitability. After standard tetanic stimulation (1000 impulses at 100/sec) the excitability of the terminals was depressed for an average duration of 60-70 sec, during most of which time no antidromic responses to stimuli of pretetanic intensity were recorded. There was no significant interaction between stimuli to the terminals at rates of 1 or 0.5/sec.2. Potassium-free solutions at first increased, then decreased, the post-tetanic depression of excitability. Raising [K](o) threefold (15 mM) abolished the post-tetanic depression and often converted it to an exaltation of excitability.3. Polarizing currents were applied to the terminals with a second electrode. Depolarizing currents increased, while hyperpolarizing currents decreased, the post-tetanic depression of excitability.4. In solutions with 70% of the normal NaCl content replaced by sucrose, the post-tetanic depression of excitability was reversibly prolonged.5. In the presence of 7.7 x 10(-6)M digoxin or 0.42 mM ouabain there was a small reversible reduction of post-tetanic excitability.6. After exposure to solutions containing no glucose or to solutions containing 3-5 mM sodium azide the excitability of the terminals was not altered by the tetanus. After washing with the control solution, post-tetanic depression of excitability returned. Antimycin-A (1.8 x 10(-6)M) had little or no effect upon post-tetanic excitability.7. It was concluded that the post-tetanic depression of excitability reflected hyperpolarization of the terminals and that this hyperpolarization was caused by a shift of the membrane potential towards the potassium equilibrium potential because of an increase in potassium permeability.     

Transmitter release in tetanus and botulinum A toxin-poisoned mammalian motor endplates and its dependence on nerve stimulation and temperature

The effects of tetanus toxin (TeTx) and botulinum A toxin (BoTx) on spontaneous and nerve-evoked transmitter release have been compared in mouse hemidiaphragms poisoned in vitro. At 37 degrees C endplates poisoned with either of these agents were characterized by (1) a decrease of miniature endplate potential (m.e.p.p.)-frequency to less than 30/min for TeTx and 3/min for BoTx, (2) reduced mean m.e.p.p.-amplitude and (3) 100% failure to show endplate potentials (e.p.p.s) in response to single nerve stimuli. In addition (4) tetanic nerve stimulation and/or reduction of temperature to about 20 degrees C caused a remarkable increase in the nerve-evoked transmitter release, but did not affect the low frequency of spontaneous m.e.p.p.s. However, several important differences exist between the effects of both toxins. (1) At room temperature even single nerve stimuli could elicit e.p.p.s in BoTx-muscles the failure rate being about 80%. For TeTx the failure was 100%. However, if the nerve was stimulated with higher frequencies (greater than 5 Hz), the probability of quantal release increased, the delay for release from the onset of stimulation being several seconds and similar to that observed at 37 degrees C. (2) TeTx distorted the synchronous release of quanta increasing the distribution of their synaptic delays. BoTx did not influence the time course of the phasic secretion process in response to nerve action potentials. (3) TeTx preferentially blocked the release of spontaneous m.e.p.p.s of large amplitude without affecting the frequency of the small amplitude ones, while BoTx inhibited both the small and large amplitude m.e.p.p.s. The distribution of the amplitudes of the nerve-evoked m.e.p.p.s were similar to those of spontaneous m.e.p.p.s before the blockade with the toxins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Coupling of L-type calcium channels to neurotransmitter release at mouse motor nerve terminals

Previously, we have presented evidence for the presence of L-type voltage-dependent Ca2+ channels (VDCC) in 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, (acetoxymethyl)ester (BAPTA-AM)-incubated motor nerve terminals (MNTs) of the levator auris muscle of mature mice. The aim of the present work was to study the coupling of these L-type VDCC to neurotransmitter release by inhibiting protein phosphatases. We thus studied the effects of the protein phosphatase inhibitors okadaic acid (OA) and pervanadate on quantal content (QC) of transmitter release with the P/Q-type channels fully blocked. The QC was not significantly different under the three experimental conditions tested: incubation with dimethylsulphoxide (DMSO), ethylene-glycol-bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid, (acetoxymethyl)ester (EGTA-AM) and BAPTA-AM. After preincubation with OA (1 microM), but not with pervanadate, QC increased substantially in the BAPTA-AM-incubated (up to 400%) MNT, but not in those incubated with DMSO or EGTA-AM. The OA-induced increment of QC was attenuated greatly (approximately 95% reduction) by preincubation with either nitrendipine (10 microM) or calciseptine (300 nM). The effect of OA (1 microM) and pervanadate (0.1 mM) on spontaneous neurotransmitter release was also studied. After preincubation with OA, but not per-vanadate, miniature end-plate potential (MEPP) frequency increased only in the BAPTA-AM-incubated MNT (up to 700% increment). This response was attenuated (by approximately 80%) by nitrendipine (10 microM) or calciseptine (300 nM). In contrast, neither omega-agatoxin IVA (120 nM) nor omega-conotoxin GVIA (1 microM) affected this OA-induced increment significantly. We also evaluated the relationship between QC and extracellular [Ca2+] ([Ca2+]o) in BAPTA-AM-incubated MNT. Under conditions in which only P/Q-type VDCC were available to participate in neurotransmitter release, QC increased as [Ca2+]o was raised from 0.5 to 2 mM. However, when only L-type VDCC were available, QC increased when [Ca2+]o increased from 0.5 to 1 mM, but decreased significantly at 2 mM. The mean latency for P/Q-type VDCC-mediated EPP was 1.7-1.9 ms; for L-type VDCC-mediated EPP, 1.9-2.5 ms. The rise time of the L-type VDCC mediated EPP was significantly slower than that mediated by P/Q-type VDCC. Preincubation with H-7 (100 microM), a potent inhibitor of protein kinase C (PKC) and adenosine 3',5'cyclic monophosphate (cAMP)-dependent protein kinase (PKA), attenuated the OA-induced increment of both QC and MEPP frequency (50% and 70% decrement, respectively), suggesting the participation of at least these two protein kinases in the coupling of L-type VDCC. In summary, our results show coupling of L-type VDCC to neurotransmitter release when protein phosphatases are inhibited and intracellular [Ca2+] is buffered by the fast chelator BAPTA.     

The effects of depolarization of motor nerve terminals upon the release of transmitter by nerve impulses

1. Depolarizing currents were applied to motor nerve terminals in the rat phrenic nerve-diaphragm muscle preparation in vitro.2. During the passage of depolarizing currents the amplitude of the presynaptic nerve action potentials and of end-plate potentials (e.p.p.s) was reduced in proportion to the current strength.3. The reduction in e.p.p. amplitudes was shown to be due to a reduction in the number of quanta released.4. An excess of Mg ions or the previous application of a hyperpolarizing current could prevent the reduction of e.p.p. amplitudes and quantal contents by depolarizing current.5. Depolarizing current application prevented later hyperpolarizing currents affecting e.p.p. amplitudes.