Long-term facilitation alters transmitter releasing properties at the crayfish neuromuscular junction

Synaptic transmission at the neuromuscular junction of the excitatory axon supplying the crayfish opener muscle was examined before and after induction of long-term facilitation (LTF) by a 10-min period of stimulation at 20 Hz. Induction of LTF led to a period of enhanced synaptic transmission, which often persisted for many hours. The enhancement was entirely presynaptic in origin, since quantal unit size and time course were not altered, and quantal content of transmission (m) was increased. LTF was not associated with any persistent changes in action potential or presynaptic membrane potential recorded in the terminal region of the excitatory axon. The small muscle fibers of the walking-leg opener muscle were almost isopotential, and all quantal events could be recorded with an intracellular microelectrode. In addition, at low frequencies of stimulation, m was small. Thus it was possible to apply a binomial model of transmitter release to events recorded from individual muscle fibers and to calculate values for n (number of responding units involved in transmission) and p (probability of transmission for the population of responding units) before and after LTF. In the majority of preparations analyzed (6/10), amplitude histograms of evoked synaptic potentials could be described by a binomial distribution with a small n and moderately high p. LTF produced a significant increase in n, while p was slightly reduced. The results can be explained by a model in which the binomial parameter n represents the number of active synapses and parameter p the mean probability of release at a synapse. Provided that a pool of initially inactive synapses exists, one can postulate that LTF involves recruitment of synapses to the active state.     

Changes in statistical parameters during facilitation at the crayfish neuromuscular junction

1. Transmitter release at excitatory neuromuscular junctions of the crayfish was studied at different frequencies of stimulation ranging from 1/sec to 20/sec.2. Over this frequency range the average number of quanta released per stimulus (m) increased with frequency by a factor of 6-7.3. Analysis of the fluctuations in quantal release using binomial statistics indicated that the increase in m was associated with increase in the average quantal release probability (p) at stimulation frequencies between 5/sec and 20/sec. Between 1/sec and 5/sec there was an apparent increase in the number of quanta available for release (n).     

Phosphatidylinositol system's role in serotonin-induced facilitation at the crayfish neuromuscular junction

1. In a crustacean neuromuscular preparation, the walking leg opener muscle of the freshwater crayfish Procambarus clarkii, application of serotonin (1 microM) produces presynaptic depolarization and long-lasting facilitation of excitatory postsynaptic potentials (EPSPs). The frequency of spontaneously released transmitter quanta also increases. Facilitation of evoked EPSPs declines after serotonin application in two phases. 2. Serotonin-induced facilitation was examined using simultaneous pre- and postsynaptic intracellular microelectrode recording. A presynaptic microelectrode recorded action potentials and membrane potential of a presynaptic axonal branch, and one or more postsynaptic microelectrodes recorded EPSPs in muscle fibers innervated by the excitatory motor axon. Components of the phosphatidylinositol second messenger system and pharmacologic agents affecting this system were injected through the presynaptic electrode, and changes in synaptic transmission were measured. 3. Presynaptic injection of inositol 1,4,5-triphosphate (IP3) causes presynaptic depolarization, increases the frequency of spontaneously released transmitter quanta, and promotes a relatively short-lasting facilitation of evoked EPSPs. These actions are consistent with elevation of intracellular Ca2+ and resemble the early phase of serotonin-induced facilitation. 4. Application of a phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), that activates protein kinase C (C-kinase), produces a long-lasting, low-level facilitation of evoked EPSPs. Application of another phorbol ester, phorbol-12-monoacetate (PTMA), which does not activate C-kinase has no effect. 5. Presynaptic injection of RA 233, a phospholipase C (PLP-C) inhibitor, blocks all aspects of serotonin-induced facilitation. This compound was found to have no general deleterious effects on synaptic transmission and does not block other forms of synaptic facilitation in this preparation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrophysiological characteristics of crayfish inhibitory neuromuscular synapses at different rhythms of activity

Parameters of IPSPs and characteristics of inhibitory neuromuscular transmission in the dactylopodite-opener muscle of the crayfish pincer at various rhythms of activity were studied by intracellular recording. The presynaptic conduction nature of high-frequency (pessimal) blockade of the inhibitory system was demonstrated. Transformation of the rhythm of stimulation correlated with a decrease in conduction velocity along the inhibitory axon and (or) its branches. At high frequencies of stimulation the mean quantum composition of IPSPs of the potentiation plateau before the beginning of rhythm transformation was lower than at lower frequencies. No signs of exhaustion of mediator reserves were found in the course of high-frequency activity. It is concluded that the mechanism of secretion of inhibitory mediator is more reliable than the function of conduction of the presynaptic nervous impulse. The possible functional significance of the comparatively low reliability of function of crustacean nerve fibers is discussed.Translated from Fiziologicheskii Zhurnal SSSR imeni I. M. Sechenova, Vol. 63, No. 11, pp. 1535–1540, November, 1977.     

Early facilitation at corticomotoneuronal synapses

1. Corticomotoneuronal EPSPs have been generated in lumbar motoneurones of the monkey by single and paired corticospinal volleys. The facilitation of the second of a pair of EPSPs with respect to the size of the first has been measured.2. The relationship between the degree of facilitation of the second response and the interval between the two volleys has been studied. Average facilitation of minimal EPSPs was found to be maximal about 2 msec after the arrival of the corticospinal volley and to decay roughly exponentially thereafter with a time constant of about 10 msec.3. The degree of facilitation varied from one minimal corticomotoneuronal EPSP to another but this facilitation was not statistically correlated with the time course of the individual EPSPs.4. Significant facilitation (0.4) was still present 10 msec after a corticospinal volley so that this phenomenon could play a part in the initiation of motoneuronal discharge by corticospinal activity at natural frequencies of the order of 100 impulses/sec.     

Mechanisms involved in persistent facilitation of neuromuscular synapses in aplysia

Synaptic plasticity can last from a fraction of a second to weeks depending on how it was induced. The mechanisms that underlie short-, intermediate-, and long-term plasticity have been intensively studied at central synapses of both vertebrates and invertebrates; however, peripheral plasticity has not received as much attention. In this study, we investigated the mechanisms that contribute to a persistent form of plasticity at neuromuscular synapses in buccal muscle I3a of Aplysia. These synapses are reversibly facilitated by the small cardioactive peptide (SCP), a peptide cotransmitter that is intrinsic to the motor neurons, and persistently facilitated by serotonin (5HT) released from modulatory neurons that are extrinsic to the motor circuit. Many of the short-term effects of 5HT and SCP are mediated by the cAMP pathway, but little is known about the mechanisms that underlie persistent modulation. We were able to eliminate several possible mechanisms. One of these was the possibility that the apparent reversal of SCP's effects was due to desensitization of the SCP receptor. Superfusion for longer periods or with higher concentrations of SCP indicate that the SCP receptors do not desensitize. We also determined that new protein synthesis is not required for the persistent facilitation of EJPs. Another possibility was that 5HT was taken up and slowly re-released. Our results suggest that this mechanism is also unlikely. Activation of the cAMP pathway does not appear to mediate persistent effects; however, 5HT as well as SCP does cause persistent increases in cAMP levels that can prime I3a synapses and increase the effectiveness of activators of the cAMP pathway. Instead, the persistent effects of 5HT are mimicked by phorbol, suggesting that protein kinase C or an Aplysia homologue of unc13 may mediate these effects. These results, in combination with results from experiments on the sensory neurons that contribute to withdrawal reflexes in Aplysia, suggest that the mechanisms for intermediate- and long-term facilitation may reside in all of the synapses involved in the sensory to motor response reflex.     

Correlation of presynaptic and postsynaptic events during establishment of long-term facilitation at crayfish neuromuscular junction

Repetitive stimulation (10-20 Hz) of the motor axon supplying the opener muscle in the crayfish leg produces long-lasting enhancement of excitatory postsynaptic potentials. This long-term facilitation (LTF) was investigated by recording simultaneously from the presynaptic nerve terminal and from the innervated muscle fiber with intracellular microelectrodes. On cessation of stimulation, the facilitated postsynaptic potential declines in amplitude when monitored with low-frequency test stimuli. A rapid decline (phase I) occurs over the first 30 s and is succeeded by a more gradual decline lasting several minutes (phase II). Finally, a residual potentiation with a very slow decay (phase III) persists for several hours. Simultaneous pre- and postsynaptic recordings were made during induction of LTF with stimuli delivered at 20 Hz for 10 min. During the tetanus, excitatory postsynaptic potentials were enhanced 20-fold, while action potentials in the presynaptic terminal declined in amplitude from 108.6 to 97.2 mV, and the presynaptic membrane became hyperpolarized by 6.4 mV. The Na+ pump inhibitor ouabain (0.5-1.0 mM) abolished the hyperpolarization, indicating that the latter resulted from activation of an electrogenic Na+ pump. The reduction in amplitude of the presynaptic action potential was consistent with a reduced transmembrane concentration gradient for Na+. Thus, it is suggested that a significant accumulation of Na+ occurs during repetitive stimulation of crayfish motor axons. Decay of phase II of LTF, but not of phases I or III, had approximately the same time course as the decay of Na+ accumulation in the terminals, monitored by changes in the presynaptic action potential. Thus it is probable that in crayfish this phase of LTF is linked to an increased intraterminal Na+ concentration. Injection of Na+ from a microelectrode into the presynaptic terminal produced enhancement of the excitatory postsynaptic potential lasting for many minutes, as well as changes in presynaptic membrane potential and action potential similar to those seen during repetitive stimulation. The results provide the first direct measurements of electrical and ionic changes in axonal terminals during prolonged periods of activity leading to LTF, and support the hypothesis that accumulation of intraterminal Na+ is associated with one phase of LTF.     

Recruitment of synapses in the neurosecretory process during long-term facilitation at the lobster neuromuscular junction

We investigated long-term facilitation at the lobster neuromuscular synapse employing a combination of FM1-43 staining of synaptic vesicles, electron microscopy analysis, and electrical recordings of synaptic activity. Synaptic terminals were loaded with the fluorescent dye FM1-43 producing clusters of activity-dependent fluorescent spots. Electron microscopy analysis of synaptic ultrastructure suggested that fluorescent spots represent compartments of synaptic terminals filled with vesicles. Excitatory postsynaptic currents were recorded from the stained synaptic terminals using focal macropatch electrodes. Terminals were stained during the nerve stimulation at a low stimulation frequency (2, 5 or 10 Hz) before and after long-term facilitation was elicited by high-frequency stimulation (20 or 30 Hz for 5 min). We found that staining after long-term facilitation results in the appearance of new fluorescent spots, as well as in the increase in fluorescence of the spots that appeared before long-term facilitation. This increase in fluorescence accounted for the increase in quantal release. Activation of individual fluorescent spots was found to be non-uniform. In spite of overall increase in fluorescence, some synaptic compartments decreased their staining after long-term facilitation. Thus, our study demonstrates that long-term facilitation produces non-uniform activation of FM1-43 uptake in synaptic compartments that correlates with the increase in quantal neurosecretion.     

Conjoint action of phosphatidylinositol and adenylate cyclase systems in serotonin-induced facilitation at the crayfish neuromuscular junction

1. Pulsatile application of serotonin (5-HT) leads to facilitation of excitatory postsynaptic potentials (EPSPs) in crayfish "opener" neuromuscular preparations. The facilitation resulting from a single application of serotonin shows two phases: an early, rapidly decaying phase, and a less intense, long-lasting phase of 1- to 2-h duration. A previous study implicated the phosphatidylinositol system as an essential component in serotonin-induced facilitation, especially the early phase. The present study was conducted to determine the roles of the adenylate cyclase and phosphatidylinositol systems in both phases of serotonin-induced facilitation. 2. Relatively brief applications of agents known to affect the intracellular concentration of cAMP (forskolin, 1 microM; and IBMX, 100 microM) cause an increase in EPSP amplitude, which persists for 1-2 h. 3. The duration of the less intense, long-lasting phase of serotonin-induced facilitation is prolonged in the presence of 1 microM IBMX. This concentration of IBMX does not affect EPSP amplitude by itself. A membrane-permeant analog of cAMP (applied in concentrations less than or equal to 1 mM) is also not effective in altering EPSP amplitude. However, when dibutyryl cAMP is applied in the presence of 1 microM IBMX, EPSP amplitude is increased (60-80%). 4. Localized presynaptic injection of the "Walsh Inhibitor" (PKI), which inhibits cAMP-activated protein kinase, blocks the less intense, long-lasting phase of serotonin-induced facilitation at synapses near the site of injection. Normal facilitation develops at synapses within the same preparation remote from the site of injection. Distribution of the injected inhibitor within the axon can be visualized by tagging PKI with a fluorescent marker.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stochastic properties of spontaneous transmitter release at the crayfish neuromuscular junction

1. Miniature excitatory junctional potentials (min.e.j.p.s) were recorded with an intracellular electrode from the adductor muscle of the dactyl of the first or second walking leg of the crayfish, Orconectes virilis.

2. The intervals between the min.e.j.p.s were compared to the exponential prediction by five goodness of fit tests. The results indicate that the intervals are not exponentially distributed.

3. The autocorrelogram of intervals shows that the intervals are unlikely to be independent.

4. A stochastic analysis that includes the power spectrum of intervals, the variance—time curve, and the ln-survivor curve suggest that there is a clustering of min.e.j.p.s. The results are similar to those on the frog neuromuscular junction.

5. An autocorrelogram of the min.e.j.p. amplitudes suggests that sizes are not independently distributed.

6. These results, which are similar to those previously reported from the frog neuromuscular junction, support the use of the branching Poisson process as a theoretical model for the stochastic properties of spontaneous quantal release of transmitter.

     

Effects of increasing Ca2+ channel-vesicle separation on facilitation at the crayfish inhibitory neuromuscular junction

We investigated the mechanism of facilitation at the crayfish inhibitory neuromuscular junction before and after blocking P-type Ca(2+) channels. P-type channels have been shown to be closer to releasable synaptic vesicles than non-P-type channels at this synapse. Prior to the block of P-type channels, facilitation evoked by a train of 10 action potentials at 100 Hz was increased by application of 40 mM [Mg(2+)](o), but decreased by pressure-injected EGTA. Blocking P-type channels with 5 nM omega-Aga IVA, which reduced total Ca(2+) influx and release to levels comparable to that recorded in 40 mM [Mg(2+)](o), did not change the magnitude of facilitation. We explored whether this observation could be attributed to the buffer saturation model of facilitation, since increasing the Ca(2+) channel-vesicle separation could potentially enhance the role of endogenous buffers. The characteristics of facilitation in synapses treated with omega-Aga IVA were probed with broad action potentials in the presence of K(+) channel blockers. After Ca(2+) channel-vesicle separation was increased by omega-Aga IVA, facilitation probed with broad action potential was still decreased by EGTA injection and increased by 40 mM [Mg(2+)](o). EGTA-AM perfusion was used to test the impact of EGTA over a range of concentration in omega-Aga IVA-poisoned preparations. The results showed a concentration dependent decrease in facilitation as EGTA concentration rose. Thus, probing facilitation with EGTA and reduced Ca(2+) influx showed that characteristics of facilitation are not changed after the role of endogenous buffer is enhanced by increasing Ca(2+) channel-vesicle separation. There is no clear indication that buffer saturation has become the dominant mechanism for facilitation after omega-Aga IVA poisoning. Finally, we sought correlation between residual Ca(2+) and the magnitude of facilitation. Using fluorescence transients of a low affinity Ca(2+) indicator, we calculated the ratio of fluorescence amplitude measured immediately before test pulse (residual Ca(2+)) to that evoked during action potential (local Ca(2+)). This ratio provides an estimate of relative changes between residual Ca(2+) and local Ca(2+) important for release. There is a significant increase in the ratio when Ca(2+) influx is reduced by 40 mM [Mg(2+)](o). The magnitude of facilitation exhibited a clear and positive correlation with the ratio, regardless of separation between Ca(2+) channels and releasable vesicles. This correlation suggests the importance of relative changes between residual and local Ca(2+) and lends support to the residual Ca(2+) hypothesis of facilitation.     

The mechanism of presynaptic inhibition at the crayfish neuromuscular junction

Summary After stimulation of the excitatory nerve fiber potential changes (e.n.t.p.s) were recorded extracellularly from the excitatory nerve terminals on crayfish muscle. The influence of neural presynaptic inhibition on these e.n.t.p.s was analysed.Diphasic e.n.t.p.s recorded at some distance from the nerve terminal were increased by inhibition. Diphasic e.n.t.p.s recorded nearer to the terminal could be abolished by inhibition, their negative phase was more affected than the positive one and sometimes they were made monophasic positive. These findings were interpreted to represent a block of conduction or a central shift of a preexisting block of conduction by inhibition of the excitatory nerve terminal.Monophasic positive e.n.t.p.s recorded at the ultimate terminal were much reduced by inhibition. The release of the excitatory transmitter substance is not decreased proportional to the reduction of the positive e.n.t.p.s by presynaptic inhibition.All effects of presynaptic inhibition on shape and amplitude of the e.n.t.p. can be explained by an increased membrane conductance for chloride and/or potassium ions in the terminal.

---

Zusammenfassung Am Krebsmuskel wurden mit Hilfe von extracellulären Mikroelektroden nach Reiz der erregenden Nervenfaser von ihren Endigungen Potentialänderungen (e.n.t.p.) abgeleitet. Die Effekte der neuralen präsynaptischen Hemmung auf diese e.n.t.p. wurden analysiert.Diphasische e.n.t.p., die in einiger Entfernung von der Endigung abgeleitet wurden, wurden durch die Hemmung vergrößert. Dagegen konnten diphasische e.n.t.p., die näher der Endigung gemessen wurden, durch die Hemmung sehr stark reduziert werden; ihre negative Phase wurde mehr betroffen als die positive, und diphasische e.n.t.p. konnten so zu monophasisch-positiven werden. Diese Befunde wurden als Anzeichen einer Blockade der Erregungsfortleitung, oder einer zentralen Verschiebung einer schon bestehenden solchen Blockade, durch die Hemmung interpretiert.An der äußersten Nervenendigung wurden monophasisch positive e.n.t.p. registriert. Diese wurden durch die Hemmung sehr stark verkleinert. Die Freisetzung des erregenden Überträgerstoffes durch die Nervenendigung wurde durch die Hemmung nicht proportional zur Amplitude der positiven e.n.t.p. herabgesetzt.Alle durch präsynaptische Hemmung bewirkten Veränderungen der Form und Amplitude der e.n.t.p. können durch eine Erhöhung der Membranleitfähigkeit der Nervenendigung für Chlorid- und/oder Kaliumionen erklärt werden.

---

---

With 8 Figures in the Text

---

This investigation was supported by grants from the Deutsche Forschungsgemeinschaft.     

The binomial nature of transmitter release at the crayfish neuromuscular junction

1. Transmitter release at excitatory junctions on the opener muscle of the crayfish dactyl was studied by recording junctional potentials with extracellular micro-electrodes.2. At low temperatures, evoked release was dispersed sufficiently in time for potentials produced by individual quanta to be counted, and the mean (m) and variance (sigma(2)) of the quantum content distribution for a series of trials measured directly. These values were used to calculate the average probability of quantal release (p), assuming a binomial distribution.3. For all values of m and p, the observed release pattern (number of 0, 1, 2, 3,... quantal releases during a series of trials) was approximated closely by the corresponding binomial distribution. However, Poisson predictions differed significantly from the observed quantal distribution for values of p > 0.2.     

The effect of a preceding stimulus on temporal facilitation at corticomotoneuronal synapses

1. Intracellular recordings were made of minimal corticomotoneuronal e.p.s.p.s in lumbar motoneurones of anaesthetized monkeys. For intervals of 2 msec and greater between paired cortical shocks, the average time course of facilitation of the second e.p.s.p. with respect to the first could be fitted closely by a negative exponential with a time constant of 10 msec.2. In the same motoneurones, ;triplets' of corticomotoneuronal e.p.s.p.s were generated by delivering three identical stimuli to the motor cortex. Considering the triplet as a conditioning e.p.s.p. followed by a test pair, the facilitation of the third e.p.s.p. with respect to the second was measured for various combinations of test and conditioning intervals. In each case the amplitude of the third e.p.s.p. was also compared with that of the first (conditioning) e.p.s.p.3. The effect of a brief conditioning interval was to reduce considerably the facilitation of the third e.p.s.p. with respect to the second at all test intervals from 2 to 50 msec. Combinations of brief conditioning intervals (e.g. 2 or 5 msec) and long test intervals (e.g. 20 or 50 msec) caused the third e.p.s.p. to be smaller than the second. As the conditioning interval lengthened, facilitation in the test pair increased towards the unconditioned values at all test intervals.4. Facilitation of the third e.p.s.p. with respect to the first could be described approximately as the linear addition of two facilitation components, one due to the conditioning input and one due to the first stimulus of the test pair. Each component followed the same negative exponential time course as found for an isolated pair of e.p.s.p.s and each of the first two inputs contributed to the facilitation of the third e.p.s.p. as if the other of these two inputs had not occurred.