ATP potentiates spontaneous transmitter release at developing neuromuscular synapses

Extracellular application of ATP, a substance co-stored and co-released with acetylcholine in peripheral nervous systems, potentiates the spontaneous secretion of acetylcholine at developing neuromuscular synapses in Xenopus cell culture, as shown by a marked increase in the frequency of spontaneous synaptic currents recorded in the postsynaptic muscle cell. The effect of ATP is apparently mediated by the activation of cytosolic protein kinases and requires the influx of Ca2+ through the plasma membrane. Since spontaneous acetylcholine release is known to regulate the development of contractile properties of the postsynaptic muscle cell, extracellular ATP may serve as a positive trophic factor at developing neuromuscular synapses.     

Repetitive impulse activity potentiates spontaneous acetylcholine secretion at developing neuromuscular synapses.

The effects of presynaptic impulse activity on the transmitter secretion at developing neuromuscular junctions were examined in Xenopus nerve-muscle cultures. Repetitive suprathreshold stimulation of the presynaptic neuron results in marked potentiation of spontaneous synaptic activity, as shown by whole-cell voltage-clamp recording of synaptic currents in the postsynaptic muscle cell. Our results are consistent with the notion that synaptic efficacy of the developing synapse is potentiated by the presence of electrical activity. Such activity-dependent synaptic modulation enables the early neuronal activity to play a regulatory role during the maturation of synaptic connections.     

Strategic location of calcium channels at transmitter release sites of frog neuromuscular synapses

The localization of Ca2+ channels relative to the position of transmitter release sites was investigated at the frog neuromuscular junction (NMJ). Ca2+ channels were labeled with fluorescently tagged omega-conotoxin GVIA, an irreversible Ca2+ channel ligand, and observed with a confocal laser scanning microscope. The Ca2+ channel labeling almost perfectly matched that of acetylcholine receptors which were labeled with fluorescent alpha-bung-arotoxin. This indicates that groups of Ca2+ channels are localized exclusively at the active zones of the frog NMJ. Cross sections of NMJs showed that Ca2+ channels are clustered on the presynaptic membrane adjacent to the postsynaptic membrane.     

Facilitation of transmitter release at squid synapses

Facilitation is shown to decay as a compound exponential with two time constants (T1, T2) at both giant and non-giant synapses in squid stellate ganglia bathed in solutions having low extracellular calcium concentrations ([Ca++]o). Maximum values of facilitation (F1) were significantly larger, and T1 was significantly smaller in giant than non-giant synapses. Decreases in [Ca++]o or increases in [Mn++]o had variable effects on T1 and F1, whereas decreases in temperature increased T1 but had insignificant effects on F1. The growth of facilitation during short trains of equal interval stimuli was adequately predicted by the linear summation model developed by Mallart and Martin (1967. J. Physiol. (Lond.). 193:676--694) for frog neuromuscular junctions. This result suggests that the underlying mechanisms of facilitation are similar in squid and other synapses which release many transmitter quanta.     

Receptor kinetics pertaining to blockade of nerve-released transmitter at synapses

The question is raised as to whether competitive inhibitors should block responses of tissue to nerve-released neurotransmitter to the same extent as they block equivalent responses to exogenous agonist. From a simple dynamic model of synaptic events, which takes into account non-constancy of transmitter concentration in space and time, it is deduced that equal blockade of responses to nerve-released and exogenous transmitter substance will occur if: (i) there are locally many more receptor molecules than transmitter molecules; (ii) the active agonist-receptor complex, AnR, has n = 1; and (iii) tissue response is insensitive to spatial or temporal inhomogeneity of AR. In such a case there will also be equal sensitivity of responses to other modes of inhibition: irreversible competitive, uncompetitive, and non-competitive. Equal blockade of responses to equi-effective endogenous and exogenous agonist will also occur if nerve stimulation gives rise to a steady uniform concentration of agonist, so that equilibrium kinetics are applicable. When n greater than 1 and/or when tissue responses reflect local peak AnR, response to nerve-released transmitter will be relatively insensitive to receptor blockade by a competitive inhibitor. The same is true for irreversible competitive blockade or for modulation of receptor density. However, an uncompetitive inhibitor (e.g. a 'channel blocker') may be more effective against nerve-released agonist than against exogenous agonist.     

Structural plasticity at crustacean neuromuscular synapses

Crustacean motor axons innervate muscle fibers via a multiplicity of synaptic terminals which release small but variable amounts of transmitter. Differences in release performance appear to be correlated with the size of synaptic contacts and presynaptic dense bars (active zones). These structural parameters proliferate via sprouting from existing synaptic terminals and relocate to ever more distal sites during development and growth of an identified axon. Moreover, alterations in number of synaptic contacts and active zones occur in adults following stimulation or decentralization, demonstrating structural plasticity of crustacean neuromuscular synapses.     

Estimation of parameters for a model of transmitter release at synapses.

We consider models for the release of transmitter in response to nerve impulses, where it is assumed that quanta of transmitter are released from some of n sites, the probability of release from any site being p. It is assumed that the quantal size is either a constant or is distributed as a normal or a gamma variate. Observations on both spontaneous potentials and evoked potentials are used to obtain moment estimated of n and p. Large sample estimates of the standard errors of these estimates are given.     

The mode of spontaneous transmitter release at the insect neuromuscular junction.

The time intervals between miniature excitatory postsynaptic potentials and the counts of them in the cockroach, Periplaneta americana, were analyzed, using a computer program to test for properties of a Poisson process. The miniature potentials occurred basically in random manner at this neuromuscular junction. Although the distribution of the potentials did not fit the criteria for a Poisson process when the muscle fiber exhibited the short burst of high-frequency discharges, it was suggested that the primary process of such a distribution is Poisson, which is occasionally contaminated by the burst phase of the release rates.     

Effect of tetanus toxin on mechanisms by which calcium ions regulate transmitter secretion at the neuromuscular junction

Phrenicodiaphragmal rat preparations were used to study the transmitter secretion by intracellular recording of end plate potentials (EPP) and miniature EPP (MEPP). In tetanus toxin-poisoned terminal, the regulatory effect of the external gradient of Ca2+ was abolished as evidenced by the fact that spontaneous secretion did not differ from that in calcium-free solution in health, as the external concentration of Ca2+ rose from 0 to 20 mM. Calcium ionophore A 23187 in intact terminals activated spontaneous release of the transmitter, but did not affect the poisoned terminal. Ouabain enhanced spontaneous secretion both in health and in poisoning. 4-Aminopyridine (4-AP) did not change the frequency of MEPP, while "giant" MEPPs that reflect spontaneous synchronization of the release of quants occurred both in health and in poisoning. 4-AP potentiated the reactivation effects of rhythmic stimulation of poisoned synapses, particularly with reference to the evoked release and led to the recovery of transmission. It is likely that tetanus toxin fixed by gangliosides of the presynaptic membrane prevents, in this particular case, the functioning of both endo- and exogenous ionophoroses that transport Ca2+ to the "active zones", without affecting their asynchronous supply from the intracellular depots.     

Functional roles of peptide cotransmitters at neuromuscular synapses inAplysia

Neuromuscular synapses inAplysia have been used as model systems to study peptidergic cotransmission. Here we describe neuromuscular preparations in which it has been possible to investigate the physiological consequences of peptide transmitter release in detail. In the first preparation, the release of peptide cotransmitters from identified motor neuron B15 has been shown to be sensitive to the pattern of stimulation. High frequencies and long burst durations evoke peptide release that modulates muscle contractions in a manner similar to that produced by exogenous cotransmitter. By contrast, the release of the same peptide transmitters from motor neuron B1 show little dependence on pattern. We conclude that there are no stimulation patterns that are prerequisites for peptide release. Peptide cotransmitter release from motor neuron B47 has also been studied. B47, depending on the stimulation pattern, uses either ACh, which acts as a conventional inhibitory transmitter, or Ach plus neuropeptides, which act as excitatory modulatory cotransmitters. Thus, neuropeptide cotransmitters have the capability to greatly increase synaptic plasticity at neuromuscular synapses.     

Structural changes after transmitter release at the frog neuromuscular junction

The sequence of structural changes that occur during synaptic vesicle exocytosis was studied by quick-freezing muscles at different intervals after stimulating their nerves, in the presence of 4-aminopyridine to increase the number of transmitter quanta released by each stimulus. Vesicle openings began to appear at the active zones of the intramuscular nerves within 3-4 ms after a single stimulus. The concentration of these openings peaked at 5-6 ms, and then declined to zero 50-100 ms late. At the later times, vesicle openings tended to be larger. Left behind at the active zones, after the vesicle openings disappeared, were clusters of large intramembrane particles. The larger particles in these clusters were the same size as intramembrane particles in undischarged vesicles, and were slightly larger than the particles which form the rows delineating active zones. Because previous tracer work had shown that new vesicles do not pinch off from the plasma membrane at these early times, we concluded that the particle clusters originate from membranes of discharged vesicles which collapse into the plasmalemma after exocytosis. The rate of vesicle collapse appeared to be variable because different stages occurred simultaneously at most times after stimulation; this asynchrony was taken to indicate that the collapse of each exocytotic vesicle is slowed by previous nearby collapses. The ultimate fate of synaptic vesicle membrane after collapse appeared to be coalescence with the plasma membrane, as the clusters of particles gradually dispersed into surrounding areas during the first second after a stimulus. The membrane retrieval and recycling that reverse this exocytotic sequence have a slower onset, as has been described in previous reports.     

Modulation of transmitter release at giant synapses of the auditory system

The relay nuclei of the auditory brainstem contain some of the largest nerve terminals in the mammalian brain. Endbulb and calyceal synapses convey signals with a high degree of precision and reliability. However, recent studies reveal that these synapses possess numerous and remarkably diverse mechanisms for the modulation of transmitter release. The implication is that successful relay of signals in vivo may require the ability to fine-tune synaptic transmission.     

Neural factors regulate AChR subunit mRNAs at rat neuromuscular synapses

To elucidate the nature of signals that control the level and spatial distribution of mRNAs encoding acetylcholine receptor (AChR), alpha-, beta-, gamma-, delta- and epsilon-subunits in muscle fibers chronic paralysis was induced in rat leg muscles either by surgical denervation or by different neurotoxins that cause disuse of the muscle or selectively block neuromuscular transmission pre- or postsynaptically and cause an increase of AChRs in muscle membrane. After paralysis, the levels and the spatial distributions of the different subunit-specific mRNAs change discoordinately and seem to follow one of three different patterns depending on the subunit mRNA examined. The level of epsilon-subunit mRNA and its accumulation at the end-plate are largely independent on the presence of the nerve or electrical muscle activity. In contrast, the gamma-subunit mRNA level is tightly coupled to innervation. It is undetectable or low in innervated normally active muscle and in innervated but disused muscle, whereas it is abundant along the whole fiber length in denervated muscle or in muscle in which the neuromuscular contact is intact but the release of transmitter is blocked. The alpha-, beta-, and delta-subunit mRNA levels show a different pattern. Highest amounts are always found at end-plate nuclei irrespective of whether the muscle is innervated, denervated, active, or inactive, whereas in extrasynaptic regions they are tightly controlled by innervation partially through electrical muscle activity. The changes in the levels and distribution of gamma- and epsilon-subunit-specific mRNAs in toxin-paralyzed muscle correlate well with the spatial appearance of functional fetal and adult AChR channel subtypes along the muscle fiber. The results suggest that the focal accumulation at the synaptic region of mRNAs encoding the alpha-, beta-, delta-, and epsilon-subunits, which constitute the adult type end-plate channel, is largely determined by at least two different neural factors that act on AChR subunit gene expression of subsynaptic nuclei.     

Calcium and facilitation at two classes of crustacean neuromuscular synapses

The closer muscle of the crab, Chionoecetes, has at least two classes of excitatory neuromuscular synapses. In one class of synapses an action potential depolarizing the synaptic region releases much more transmitter if it has been preceded recently by another action potential. The other class of synapses shows this property, called facilitation, to a far lesser extent. Immediately after one conditioning stimulus the level of facilitation is similar in both classes. The rate of the ensuing decay of the facilitation is the critical factor differentiating the two classes of synapses. The relationship between external Ca⁺⁺ concentration and transmitter release is similar for both classes of synapses. The slope of a double logarithmic plot of this relationship varies from 3.1 between 5 and 10 mM Ca⁺⁺ to 0.9 between 30 and 40 mM Ca⁺⁺. Facilitation does not significantly change when tested in external Ca⁺⁺ concentrations ranging from 7 to 30 mM. The extracellularly recorded nerve terminal action potential does not increase in amplitude during facilitation. The results suggest that the mechanism of synaptic facilitation is similar for both classes of synapses and occurs after the stage in transmitter release involving Ca⁺⁺.