Transmitter release increases intracellular calcium in perisynaptic Schwann cells in situ.

Glial cells isolated from the nervous system are sensitive to neurotransmitters and may therefore be involved in synaptic transmission. The sensitivity of individual perisynaptic Schwann cells to activity of a single synapse was investigated, in situ, at the frog neuromuscular junction by monitoring changes in intracellular Ca2+ in the Schwann cells. Motor nerve stimulation induced an increase in intracellular Ca2+ in these Schwann cells; this increase was greatly reduced when transmitter release was blocked. Furthermore, local application of the cotransmitters acetylcholine and ATP evoked Ca2+ responses even in the absence of extracellular Ca2+. Successive trains of nerve stimuli or applications of transmitters resulted in progressively smaller Ca2+ responses. We conclude that transmitter released during synaptic activity can evoke release of intracellular Ca2+ in perisynaptic Schwann cells. This Ca2+ signal may play a role in the maintenance or modulation of a synapse. These data show that synaptic transmission involves three cellular components with both postsynaptic and glial components responding to transmitter secretion.     

Presynaptic actions of botulinal neurotoxins at vertebrate neuromuscular junctions

1. In the present paper we review some presynaptic aspects of the mode of action of botulinal toxins (BoTxs) at vertebrate neuromuscular junctions with emphasis on studies carried out in our laboratories using electrophysiological and morphological techniques. 2. Spontaneous quantal transmitter release recorded as miniature end-plate potentials is drastically affected by BoTxs. The low probability of release at poisoned terminals can be enhanced by carbonyl cyanide m-chlorophenylhydrazone (CCCP), Cd2+ and La3+. However, CCCP and La3+ which drastically deplete clear synaptic vesicles from unpoisoned terminals failed to markedly affect the density of synaptic vesicles at poisoned terminals. It is concluded that poisoned terminals have a reduced sensitivity to the release-promoting action of Ca2+, Cd2+ and La3+. 3. When comparing the effect of the various BoTxs on nerve-impulse evoked transmitter release it appears that increasing phasic Ca2+ entry into the terminals enhances evoked synchronized quantal release only from terminals poisoned with serotypes A and E. In contrast, enhanced Ca2+ entry into terminals poisoned with serotypes B, D and F induced a period of high frequency asynchronous release suggesting that these BoTxs may affect a presynaptic step beyond the influx of Ca2+, that may be involved in the synchronization of transmitter quanta. These data suggest that the actions of BoTxs involve several steps of the acetylcholine release process. 4. The analysis of presynaptic currents which depend on both Ca2+ entry and intraterminal background Ca2+ levels strongly suggests that neither Ca2+ entry nor intraterminal Ca2+ levels are altered by BoTxs. Furthermore, poisoned terminals are no more efficient than unpoisoned ones in dealing with Ca2+ overloads. 5. Finally, the morphological examination of junctions paralysed by BoTx-A indicates that the toxin triggers a particularly important overgrowth of the nerve terminals and suggests that the in vivo functional recovery may occur from an extension of the original nerve terminal arborization and the concomitant remodelling of postsynaptic structures.     

Differences in presynaptic action of 4-aminopyridine and tetraethylammonium at frog neuromuscular junction

4-Aminopyridine markedly potentiates transmitter release at the frog pectoris neuromuscular junction by increasing the quantal content even when applied at low concentrations (5-20 microM). This enhancement of transmitter release is associated with greater minimum synaptic latency, but the dispersion of the synaptic latencies does not appear much affected. This is in contrast with the action of tetraethylammonium (0.2-0.5 mM) in which case similar enhancement of transmitter release results not only in larger minimum synaptic latency but also in greater dispersion of the synaptic latencies. The time course of transmitter release associated with enhanced transmitter output is hence much more prolonged in the presence of tetraethylammonium than 4-aminopyridine, at least for low concentrations of 4-aminopyridine (5-20 microM). This indicates that their presynaptic actions differ significantly. This conclusion is further strengthened by the finding that unlike tetraethylammonium, 4-aminopyridine induces bursts of release, presumably by producing multiple action potentials in the nerve terminal. Tetraethylammonium probably acts by blocking the delayed potassium conductance, but the blockade of Ca2+-activated K+ conductance cannot be excluded. 4-Aminopyridine, however, probably blocks the fast inactivating (IA) K+ current, but it also may be acting directly on the voltage-dependent Ca2+ conductance or on the intracellular Ca2+ buffering.     

The effects of pentachlorophenol (PCP) at the toad neuromuscular junction

1. Effects of PCP at the frog neuromuscular junction were studied in vitro in sciatic nerve sartorius muscle of the toad Pleurodema-thaul. 2. Within the concentration 0.003-0.1 mM, PCP caused a dose-time-dependent block of evoked transmitter release acompanied by an increase in the rate of spontaneous quantal release. 3. PCP induced an increase in miniature endplate potential (MEPP) frequency and it was not antagonized in a Ca2(+)-free medium, indicating that it does not depend upon Ca2+ influx from the external medium, but may act by releasing Ca2+ from intraterminal stores. 4. The present data, together with previous results concerning PCP at eighth sympathetic ganglia indicate that 3,4-diaminopyridine (3,4-DAP) counteracts the effects of PCP on synaptic transmission. This result suggests that PCP interfering Ca2+ influx occurs during depolarization of motor nerve terminals.     

Mechanism of long-term potentiation of transmitter release induced by adrenaline in bullfrog sympathetic ganglia

A mechanism of the long-term potentiation of transmitter release induced by adrenaline (ALTP) was studied by recording intracellularly the fast excitatory postsynaptic potentials (fast EPSPs). The ALTP was produced during the blockade of K+ channels at the presynaptic terminals by tetraethylammonium (TEA). The synaptic delay, possibly reflecting a relative change in the duration of an action potential at the presynaptic terminal, was not changed during the course of the ALTP. By contrast, it was significantly lengthened by TEA and other K+ channel inhibitors (4-aminopyridine and Cs+) that markedly enhanced the evoked release of transmitter. The magnitude of facilitation of the fast EPSP, induced by a conditional stimulus to the preganglionic nerve, was decreased during the generation of the ALTP, but was unchanged during the potentiation of transmitter release caused by TEA. These results, together with theoretical considerations applying the residual Ca2+ hypothesis to the facilitation, suggest that the enhancement of transmitter release during the ALTP is not caused by an increased Ca2+ influx during a presynaptic impulse owing to the blockade of K+ channel or the modulation of Ca2+ channel, but presumably is induced by a rise in the basal level of free Ca2+ in the presynaptic terminal.     

Localization and characterization of nitric oxide synthase at the frog neuromuscular junction

Summary. The frog neuromuscular junction is sensitive to nitric oxide (NO), since exogenously applied NO reduces the release of transmitter by presynaptic terminals and the size of ATP-induced Ca²⁺ responses in perisynaptic Schwann cells. This study aimed at determining whether an NO synthase (NOS) is present at the neuromuscular junction, notably in perisynaptic Schwann cells, the glial cells at this synapse. The NADPH-diaphorase (NADPH-d) histochemical technique revealed the presence of NOS in cell bodies and presumed processes of perisynaptic Schwann cells. Incubation with NOS inhibitors, N^G-nitro-L-arginine methyl ester or N^G-monomethyl-L-arginine-acetate, abolished the NADPH-d staining. Moreover, L-arginine, the precursor of NO, impeded the blockade by NOS inhibitors, establishing the NOS specificity of NADPH-d staining in frog tissue. The pattern of labelling with a polyclonal antibody against the neuronal form of NOS was similar to the NADPH-d staining, also suggesting the presence of a neuronal NOS in perisynaptic Schwann cells. Using electron microscopy, the NOS immunostaining was found at the membrane and occasionally in the cytoplasm of perisynaptic Schwann cells and was not detected in the nerve terminal or muscle. There was no enzymatic or immunocytochemical labelling of NOS 6 days after denervation. It is concluded that NOS is present in frog perisynaptic Schwann cells. The presence of this endogenous NOS suggests that NO may act as a diffusible glial messenger to modulate synaptic activity and synapse formation at the neuromuscular junction.     

Phosphatases modulate transmission and serotonin facilitation at synapses: studies with the inhibitor okadaic acid.

We examined the role of phosphatases in synaptic transmission using the permeant phosphatase inhibitor okadaic acid (OA). In the crayfish neuromuscular junction (NMJ), postsynaptic effects including increases in input resistance occurred at doses greater than 5 microM OA. At lower doses (0.5-5 microM) the effects were solely presynaptic and transmitter release increased over three-fold despite small reductions in amplitude and duration of presynaptic action potentials. Potentiating effects of serotonin on transmitter release, which depend on phosphorylation, were increased by OA. Frequency facilitation was reduced but its decay was not affected. In frog NMJs, OA increased spontaneous and evoked release two-fold through presynaptic mechanisms. An inactive analog of OA, OA tetra-acetate, had no effect on transmitter release at frog and crayfish NMJ. Therefore, phosphatases have a strong modulating influence on synaptic transmission.     

Role of intracellular Ca2+ in stimulation-induced increases in transmitter release at the frog neuromuscular junction

Under conditions of reduced quantal content, repetitive stimulation of a presynaptic nerve can result in a progressive increase in the amount of transmitter released by that nerve in response to stimulation. At the frog neuromuscular junction, this increase in release has been attributed to four different processes: first and second components of facilitation, augmentation, and potentiation (e.g., Zengel, J. E., and K. L. Magleby. 1982. Journal of General Physiology. 80:583-611). It has been suggested that an increased entry of Ca2+ or an accumulation of intraterminal Ca2+ may be responsible for one or more of these processes. To test this hypothesis, we have examined the role of intracellular Ca2+ in mediating changes in end-plate potential (EPP) amplitude during and after repetitive stimulation at the frog neuromuscular junction. We found that increasing the extracellular Ca2+ concentration or exposing the preparation to carbonyl cyanide m- chlorophenylhydrazone, ionomycin, or cyclopiazonic acid all led to a greater increase in EPP amplitude during conditioning trains of 10-200 impulses applied at a frequency of 20 impulses/s. These experimental manipulations, all of which have been shown to increase intracellular levels of Ca2+, appeared to act by increasing primarily the augmentation component of increased release. The results of this study are consistent with previous suggestions that the different components of increased release represent different mechanisms, and that Ca2+ may be acting at more than one site in the nerve terminal.     

Glial cells maintain synaptic structure and function and promote development of the neuromuscular junction in vivo

To investigate the in vivo role of glial cells in synaptic function, maintenance, and development, we have developed an approach to selectively ablate perisynaptic Schwann cells (PSCs), the glial cells at the neuromuscular junction (NMJ), en masse from live frog muscles. In adults, following acute PSC ablation, synaptic structure and function were not altered. However, 1 week after PSC ablation, presynaptic function decreased by approximately half, while postsynaptic function was unchanged. Retraction of nerve terminals increased over 10-fold at PSC-ablated NMJs. Furthermore, nerve-evoked muscle twitch tension was reduced. In tadpoles, repeated in vivo observations revealed that PSC processes lead nerve terminal growth. In the absence of PSCs, growth and addition of synapses was dramatically reduced, and existing synapses underwent widespread retraction. Our findings provide in vivo evidence that glial cells maintain presynaptic structure and function at adult synapses and are vital for the growth and stability of developing synapses.     

Xestospongin C is a potent inhibitor of SERCA at a vertebrate synapse

The specificity of action of Xestospongin C (XeC) towards the inositol 1,4,5-trisphosphate (IP3) receptor has been studied using the frog neuromuscular junction. In perisynaptic Schwann cells (PSCs), glial cells at this synapse, Ca2+ stores are dependent upon IP3 activation. Bath application of XeC (700 nM) caused a transient calcium elevation and blocked Ca2+ responses evoked in PSCs by synaptic activity or various agonists (ATP, muscarine, adenosine) only when Ca2+ stores had previously been challenged with local application of agonists. Moreover, XeC occluded the effects of thapsigargin (tg; 2 microM), a blocker of the Ca2+ ATPase pump of internal stores, which failed to evoke Ca2+ transients following 20 min of exposure to XeC. In nerve terminals, where the Ca2+ stores are ryanodine-sensitive, application of XeC (700 nM) prolonged the recovery phase of Ca2+ transients evoked by single action potentials, due to a prolonged Ca2+ clearance in the nerve terminal. No effects of tg (2 microM) were observed on Ca2+ response evoked by nerve stimulation when applied on the preparation after XeC (700 nM). Conversely, XeC (700 nM) had no effect on the shape and duration of Ca2+ entry in nerve terminals when tg was applied before XeC. These results indicate that XeC acts as an inhibitor of the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) pump of internal stores.     

Quantitative freeze-fracture analysis of the frog neuromuscular junction synapse – I. Naturally occurring variability in active zone structure

The orderly arrays of intramembranous particles (IMPs) found in the p-face of freeze-fracture replicas of the frog neuromuscular junction (‘active zones’) are believed to be involved in transmitter release. Some or all of the particles represent voltage-dependent Ca2+ channels. Since there is a great heterogeneity in the amount of transmitter released by different frog motor nerve terminals we sought to determine whether active zone (AZ) structure displayed a similar heterogeneity by using a novel freeze-fracture procedure providing large, intact replicas containing significant portions of motor nerve terminals from the cutaneous pectoris muscle of the frog, Rana pipiens. Using only junctions in which more than 50 AZs or more than 50 μm of nerve terminal were included in the fractures, we measured AZ length, AZ intramembranous particle density, terminal width at each AZ, space between AZs, the angle of AZ orientation with respect to the longitudinal axis of the nerve terminal, exposed pre-synaptic nerve terminal surface area and a calculated value for mean AZ length per unit terminal length. The analysis led to the following conclusions. There is an approximate 5-fold range in mean AZ length/micrometre terminal length. Terminal width is a good predictor of AZ length. Particle density does not vary significantly within a given AZ, nor between AZs from the same or different junctions. The distance between AZs is not related to AZ length, i.e. shorter AZs are no more or less likely to be closer to the adjacent AZ. The probability of release from any AZ on action potential invasion is small. If most of the IMPs are Ca2+ channels, either the probability of channel opening or the efficacy of triggering release is very low or both. That the variability in release efficacy in different terminals is much greater than ultrastructural variability in terminals suggests that regulation of release is dominated by physiological processes that do not have obvious ultrastructural correlates. On the other hand, the apparent excess of AZ relative to the number of vesicles released indicates that the amount and variability in amount of AZ is important in ways that need to be elucidated.     

Facilitation, augmentation, and potentiation of synaptic transmission at the superior cervical ganglion of the rabbit

The effect of repetitive stimulation on synaptic transmission was studied in the isolated superior cervical ganglion of the rabbit under conditions of reduced quantal content. Excitatory postsynaptic potentials (EPSP) were recorded with the sucrose gap technique to obtain estimates of transmitter release. Four components of increased transmitter release, with time constants of decay similar to those observed at the frog neuromuscular junction at 20 degrees C, were found in the ganglion at 34 degrees C: a first component of facilitation, which decayed with a time constant of 59 +/- 14 ms (mean +/- SD); a second component of facilitation, which decayed with a time constant of 388 +/- 97 ms; augmentation, which decayed with a time constant of 7.2 +/- 1 s; and potentiation, which decayed with a time constant of 88 +/- 25 s. The addition of 0.1-0.2 mM Ba2+ to the Locke solution increased the magnitude but not the time constant of decay of augmentation. Ba2+ had little effect on potentiation. The addition of 0.2-0.8 mM Sr2+ to the Locke solution appeared to increase the magnitude of the second component of facilitation. Sr2+ had little effect on augmentation or potentiation. These selective effects of Ba2+ and Sr2+ on the components of increased transmitter release in the rabbit ganglion are similar to the effects of these ions at the frog neuromuscular junction. Although the effects of Ba2+ and Sr2+ are similar in the two preparations, the magnitudes of augmentation and the second component of facilitation after a single impulse were about 6-10 times greater in the rabbit ganglion than at the frog neuromuscular junction. These results suggest that the underlying mechanisms in the nerve terminal that give rise to the components of increased transmitter release in the rabbit ganglion and frog neuromuscular junction are similar but not identical.     

Binding of a Glycera convoluta neurotoxin to cholinergic nerve terminal plasma membranes

The crude extract of venom glands of the polychaete annelid Glycera convoluta triggers a large Ca2+-dependent acetylcholine release from both frog motor nerve terminals and Torpedo electric organ synaptosomes. This extract was partially purified by Concanavalin A affinity chromatography. The biological activity was correlated in both preparations to a 300,000-dalton band, as shown by gel electrophoresis. This confirmed previous determinations obtained with chromatographic methods. This glycoprotein binds to presynaptic but not postsynaptic plasma membranes isolated from Torpedo electric organ. Pretreatment of intact synaptosomes by pronase abolished both the binding and the venom- induced acetylcholine release without impairing the high K+-induced acetylcholine release. Pretreatment of nerve terminal membranes by Concanavalin A similarly prevented the binding and the biological response. Binding to Torpedo membranes was still observed in the presence of EGTA. An antiserum directed to venom glycoproteins inhibited the neurotoxin so we could directly follow its binding to the presynaptic membrane. Glycera convoluta neurotoxin has to bind to a ectocellularly oriented protein of the presynaptic terminal to induce transmitter release.     

Adenosine depresses spontaneous transmitter release from frog motor nerve terminals by acting at an A1-like receptor

Adenosine (1 microM to 1 mM) depressed spontaneous transmitter release from frog motor nerve terminals without producing any observable postsynaptic effects. Since this action of adenosine was blocked by 20 microM theophylline and 1 microM 8-phenyltheophylline, adenosine probably acts at a specific receptor on motor nerve terminals to reduce spontaneous transmitter output. The effects of the adenosine analogs, L-N6-phenylisopropyladenosine (L-PIA, 100 pM to 1 microM), D-PIA (100 nM to 100 microM), and 5'-N-ethylcarboxamidoadenosine (NECA, 10nM to 100 microM), were tested on spontaneous transmitter release at the frog neuromuscular junction. L-PIA depressed mepp frequency at a threshold concentration of about 1 nM, was thirteen times more potent than NECA, and was 294 times more effective than D-PIA. The rank-order potency of these analogs indicates that adenosine acts at an A1-like receptor to depress spontaneous transmitter release. Inhibitory actions of maximally effective concentrations of adenosine and L-PIA were also blocked by the A1-specific antagonist, 1-3-dipropyl-8-cyclopentylxanthine (DPCPX) at a concentration of 100 nM. Micromolar concentrations of NECA, an agonist with approximately equal affinity for the A1 and A2 receptors, produced biphasic effects on mepp frequency. Thus, a second adenosine receptor, perhaps of the A2 subtype, may be present on motor nerve terminals and may mediate an increase in spontaneous transmitter release.     

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.     

Presynaptic mechanisms of zinc effects on the neuromuscular transmission]

The effect of zinc ions on presynaptic currents and transmitter release was studied at the neuromuscular junction of the frog cutaneous pectoris muscle preparation with using an extracellular microelectrode. It has been shown that zinc (100 mkM) amplified MEPP frequency at first, but suppressed it later. Zinc affected the presynaptic spike waveform and transmitter release in a concentration-dependent manner. Depending on concentration and time of exposure zinc increased or suppressed transmitter release. Increase of transmitter release was shown to be resulted by blockade voltage gated and calcium activated potassium channels in nerve ending, leading to broad of both presynaptic spike and action potential. Strong change of presynaptic spike waveform after high concentration zinc treatment supposed that under this condition zinc depressed voltage gated calcium and sodium channel leading to decrease of transmitter release. It was concluded that the final and irreversible depression of acetylcholine release by zinc was due to alteration of whole ion conductances in nerve ending and to change of configuration of proteins included in structure of ion channels. It is discussed possible mechanisms of various effects of zinc ions at the neuromuscular synapse.     

Can presynaptic depolarization release transmitter without calcium influx?

Recent experimental evidence suggesting that presynaptic depolarization can evoke transmitter release without calcium influx has been re-examined. The presynaptic terminal of the squid giant synapse can be depolarized by variable amounts while recording presynaptic calcium current under voltage clamp and postsynaptic responses. Small depolarizations open few calcium channels with large single channel currents. Large depolarizations approaching the calcium equilibrium potential open many channels with small single channel currents. When responses to small and large depolarizations eliciting similar total macroscopic calcium currents are compared, the large pulses evoke more transmitter release. This apparent voltage-dependence of transmitter release may be explained by the greater overlap of calcium concentration domains surrounding single open calcium channels when many closely apposed channels open at large depolarizations. This channel domain overlap leads to higher calcium concentrations at transmitter release sites and more release for large depolarizations than for small depolarizations which open few widely dispersed channels. At neuromuscular junctions, a subthreshold depolarizing pulse to motor nerve terminals may release over a thousand times as much transmitter if it follows a brief train of presynaptic action potentials than if it occurs in isolation. This huge synaptic facilitation has been taken as indicative of a direct effect of voltage which is manifest only when prior activity raises presynaptic resting calcium levels. This large facilitation is actually due to a post-tetanic supernormal excitability in motor nerve terminals, causing the previously subthreshold test pulse to become suprathreshold and elicit a presynaptic action potential. When motor nerve terminals are depolarized by two pulses, as the first pulse increases above a certain level it evokes more transmitter release but less facilitation of the response to the second pulse.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dynamic aspects of presynaptic calcium currents mediating synaptic transmission

Ca2+ entry through voltage-gated Ca2+ channels (VGCC) triggers transmitter release. Direct recording of Ca2+ currents from the calyx of Held nerve terminal revealed that presynaptic VGCCs undergo various modulations via presynaptic G protein-coupled receptors (GPCRs), Ca2+-binding proteins and a developmental switch of their alpha1 subunits. Dynamic changes of presynaptic VGCCs alter synaptic efficacy, thereby contributing to a variety of modulations of the CNS function.     

Modulation of Synaptic Vesicle Exocytosis in Muscle-Dependent Long-Term Depression at the Amphibian Neuromuscular Junction

We have labeled recycling synaptic vesicles at the somatic Bufo marinus neuromuscular junction with the styryl dye FM2-10 and provide direct evidence for refractoriness of exocytosis associated with a muscle activity-dependent form of long-term depression (LTD) at this synapse. FM2-10 dye unloading experiments demonstrated that the rate of vesicle exocytosis from the release ready pool (RRP) of vesicles was more than halved in the LTD (induced by 20 min of low frequency stimulation). Recovery from LTD, observed as a partial recovery of nerve-evoked muscle twitch amplitude, was accompanied by partial recovery of the refractoriness of RRP exocytosis. Unexpectedly, paired pulse plasticity, another routinely used indicator of presynaptic forms of synaptic plasticity, was unchanged in the LTD. We conclude that the LTD induces refractoriness of the neuromuscular vesicle release machinery downstream of presynaptic calcium entry.     

Changes in the asynchronism of transmitter release in a neuromuscular synapse and the time course of evoke postsynaptic signals during growth and branching og frog nerve ending

The influence of both growth and branching of a nerve terminal on the asynchronism of transmitter release and the time-course of evoked postsynaptic responses was investigated using a model of a frog neuromuscular synapse in which the nerve terminal represents a population of spatially isolated active zones. It was shown that the appearance of additional branching in proximal parts of the nerve ending leads to decrease in the asynchronism of transmitter release, an increase in quantum content and the amplitude of the postsynaptic signal, and the shortening of its phase of growth. It was found that the asynchronism of transmitter release has a much stronger influence on the time-course of end plate currents compared with end plate potentials. The factors strengthening and weakening the asynchronism of transmitter release in a neuromuscular synapse and the reasons for various length and branching of vertebrate nerve terminals are considered.