The formation and function of single transmitter release sites at mature amphibian motor-nerve terminals

The extent of quantal transmitter release from single sites of synaptic vesicle accumulations along the length of motor-nerve terminal branches at the amphibian neuromuscular junction has been investigated. Such a determination involves development of a model for the generation of quantal potential fields at single styryl-dye stained sites along the length of a branch. Successful testing and application of this model indicates that the extent of quantal release at a dye-stained site is proportional to the total length of active zone at the site.The stability of these sites and of their ensheathing terminal Schwann cell processes was also investigated. Following simultaneous injection of the terminal Schwann cell and nerve terminal with different fluorescent dyes, terminal branches were observed to show dynamic changes in their length, with these occurring in relatively short periods of hours or less. Redistribution of styryl dye stained sites at the ends of branches also occurred in such short periods of time. These were accompanied by changes in the configuration of terminal Schwann cells, which generally occurred prior to changes in the length of nerve terminal branches.     

Quantal secretion and nerve-terminal cable properties at neuromuscular junctions in an amphibian (Bufo marinus)

The effect of a conditioning depolarizing current pulse (80-200 micros) on quantal secretion evoked by a similar test pulse at another site was examined in visualized motor-nerve terminal branches of amphibian endplates (Bufo marinus). Tetrodotoxin (200 nM) and cadmium (50 microM) were used to block voltage-dependent sodium and calcium conductances. Quantal release at the test electrode was depressed at different distances (28-135 microm) from the conditioning electrode when the conditioning and test pulses were delivered simultaneously. This depression decreased when the interval between conditioning and test current pulses was increased, until, at an interval of approximately 0.25 ms, it was negligible. At no time during several thousand test-conditioning pairs, for electrodes at different distances apart (28-135 microm) on the same or contiguous terminal branches, did the electrotonic effects of quantal release at one electrode produce quantal release at the other. Analytic and numerical solutions were obtained for the distribution of transmembrane potential at different sites along terminal branches of different lengths for current injection at a point on a terminal branch wrapped in Schwann cell, in the absence of active membrane conductances. Solutions were also obtained for the combined effects of two sites of current injection separated by different time delays. This cable model shows that depolarizing current injections of a few hundred microseconds duration produce hyperpolarizations at approximately 30 microm beyond the site of current injection, with these becoming larger and occurring at shorter distances the shorter the terminal branch. Thus the effect of a conditioning depolarizing pulse at one site on a subsequent test pulse at another more than approximately 30 microm away is to substantially decrease the absolute depolarization produced by the latter, provided the interval between the pulses is less than a few hundred microseconds. It is concluded that the passive cable properties of motor nerve terminal branches are sufficient to explain the effects on quantal secretion by a test electrode depolarization of current injections from a spatially removed conditioning electrode.     

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.     

Schwann cell dynamics with respect to newly formed motor-nerve terminal branches on mature (Bufo marinus) muscle fibers

A study has been made of the formation of synaptic terminals from long processes formed at the end of motor nerve branches of endplates in mature amphibian (Bufo marinus) muscle. Injection of fluorescent dyes into individual motor axons showed the full extent of their branches at single endplates. Synaptic vesicle clusters at these branches were identified with styryl dyes. Some terminal branches consisted of well separated varicosities, each possessing a cluster of functioning synaptic vesicles whilst others formed by the same axon consisted of closely spaced clusters of vesicles in a branch of approximately uniform diameter. All the varicosities gave rise to calcium transients on stimulation of their parent axon. Both types of branches sometimes possessed short processes (<5 microm long) or very long thin processes (>10 microm long) which ended in a bulb that possessed a functional synaptic vesicle cluster. These thin processes could move and form a varicosity along their length in less than 30 min.Injection of a fluorescent dye into terminal Schwann cells (TSCs) at an endplate showed that they also possessed very long thin processes (>10 microm long) which could move over relatively short times (<30 min). Injecting fluorescent dyes into both axons and their associated TSCs showed that on some occasions long TSC processes were accompanied by a long nerve terminal process and at other times they were not. It is suggested that the mature motor-nerve terminal is a dynamic structure in which the formation of processes by TSCs guides nerve terminal sprouting.     

Postsynaptic production of nitric oxide implicated in long-term depression at the mature amphibian (Bufo marinus) neuromuscular junction

We report here evidence for endogenous NO signalling in long-term (> 1 h) synaptic depression at the neuromuscular junction induced by 20 min of 1 Hz nerve stimulation. Synaptic depression was characterized by a 46% reduction in the end-plate potential (EPP) amplitude and a 21% decrease in miniature EPP (MEPP) frequency, but no change to MEPP amplitude, indicating a reduction in evoked quantal release. Both the membrane-impermeant NO scavenger cPTIO and the NOS inhibitor⁺ release from the sarcoplasmic reticulum and muscle contraction were blocked with dantrolene. These data suggest that the depression depends on transmission, but not muscle contraction. The calcineurin inhibitors cyclosporin A and FK506, as well as ODQ, an inhibitor of NO-sensitive soluble guanylyl cyclase, Rp-8-pCPT-cGMPS, an inhibitor of cGMP-dependent protein kinase, and the calmodulin antagonist phenoxybenzamine also blocked depression. We propose that low frequency synaptic transmission leads to production of NO at the synapse and depression of transmitter release via a cGMP-dependent mechanism. The NO could be generated either directly from the muscle, or possibly from the Schwann cell in response to an unidentified muscle-derived messenger. We showed that the long-lasting depression of transmitter release was due to sustained activity of the NO signalling pathway, and suggest dephosphorylation of NOS by calcineurin as the basis for continued NO production.     

Vesicle-associated proteins and P2X receptor clusters at single sympathetic varicosities in mouse vas deferens

1. Antibodies against vesicle-associated proteins of the SNARE complex (syntaxin (AbS), SNAP 25 (AbS25), synaptobrevin (VAMP; AbV) and the 1B subunit of calcium channels (Ab_1B) were located with respect to sympathetic varicosities (labelled with the ubiquitous vesicle proteoglycan antibody AbSV2) and to clusters of P2X receptor subunits (labelled with antibodies AbP2X₁ to AbP2X₆). In addition, these receptor clusters were located with respect to Schwann cells labelled with antibodies to S100 (AbS100).2. The spatial relation between proteins of the SNARE complex and calcium channels was determined. AbS25 patches ranged from 250–500 nm in size and were often colocalised with smaller AbS patches (250–350 nm). Ab_1B patches (300–700 nm diameter) were always coincidental with AbS patches. AbV patches (400–1000 nm in diameter) also coincided with AbS patches.3. The spatial relation between different P2X subunit clusters and varicosities labelled with AbSV2 was ascertained. Large (500–700 nm diameter) AbP2X₁ receptor clusters were found colocalised with many (91%) AbSV2 labelled varicosities, although small diameter (250–350 nm) AbP2X₁ clusters occurred at random over the muscle. Small AbP2X₂ clusters were found uniquely in the vicinity of AbSV2 labelled varicosities, but were not entirely coincidental with these. Small AbP2X₃ receptor clusters were found in close association with AbSV2 labelled nerves. Small diameter AbP2X₄ clusters (250–350 nm) were found throughout the muscle with some of these coincidental with AbSV2 labelling. Small diameter AbP2X₅ (250–350 nm) cluster labelling was found in juxtaposition to strings of AbSV2 labelled varicosities but were not coincidental with these. Small (250–350 nm) diameter AbP2X₆ clusters were also found in close juxtaposition to AbSV2 labelled nerves.4. The spatial relation between different P2X subtype clusters and Schwann cells labelled with AbS100 was examined. Both AbP2X₁ and AbP2X₃ receptor clusters were found in close apposition with AbS100, with clusters of the former sometimes coincidental with patches of the latter. On the other hand AbP2X₂ was found in association with AbS100 at low levels while AbP2X₄ labelling was generally not coincidental with AbS100. AbP2X₅ and AbP2X₆ labelling was often colocalised with AbS100 labelling.5. The spatial relation between proteins of the SNARE complex and P2X₁ receptors was determined. Large AbP2X₁ clusters were often found apposed by AbS, AbV and Ab_1B labelled patches.6. Destruction of the sympathetic varicosities with 6-hydroxydopamine led to the virtual disappearance of AbP2X₂ labelling, but to a large increase in the number of small AbP2X₁ receptor clusters and a reduction in the number of large AbP2X₁ clusters. AbS100 Schwann cell labelling was largely unaffected.7. These observations are interpreted as showing that most terminal sympathetic varicosities possess active zones about 250–700 nm diameter, delineated by syntaxin, SNAP 25 and N-type calcium channels and that synaptic vesicles are concentrated at these sites as indicated by the localisation of VAMP. Most of these terminal varicosities possess active zones that are precisely apposed to large clusters of P2X₁ receptors. However small clusters of P2X₂ to P2X₆ receptors can be found that are near the strings of varicosities but not usually coincidental with them except P2X₃. The functional significance of this arrangement of vesicle-associated proteins and P2X receptors for the generation of synaptic potentials at the autonomic neuromuscular junction is discussed.     

Functional morphology of lingual protrusion in marine toads (Bufo marinus)

Bufo marinus catches its prey by stiffening the intrinsic muscles of the tongue, rapidly flipping the tongue out of the mouth. High-speed cinematography synchronized with computer-analyzed electromyograms (EMGs) shows that during the flip the tongue is supported by the M. genioglossus medialis and that this muscle stiffens into a rod when stimulated. Coincident stiffening of the transversely arranged M. genioglossus basalis provides a wedge under the anterior tip of this rod. Stiffening of the M. submentalis depresses the mandibular symphysis and brings the dentary tips together. The M. submentalis also acts on the wedge of the basalis to raise and rotate the rigid rod of the medialis over the symphysial attachment. The tip of this lingual rod carries along the pad and soft tissues of the tongue. The lingual pad, positioned in the posterodorsal portion of the resting tongue, rotates during eversion so that its dorsal surface impacts onto the prey object. Retraction starts by contraction of the elongate, parallel fibers of the M. hyoglossus; this retracts the medial sulcus of the pad and holds the prey by a suction cup-like effect. The extensibility of the buccal membranes allows the pad to be retracted first; it reaches the posterior portion of the buccal cavity before the still-rigid, backward rotating M. genioglossus has reached the level of the symphysis. Protraction of the hyoid facilitates the extension of the M. hyoglossus. The M. sternohyoideus only retracts the hyoid and stabilizes it when the tongue starts to pull posteriorly; it does not assist tongue protrusion. The Mm. petrohyoideus and omohyoideus show only incidental activity, and the M. depressor mandibulae participates in mouth opening but is not otherwise involved in the flip. Previous hypotheses of the flipping mechanism are reviewed and evaluated.     

Z and W sex chromosomes in the cane toad (Bufo marinus)

The cane toad (Bufo marinus) is one of the most notorious animal pests encountered in Australia. Members of the genus Bufo historically have been regarded as having genotypic sex determination with male homogamety/female heterogamety. Nevertheless, as with many toads, karyotypic analyses of the cane toad have so far failed to identify heteromorphics sex chromosomes. In this study, we used comparative genomic hybridization, reverse fluorescence staining, C-banding, and morphometric analyses of chromosomes to characterize sex chromosome dimorphism in B. marinus. We found that females consistently had a length dimorphism associated with a nucleolus organizer region (NOR) on one of the chromosome 7 pair. A strong signal over the longer NOR in females, and the absence of a signal in males indicated sex-specific DNA sequences. All females were heterozygous and all males homozygous, indicating a ZZ/ZW sex chromosomal system. Our study confirms the existence of sex chromosomes in this species. The ability to reliably identify genotypic sex of cane toads will be of value in monitoring and control efforts in Australia and abroad.     

Occurrence and experimental infection of toads (Bufo marinus and B. granulosus) with Mycobacterium chelonei subsp. abscessus

In a survey of 234 Amazonian toads and frogs, six strains of Mycobacterium chelonei subsp. abscessus were isolated from the liver or spleen of four of 66 Bufo marinus (6.1%) and from the kidney or peritoneal fluid of two of 86 B. granulosus (2.3%). There were no histopathological lesions in the viscera of the infected animals. Experimental infection of 29 captive B. marinus and B. granulosus, by the intraperitoneal route, with a pooled inoculum of M. chelonei subsp. abscessus caused five deaths near the end of a 2-month observation period. M. chelonei subsp. abscessus was isolated from the liver, spleen, kidney, gonad, heart and lung of toads killed at various intervals after inoculation, and intracellular acid-fast bacilli were seen in these organs. Histological evidence of invasion of tissues by mycobacteria became apparent from the 45th day after infection. The susceptibility to infection of B. marinus and B. granulosus suggests that these toads may serve as a fortuitous animal host for M. chelonei subsp. abscessus.     

Thermal regulation of the immune response in South American toads (Bufo marinus)

Thermal regulation of the immune response was studied in toads following single injections of bacteriophage f2. The immune response was markedly inhibited in animals kept at 15° as compared to the controls (25°). The appearance of serum antibodies was delayed in animals kept at 15° for the first post-immunization week but their peak antibody levels were similar to those in toads maintained at 25° throughout. Transfer of animals from 25° to 15° 2 weeks after immunization only temporarily depressed the serum antibody levels but caused a marked delay in conversion from heavy to light antibodies.

Our results are in keeping with the hypothesis that lowered environmental temperatures inhibit a variety of metabolic processes which may be concerned with the utilization of antigen and/or the synthesis of antibodies.

     

Strontium and quantal release of transmitter at the neuromuscular junction

1. Previous work has shown that in calcium-free solutions nerve impulses invade the motor nerve terminals at the neuromuscular junction, but fail to release transmitter. In these conditions, strontium ions applied iontophoretically to a minute part of a junction, or to the whole muscle by bath application, restore to the nerve impulse its ability to release transmitter.2. As with calcium, the transmitter released in the presence of strontium is in the form of packages (quanta) whose release can be predicted from Poisson's Theorem.3. The mean number of quanta released by a nerve impulse increases with the concentration of strontium. Strontium is much less effective than calcium in equimolar concentrations.4. Transmitter quanta released in the presence of strontium evoke larger unit potentials than quanta released in the presence of calcium. The larger size of the Sr-unit potentials is caused by a prolongation of transmitter action, presumably due to a post-synaptic effect of strontium.5. Neuromuscular transmission was blocked in some fibres when the concentration of strontium was raised beyond 10 mM. This junctional block was presumably due to a failure in the propagation of nerve impulses.6. The post-stimulation increase in the frequency of miniature end-plate potentials, which is normally seen in calcium solutions, is also observed when calcium is substituted by strontium. The post-stimulation effect increases with the concentration of strontium.7. It is concluded that strontium can substitute for calcium in the process of quantal release of transmitter. The physico-chemical mechanism of this substitution remains unknown.     

Effects of lectins on quantal release at the frog neuromuscular junction.

This work was initiated because pretreatment with concanavalin A was reported to abolish the increase in spontaneous quantal release produced by hypertonic solutions [Gorio A. and Mauro A. (1979) J. gen. Physiol. 73, 245-263]. This suggested that lectins might be valuable tools for investigating the role of glycoproteins in the response to tonicity. We compared muscles soaked for 2 h in hypertonic solution containing concanavalin A with paired muscles soaked in hypertonic solution without lectin. The lectin treatment decreased miniature end-plate potential frequencies in Ringer and in hypertonic solutions compared with the controls. Even after lectin treatment hypertonic solutions and elevated K+ solutions increased miniature end-plate potential frequencies, and the proportional increases were the same as in controls. The lectin treatment lowered baseline frequency, but the preparation still responded to hypertonic solutions. Concanavalin A effects appeared after treatment for more than 1 h and required concentrations of 10 micrograms/ml or higher. Higher concentrations did not produce more effect. Similar results were obtained with four other lectins with different sugar specificities. Treatment in hypertonic solution without lectin produces a similar, but smaller, decrease in baseline frequency. Concanavalin A pretreatment had no detectable effects on evoked release or facilitation. We conclude that the effects of lectins on quantal release are not mediated by binding to a single sugar group. The lectins do not produce a unique effect; they exaggerate the changes produced by hypertonic pretreatment. All of the effects could be accounted for by a reduction in baseline [Ca2+] in the nerve terminal. Such reductions are produced by lectins in many cell types.     

Effect of lithium on veratridine-induced quantal and non-quantal release from inhibitory nerve terminals in crayfish muscle

Muscle fibres of small crayfish were voltage clamped and superfused for about 10 min with Li⁺ saline (Na⁺ replaced by Li⁺) which contained 5 mmol/l glutamate to desensitize excitatory postsynaptic receptors. Then 100 mol/l veratridine were added to the superfusate which caused strong asynchronous quantal release of inhibitory transmitter. However, in the presence of Li⁺ strong inhibitory quantal release was only transient. It could be activated a second time by removal of Li⁺ and readministration of Na⁺. From the total of 0.7 to 1.1 million quanta released by veratridine only about 30–35% could be released in Li⁺ saline. The voltage clamp DC-currents recorded during veratridine-induced quantal release suggested that a nonquantal release component is additionally involved. This non-quantal release component was most prominent during the period of quantal release in Li⁺ superfusate while it was less obvious during the second enhancement of quantal release in normal saline. Together with previous results (Martin and Finger 1988) it may be concluded that quantal release, but not non-quantal release, is decreased by Li⁺ in the nerve terminals.This investigation was supported by the Deutsche Forschungsgemeinschaft, SFB 220     

Inositol trisphosphate and cyclic adenosine diphosphate-ribose increase quantal transmitter release at frog motor nerve terminals: possible involvement of smooth endoplasmic reticulum

The release of chemical transmitter from nerve terminals is critically dependent on a transient increase in intracellular Ca2+. The increase in Ca2+ may be due to influx of Ca2+ from the extracellular fluid or release of Ca2+ from intracellular stores such as mitochondria. Whether Ca2+ utilized in transmitter release is liberated from organelles other than mitochondria is uncertain. Smooth endoplasmic reticulum is known to release Ca2+, e.g., on activation by inositol trisphosphate or cyclic adenosine diphosphate-ribose, so the possibility exists that Ca2+ from this source may be involved in the events leading to exocytosis. We examined this hypothesis by testing whether inositol trisphosphate and cyclic adenosine diphosphate-ribose modified transmitter release. We used liposomes to deliver these agents into the cytoplasmic compartment and binomial analysis to determine their effects on the quantal components of transmitter release. Administration of inositol trisphosphate (10(-4)M) caused a rapid, 25% increase in the number of quanta released. This was due to an increase in the number of functional release sites, as the other quantal parameters were unaffected. The effect was reversed with 40 min of wash. Virtually identical results were obtained with cyclic adenosine diphosphate-ribose (10(-4)M). Inositol trisphosphate caused a 10% increase in quantal size, whereas cyclic adenosine diphosphate-ribose had no effect. The results suggest that quantal transmitter release can be increased by Ca2+ released from smooth endoplasmic reticulum upon stimulation by inositol trisphosphate or cyclic adenosine diphosphate-ribose. This may involve priming of synaptic vesicles at the release sites or mobilization of vesicles to the active zone. Inositol trisphosphate may have an additional action to increase the content of transmitter within the vesicles. These findings raise the possibility of a role of endogenous inositol phosphate and smooth endoplasmic reticulum in the regulation of cytoplasmic Ca2+ and transmitter release.     

Inositol trisphosphate and cyclic adenosine diphosphate-ribose increase quantal transmitter release at frog motor nerve terminals: possible involvement of smooth endoplasmic reticulum

The release of chemical transmitter from nerve terminals is critically dependent on a transient increase in intracellular Ca²⁺.6., 25. The increase in Ca²⁺ may be due to influx of Ca²⁺ from the extracellular fluid¹⁵ or release of Ca²⁺ from intracellular stores such as mitochondria.1., 8., 18. Whether Ca²⁺ utilized in transmitter release is liberated from organelles other than mitochondria is uncertain. Smooth endoplasmic reticulum is known to release Ca²⁺, e.g., on activation by inositol trisphosphate or cyclic adenosine diphosphate-ribose,² so the possibility exists that Ca²⁺ from this source may be involved in the events leading to exocytosis. We examined this hypothesis by testing whether inositol trisphosphate and cyclic adenosine diphosphate-ribose modified transmitter release. We used liposomes to deliver these agents into the cytoplasmic compartment and binomial analysis to determine their effects on the quantal components of transmitter release. Administration of inositol trisphosphate (10⁻⁴ M) caused a rapid, 25% increase in the number of quanta released. This was due to an increase in the number of functional release sites, as the other quantal parameters were unaffected. The effect was reversed with 40 min of wash. Virtually identical results were obtained with cyclic adenosine diphosphate-ribose (10⁻⁴ M). Inositol trisphosphate caused a 10% increase in quantal size, whereas cyclic adenosine diphosphate-ribose had no effect. The results suggest that quantal transmitter release can be increased by Ca²⁺ released from smooth endoplasmic reticulum upon stimulation by inositol trisphosphate or cyclic adenosine diphosphate-ribose. This may involve priming of synaptic vesicles at the release sites or mobilization of vesicles to the active zone. Inositol trisphosphate may have an additional action to increase the content of transmitter within the vesicles. These findings raise the possibility of a role of endogenous inositol phosphate and smooth endoplasmic reticulum in the regulation of cytoplasmic Ca²⁺ and transmitter release.