Differential Responses of Crab Neuromuscular Synapses to Cesium Ion

Excitatory postsynaptic potentials (EPSP's) generated in crab muscle fibers by a single motor axon, differ in amplitude and facilitation. Some EPSP's are large at low frequencies of stimulation and show little facilitation; others are smaller and show pronounced facilitation. When K⁺ is replaced by Cs⁺ in the physiological solution, all EPSP's increase in amplitude, but small EPSP's increase proportionately more than large ones. Quantal content of transmission, determined by external recording at single synaptic regions, undergoes a much larger increase at facilitating synapses. The increase in quantal content of transmission is attributable to prolongation of the nerve terminal action potential in Cs⁺. After 1–2 h of Cs⁺ treatment, defacilitation of synaptic potentials occurs at synapses which initially showed facilitation. This indicates that Cs⁺ treatment drastically increases the fraction of the "immediately available" transmitter store released by each nerve impulse, especially at terminals with facilitating synapses. It is proposed that facilitating synapses normally release less of the "immediately available" store of transmitter than poorly facilitating synapses. Possible reasons for this difference in performance are discussed.     

Comparison of fast and slow synaptic terminals in lobster muscle

Summary Synaptic terminals of fast (FCE) and slow (SCE) excitatory neurons were physiologically identified on separate fibres of one muscle, the closer muscle in lobster claws. The innervation by these identified fibers was demonstrated over long distances (7–21 m) by examining serial thin sections at periodic intervals. The ultrastructure of each type of innervation was consistent both qualitatively and quantitatively in two separate samples. The FCE innervation is relatively simple in having consistently small-diameter terminals each forming a single long synapse, with few synaptic vesicles, and little if any postsynaptic apparatus. The SCE innervation is more complex in having larger-diameter but more variable terminals forming several short synapses, with many synaptic vesicles and an extensive postsynaptic apparatus. These differences in the size of the synapses and the number of synaptic vesicles parallel differences in transmitter release and fatigue sensitivity characteristic of the two types of innervation. The degree of elaboration of the postsynaptic apparatus may reflect differences in the amount of transmitter taken up after release. Our data reveal for the first time in a single muscle differences between FCE and SCE innervation previously reported in different muscles and in different species.Supported by grants from NIH (NINCDS) to A.G. Humes and the late Fred Lang and from NSERC and Muscular Dystrophy Assoc. of Canada to C.K. GovindWe thank Lena Hill for her technical expertise and critical evaluation of the study, and Dr. A.G. Humes for providing research facilities     

New Step in Transmitter Release at the Myoneural Junction

QUANTAL release of acetylcholine from vesicles in the presynaptic terminals of neuromuscular synapses is well established^1–3, even if some doubts persist⁴. The mechanism by which acetylcholine (or any other transmitter at other synapses) is transferred from the vesicles into the synaptic gap, however, is unknown. A calcium influx into the terminal is associated with release of transmitter⁵, as is an electrical field change⁶.     

Ultrastructure of synapses with different transmitter-releasing characteristics on motor axon terminals of a crab,Hyas areneas

Synaptic terminals on branches of an excitatory motor axon in a spider crab (Hyas areneas) were examined by electron microscopy to determine whether differences in size, structure, and number of synapses could be correlated with differences in transmitter release. Terminals releasing relatively large amounts of transmitter during low frequencies of nerve impulses ("high-output" terminals) had larger synapses, more prominent presynaptic dense bodies (active zones), and fewer synapses per unit length than terminals releasing relatively small amounts of transmitter ("low-output" terminals). Neither the difference in synaptic area, nor the quantitative differences in the active zones, were sufficient in themselves to explain the difference in synaptic efficacy, and it is postulated that a non-linear relationship may exist between structural features of the synapse and release of transmitter by a nerve impulse, and that differences other than those apparent from the ultrastructure could be involved. Greater facilitation at low-output terminals with high frequencies of nerve impulses may be due to greater reserves of "immediately available" transmitter, and to recruitment or activation of more individual synaptic contacts.     

Glutamate uptake by a stimulated insect nerve muscle preparation

Recent reports suggest that glutamate may be the excitatory neuromuscular transmitter in insects. In this study, glutamate uptake by isolated cockroach nerve muscle preparations was investigated by means of chemical and electron microscope radioautographic techniques. We found that the preparation had a high affinity for glutamate and that nerve stimulation enhanced glutamate uptake. Chemical studies showed that the average tissue concentration of glutamate bound during a 1 hr incubation period in 10^-5 M glutamate-³H after nerve stimulation was 2.8 x 10^-5 M. Less than 1% of the radioactivity was present in the perchloric acid-precipitated protein fraction. Using electron microscope radioautography, we observed that sheath cells showed the highest glutamate concentration of all cellular compartments. Uptake was greater at neuromuscular junctions than in other regions of the tissue. The data suggest a possible mechanism for transmitter inactivation and protection of synapses from high blood glutamate.     

Quantal Mechanism of Transmitter Release during Progressive Depletion of the Presynaptic Stores at a Ganglionic Synapse : The action of hemicholinium-3 and thiamine deprivation

In the present experiments we interfered with the mechanism of acetylcholine (ACh) synthesis in the rat superior cervical ganglion by impairing the supply of either the choline group (hemicholinium no. 3 [HC-3]treatment) or the acetyl group (thiamine deprivation). Under both conditions stimulation causes in the ganglion a progressive decline in ACh output associated with a depletion of transmitter tissue content. ACh release from the terminals of a single preganglionic fiber was estimated from the quantum content value of the evoked excitatory postsynaptic potentials (EPSP's) recorded intracellularly in the ganglion neuron under test. The present observations indicate that Poisson statistics describe transmitter release at either low or high release levels. Furthermore, the progressive decline in the rate of ACh output occurring during repetitive stimulation is shown to correspond to a progressive decrease in the number of transmitter quanta released per impulse and not to any modification in the size of individual quanta. Some 8,000 transmitter quanta proved to represent the presynaptic transmitter store initially present in those terminals on a neuron that are activated by stimulation of a single preganglionic fiber. Speculations are considered about synaptic efficacy and nerve connections in rat autonomic ganglia. It is suggested that six preganglionic fibers represent the mean input to a ganglion neuron.     

Structural-functional differences of a crab motoneuron to four stomach muscles

A motor unit in the stomach of the blue crab, Callinectes sapidus, consists of four separate muscles involved in different aspects of the trituration and filtering of food. Motor nerve terminals to two of the muscles (CPV7a and GM5) release small amounts of transmitter (low-output) while those to the other two muscles (CV2 and CV3) release between three and five-fold greater amounts (high-output). Structural features underlying the disparity in synaptic strength were analysed with thin serial-section electron microscopy. Nerve terminals were similar in their volume percent of mitochondria, clear vesicles and dense core vesicles among the four muscles. This was also the case for the number and size of synaptic contacts. However, presynaptic dense bars representing active zones were longer and occurred more frequently at high-output synapses than at low-output ones. High-output synapses were also characterized by the close spacing of adjacent dense bars. The longer and more closely spaced dense bars at high-output synapses would be factors in the generation of larger synaptic potentials in these terminals compared to their low-output counterparts. Other factors, however, need to be considered to fully account for the physiological differences in synaptic strength among the four muscles.     

Effects of high calcium solutions on glutamate sensitivity of crayfish muscle fibres

Crayfish neuromuscular preparations were studied after 18--36 h exposure to high calcium solutions. As previously reported for frog neuromuscular preparations the treatment damaged the nerve terminals and decreased junctional potentials. The resting potentials and input resistances of the muscle fibres were not affected; but their sensitivity to glutamate was significantly decreased when compared to that of control muscles. After exposure to high calcium, the sensitivity to gamma-aminobutyric acid, the putative transmitter at inhibitory synapses, was increased. Apparently normal twitches were elicited by direct stimulation, and calcium spikes could still be observed in the fibres. A decreased sensitivity to glutamate was also noted in experiments carried out on denervated muscles 8 months after section of the motor axons. Possible relations between nerve terminal damage and the decrease in sensitivity to glutamate are discussed.     

Glutamate,acetylcholine, and γ-aminobutyric acid as transmitters in the pyloric system of the stomatogastric ganglion of a stomatopod,Squilla oratoria

The neurotransmitters mediating the synaptic interactions in the pyloric system of the stomatogastric ganglion of a stomatopod, Squilla oratoria, were examined. Putative transmitters were applied iontophoretically to the pyloric cells. Glutamate and GABA produced inhibitory responses in all motoneurons but acetylcholine did not. These inhibitory responses were due to increases in conductance to either K⁺ or Cl^– or both, and blocked by picrotoxin. The inhibitory postsynaptic potentials evoked by the constrictor and dilator neurons were different in their time courses, reversal potentials, ion selectivities, and picrotoxin sensitivities. Glutamate is a transmitter candidate for inhibitory synapses made among the pyloric cells as well as for their neuromuscular junctions. In some cells, glutamate and acetylcholine evoked excitatory responses which were blocked by joro spider toxin and by tubocurare, respectively. They mediated the extrinsic inputs to modulate the pyloric rhythm. The transmitter, glutamate, is conserved in the ganglion neurons between stomatopods and decapods during evolution. Use of two transmitters, glutamate and acetylcholine, may have evolved in decapods, while the ionic mechanism is preserved in both orders. The neuromodulators, acetylcholine and -aminobutyric acid, are conserved between both orders. Glutamate may be used as the neuromodulator in stomatopods.Abbreviations ACh acetylcholine - EPSP excitatory postsynaptic potential - GABA -aminobutyric acid - Glu glutamate - IC inferior cardiac - IPSP inhibitory postsynaptic potential - JSTX joro spider toxin - LP lateral pyloric - pcp posterior cardiac plate - PTX picrotoxin     

Neurotransmitters of motor neurons in the stomatogastric ganglion of an isopod crustacean, Ligia exotica

Neurotransmitters of motor neurons in the foregut muscles of an isopod Ligia exotica were identified by recording changes in membrane potential to exogenously applied glutamate and acetylcholine. The effects of antagonists, tubocurare and joro spider toxin, on excitatory junctional potentials evoked by nerve stimulation and by iontophoretic application of glutamate and acetylcholine provided additional evidence for identification. The junctional receptors were desensitized by putative neurotransmitters. Glutamate is a candidate as an excitatory neurotransmitter at the neuromuscular junctions in intrinsic muscles of the gastric mill and pylorus, and acetylcholine is a candidate in the extrinsic muscles of the gastric mill and cardiopyloric valve.     

Differential effects of 4-aminopyridine on acetylcholine release triggered by K+ depolarization,veratridine, or A23187 in rat cerebral cortical synaptosomes

The effect of 4-aminopyridine on [³H]acetylcholine release was studied in rat cerebral cortical synaptosomes in the presence of a several secretagogues that have different mechanisms of action. As found previously, 4-aminopyridine increased [³H]acetylcholine release in a concentration-dependent manner (5–10 mM); a high concentration (10 mM) also elevated [³H]choline efflux. However, the 35 mM K⁺ induced release of [³H]acetylcholine was attenuated by 4-aminopyridine at concentrations (less than 5 mM) that had no effect on transmitter release. At no concentration of 4-aminopyridine was the release of transmitter additive with 35 mM K⁺ induced release. Veratridine-induced release was neither attenuated nor additive with low concentrations of 4-aminopyridine, even when a sub-maximal concentration of the sodium ionophore was used (10 M). In contrast, A23187-induced release was additive with that caused by 4-aminopyridine. These results suggest that: 1) 4-aminopyridine blocks potassium channels involved in regulating membrane potential in isolated cholinergic terminals; and 2) changes in the activity of these 4-aminopyridine sensitive K⁺ channels are not important in the nerve terminal's response to depolarization caused by sodium influx.     

Parameters not influenced by vesamicol: Membrane potential,calcium uptake,and internal calcium concentration of synaptosomes

In our previous study vesamicol, an inhibitor of the acetylcholine transporter of the cholinergic vesicles, inhibited veratridine-evoked external Ca²⁺-dependent acetylcholine release from striatal slices but did not influence acetylcholine release observed in Ca²⁺-free medium (4). Here we examined if the effect of veratridine on membrane potential, Ca²⁺ uptake, and intracellular Ca²⁺ concentration of synaptosomes was altered by vesamicol in parallel with the inhibition of acetylcholine release. The depolarizing effect of 10 M veratridine (from 67±2.3 mV resting membrane potential to 50.7±2.5 mV) was not significantly influenced by vesamicol (1–20 M). Vesamicol (1–20 M) had no effect on either the overall curve of the veratridine-evoked⁴⁵Ca²⁺ uptake or the amount of Ca²⁺ taken up by synaptosomes. Veratridine caused a rise in intrasynaptosomal Ca²⁺ concentration as measured by Fura2 fluorescence, and the same increase both in characteristics and in magnitude was observed in the presence of vesamicol (20 M). The K⁺-evoked (40 mM) increase of Ca²⁺ uptake and of intracellular calcium concentration were also unaltered by vesamicol. In high concentration (50 M) vesamicol inhibited both the fall in membrane potential and the elevated Ca²⁺ uptake by veratridine, indicating a possible nonspecific effect on potential-dependent Na⁺ channels at this concentration. Vesamicol, in lower concentration (20 M) when neither of the above parameters was changed, completely prevented veratridine-evoked increase of [¹⁴C]acetylcholine release. This was observed only when vesamicol was present in the media throughout the experiment after loading the preparation with [¹⁴C]choline. The results suggest that vesamicol does not interfere with veratridine-induced changes in isolated nerve terminals other than with the release of acetylcholine, thus further supporting the involvement of a vesamicol-sensitive vesicular transmitter pool in Ca²⁺-dependent veratridine-elicited acetylcholine release.     

High-level inhibition of mitochondrial complexes III and IV is required to increase glutamate release from the nerve terminal

Background

The activities of mitochondrial complex III (ubiquinol-cytochrome c reductase, EC 1.10.2.2) and complex IV (cytochrome c oxidase EC 1.9.3.1) are reduced by 30-70% in Huntington's disease and Alzheimer's disease, respectively, and are associated with excitotoxic cell death in these disorders. In this study, we investigated the control that complexes III and complex IV exert on glutamate release from the isolated nerve terminal.

Results

Inhibition of complex III activity by 60-90% was necessary for a major increase in the rate of Ca²⁺-independent glutamate release to occur from isolated nerve terminals (synaptosomes) depolarized with 4-aminopyridine or KCl. Similarly, an 85-90% inhibition of complex IV activity was required before a major increase in the rate of Ca²⁺-independent glutamate release from depolarized synaptosomes was observed. Inhibition of complex III and IV activities by ~ 60% and above was required before rates of glutamate efflux from polarized synaptosomes were increased.

Conclusions

These results suggest that nerve terminal mitochondria possess high reserves of complex III and IV activity and that high inhibition thresholds must be reached before excess glutamate is released from the nerve terminal. The implications of the results in the context of the relationship between electron transport chain enzyme deficiencies and excitotoxicity in neurodegenerative disorders are discussed.     

Innervation and vascular supply of the crayfish opener muscle: Scanning and transmission electron microscopy

Summary Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were employed to study the innervation and vascular supply of crayfish skeletal muscle.Blood vessels and nerve terminals identified by TEM were often closely associated. Synaptic regions of the nerve terminals were always located under sarcolemma and contained both dense-cored and agranular synaptic vesicles. Axo-axonal synapses of several different types were observed. Blood vessels consisted of several vessel cells or supporting cells enclosing a lumen, which was connected to the exterior by fine channels between the supporting cells.SEM of whole freeze-dried muscles revealed two types of ramifying structure, which often ran in parallel over the muscle surface. One, identified as nerve, was more cylindrical and had a smoother surface than the other, which was identified as blood vessel. Fine nerve branches disappeared under the sarcolemma, probably near synaptic regions, but synapses could not be seen. Blood vessels also had fine terminations which merged into the sarcolemma.Supported by grants from the National Research Council of Canada and The Muscular Dystrophy Association of Canada. The technical assistance of Mr. M. Uy is acknowledged. Dr. F. Lang held a Postdoctoral Fellowship from the Muscular Dystrophy Association of Canada. Acknowledgement is made for the use of the scanning electron microscope in the Royal Ontario Museum, established through a grant from N.R.C. to the Department of Zoology, University of Toronto for the development of a program in systematic and evolutionary Zoology.     

Bilirubin Inhibits Ca2+-Dependent Release of Norepinephrine from Permeabilized Nerve Terminals

Although the well-known neurotoxic agent bilirubin can induce alterations in neuronal signaling, direct effects on neurotransmitter release have been difficult to demonstrate. In the present study we have used permeabilized nerve terminals (synaptosomes) from rat brain prelabeled with [³H]norepinephrine to examine the effects of bilirubin on transmitter release. Rat cerebrocortical synaptosomes were permeabilized with streptolysin-O (2 U/ml) in the absence or presence of bilirubin (10 M–320 M) and Ca²⁺ (100 M), and the amount of radiolabeled transmitter released during 5 min to the medium was analysed. Low levels of bilirubin decreased Ca²⁺-evoked release in a dose-dependent manner, with half-maximal effect at approx 25 M bilirubin. Higher levels of bilirubin (100–320 M) increased [³H]norepinephrine efflux in the absence of Ca²⁺, suggesting that high bilirubin levels induced leakage of transmitter from vesicles. The nontoxic precursor biliverdin had no effect on Ca²⁺-dependent exocytosis. Our data indicate that bilirubin directly inhibits both exocytotic release and vesicular storage of brain catecholamines.     

THREE-DIMENSIONAL ULTRASTRUCTURE OF THE CRAYFISH NEUROMUSCULAR APPARATUS

The synapse-bearing nerve terminals of the opener muscle of the crayfish Procambarus were reconstructed using electron micrographs of regions which had been serially sectioned. The branching patterns of the terminals of excitatory and inhibitory axons and the locations and sizes of neuromuscular and axo-axonal synapses were studied. Excitatory and inhibitory synapses could be distinguished not only on the basis of differences in synaptic vesicles, but also by a difference in density of pre- and postsynaptic membranes. Synapses of both axons usually had one or more sharply localized presynaptic "dense bodies" around which synaptic vesicles appeared to cluster. Some synapses did not have the dense bodies. These structures may be involved in the physiological activity of the synapse. Excitatory axon terminals had more synapses, and a larger percentage of terminal surface area devoted to synaptic contacts, than inhibitory axon terminals. However, the largest synapses of the inhibitory axon exceeded in surface area those of the excitatory axon. Both axons had many side branches coming from the main terminal; often, the side branches were joined to the main terminal by narrow necks. A greater percentage of surface area was devoted to synapses in side branches than in the main terminal. Only a small fraction of total surface area was devoted to axo-axonal synapses, but these were often located at narrow necks or constrictions of the excitatory axon. This arrangement would result in effective blockage of spike invasion of regions of the terminal distal to the synapse, and would allow relatively few synapses to exert a powerful effect on transmitter release from the excitatory axon. A hypothesis to account for the development of the neuromuscular apparatus is presented, in which it is suggested that production of new synapses is more important than enlargement of old ones as a mechanism for allowing the axon to adjust transmitter output to the functional needs of the muscle.     

Guanylyl Cyclase Participates in the Mechanism of Glutamatergic Modulation of Acetylcholine Non-Quantum Release in the Rat Neuromuscular Junction

It was shown that glutamate (10 M to 1 M) suppresses in a dose-dependent manner the non-quantum release of acetylcholine from rat motor nerve endings; the release intensity was estimated by the H effect. The action of glutamate was completely eliminated by the blockade of guanylyl cyclase by 1 M ODQ. An increase in the intracellular cGMP concentration by 1 M dibutyryl-cGMP reduced the H effect in a similar manner as glutamate did.     

Dipalmitoylphosphatidylcholine liposomes inhibit calcium-dependent [3H]acetylcholine release

We examined the effects of small unilamellar vesicles composed of dipalmitoylphosphatidylcholine on rat cerebral cortical [³H]acetylcholine release. Synaptosomes from this region were loaded with the labeled transmitter, and then incubated with the lipid (0–6 mg/ml) for specified intervals before adding various secretagogues. Liposomes (0.4 mg/ml–6 mg/ml) inhibited the calcium-dependent release of [³H]acetylcholine induced by 50 mM K⁺, A23187 (1–5 g/ml) or 500 M ouabain; the calcium-independent release induced by ouabain was not affected by the highest liposome concentration studied (6 mg/ml). [³H]Acetylcholine levels were also reduced by the liposomes, but higher concentrations were necessary to do so than to reduce K⁺-induced release. These reductions occurred in the S₃ (cytosol) but not P₃ (microsomal) subcellular fraction of the nerve terminals. The 50 mM K⁺-induced induced release of [³H]norepinephrine and [³H]dopamine from cerebral cortical and striatal synaptosomes, respectively, were not affected by 6 mg/ml lipid. Together, these results suggest that the dipalmitoylphosphatidylcholine liposomes may modulate cholinergic transmission presynaptically at the level of the calcium-dependent transmitter-release process.     

Synaptic diversity and differentiation: Crustacean neuromuscular junctions

Crustacean motor neurons exhibit a wide range of synaptic responses. Tonically active neurons generally produce small excitatory postsynaptic potentials (EPSPs) at low impulse frequencies, and are able to release much more transmitter as the impulse frequency increases. Phasic neurons typically generate large EPSPs in their target cells, but have less capability for frequency facilitation, and undergo synaptic depression during maintained activity. These differences depend in part upon the neuron's ongoing levels of activity; phasic neurons acquire physiological and morphological features of tonic neurons when their activity level is altered. Molecules responsible for adaptation to activity can be sought in single identified phasic neurons with current techniques. The fact that both phasic and tonic neurons innervate the same target muscle fibers is evidence for presynaptic determination of synaptic properties, but there is also evidence for postsynaptic determination of specific properties of different endings of a single neuron. The occurrence of high- and low-output endings of the same tonic motor neurons on different muscle fibers suggests a target-specific influence on synaptic properties. Structural variation of synapses on individual terminal varicosities leads to the hypothesis that individual synapses have different probabilities for release of transmitter. We hypothesize that structurally complex synapses have a higher probability for release than the less complex synapses. This provides an explanation for the larger quantal contents of high-output terminals (where the proportion of complex synapses is higher), and also a mechanism for progressive recruitment of synapses during frequency facilitation.     

δ-Aminolaevulinic acid and amino acid neurotransmitters

Summary The effects of the porphyrin precursor -aminolaevulinic acid (ALA) on -aminobutyric acid (GABA) and L-glutamate transmitter systems was investigated in rat brain. It was found that ALA inhibited GABA and glutamate uptake and stimulated basal efflux of the amino acids in purified nerve endings. These effects were evident only at relatively high concentrations of ALA (at least 100 M). Such concentrations probably do not occur in the nervous systems of patients suffering from acute porphyria. In addition, it was found that ALA inhibited the stimulated release of GABA from nerve endings probably by acting as an agonist at GABA autoreceptors. This effect was found at very low concentrations of ALA (1 M). It is therefore likely that the neuropsychiatric manifestations of the acute porphyric attack are attributable, to some extent, to reduced GABA release at central synapses.