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.     

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.     

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.     

Crustacean phasic and tonic motor neurons

Crustacean motor neurons subserving locomotion are specializedfor the type of activity in which they normally participate.Neurons responsible for maintained activity (‘tonic’neurons) support moderate to high frequencies of nerve impulsesintermittently or continuously during locomotion, while thoserecruited for short-lasting rapid responses (‘phasic’neurons) generally fire a few impulses in a rapid burst duringrapid locomotion and are otherwise silent. The synaptic responsesof the two types, recorded at their respective neuromuscularjunctions, differ enormously: phasic neurons exhibit much higherquantal release per synapse and per muscle fibre, along withmore rapid synaptic depression and less short-term facilitation.We have analyzed the factors that are responsible for the largedifference in initial release of neurotransmitter. Several possibilities,including synapse and active zone size differences, entry ofcalcium at active zones, and immediately releasable vesiclepools, could not account for the large phasic-tonic differencein initial transmitter output. The most likely feature thatdifferentiates synaptic release is the sensitivity of the exocytoticmachinery to intracellular calcium. Molecular features of thephasic and tonic presynaptic nerve terminals are currently underinvestigation.     

Quantitative comparison of low- and high-output neuromuscular synapses from a motoneuron of the lobster (Homarus americanus)

Summary Representative examples of lowand high-output neuromuscular synapses between motoneuron and distal accessory flexor muscle of the lobster were selected on the basis of their mean quantal content, and subsequently analysed by serial section electron microscopy. The high-output terminal has twice as many synapses as the low-output terminal. However, since the mean surface area of synapses is significantly smaller in the high-output terminal than in the low-output one, the total synaptic surface area between the two types of terminals is similar. Also, though the high-output terminal possesses a greater number of presynaptic dense bodies than its low-output counterpart, the mean number per synapse is similar for the two terminals. The terminals, however, differ significantly in the size of their dense bodies. Thus both the mean and total surface area of these bodies is greater in the high-output terminal than in the low-output one. Moreover, the mean ratio of dense body area to synaptic area is significantly greater for the high-output terminal than for its low-output counterpart. This difference in dense body area parallels the difference in quantal content of synaptic transmission between the lowand high-output terminals and supports the hypothesis that presynaptic densities represent the ultrastructural correlates of transmitter mobilization and/or release.Supported by grants from the National Research Council and Muscular Dystrophy Association of Canada to C.K. Govind. D.E. Meiss is a post-doctoral fellow of the Muscular Dystrophy Association of Canada. We thank Eva Yap-Chung for her expert and unfailing technical assistance     

Strength of synaptic transmission at neuromuscular junction of crustaceans and insects in relation to calcium entry

Crustacean and insect neuromuscular junctions typically include numerous small synapses, each of which usually contains one or more active zones, which possess voltage-sensitive calcium channels and are specialized for release of synaptic vesicles. Strength of transmission (the number of quantal units released per synapse by a nerve impulse) varies greatly among different endings of individual neurons, and from one neuron to another. Ultrastructural features of synapses account for some of the physiological differences at endings of individual neurons. The nerve terminals that release more neurotransmitter per impulse have a higher incidence of synapses with more than one active zone, and this is correlated with more calcium build-up during stimulation. However, comparison of synaptic structure in neurons with different physiological phenotypes indicates no major differences in structure that could account for their different levels of neurotransmitter release per impulse, and release per synapse differs among neurons despite similar calcium build-up in their terminals during stimulation. The evidence indicates differences in calcium sensitivity of the release process among neurons as an aspect of physiological specialization.     

Active zones and receptor surfaces of freeze-fractured crayfish phasic and tonic motor synapses

Deep and superficial flexor muscles in the crayfish abdomen are innervated respectively by small populations of physiologically distinct phasic and tonic motoneurons. Phasic motoneurons typically produce large EPSP's, releasing 100 to 1000 times more transmitter per synapse than their tonic counterparts, and exhibiting more rapid synaptic depression with maintained stimulation. Freeze-fracturing the abdominal flexor muscles yielded images of phasic and tonic synapse-bearing terminals. The two types of synapse are qualitatively similar in ultrastructure, displaying on the presynaptic membrane's P-face synaptic contacts recognized by relatively particle-free oval plaques which are often framed by the muscle fiber's E-face leaflet with its associated receptor particles. Situated within these presynaptic plaques are discrete clusters of large intramembrane particles, forming active zone (AZ) sites specialized for transmitter release. AZs of phasic and tonic synapses are similar: 80% had a range of 15–40 large particles distributed in either paired spherical clusters or in linear form, with a few depressions denoting sites of synaptic vesicle fusion or retrieval around their perimeters. The packing density of particles is similar for phasic and tonic AZs. The E-face of the muscle membrane displays oval-shaped receptor-containing sites made up of tightly packed intramembranous particles. Phasic and tonic receptor particles are packed at similar densities and the measured values resemble those of several other crustacean and insect neuromuscular junctions. Overall, the similarity between phasic and tonic synapses in the packing density of particles at their presynaptic AZs and postsynaptic receptor surfaces suggests similar regulatory mechanisms for channel insertion and spacing. Furthermore, the findings suggest that morphological differences in active zones or receptor surfaces cannot account for large differences in transmitter release per synapse.     

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.     

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.     

The molecular mechanisms of quantum mediator secretion in the synapse

The modern condition of knowledge about the molecular mechanisms underlying the quantal transmitter release in the central and the peripheric synapses is analysed. The data about the synaptic vesicles types, their forming, transporting to the sites of release at the nerve endings, exo- and endocytosis processes are presented. Ultrastructural and molecular organization of active zone of nerve ending and transmitter release morphofunctional unit--secretosome, which includes synaptic vesicle, exocytosis protein complex and calcium channels, are described. The basic proteins involved in the exo- and endocytosis and their interactions during transmitter release are examined. The role of the intracellular buffer systems, calcium micro- and macrodomains in the quantal transmitter secretion are considered. The reasons of the active zones functional non-uniformity and plasticity and factors reduced transmitter release in the active zone to the single quantum are analysed.     

Differential facilitation of high- and low-output nerve terminals from a single motoneuron.

In the crayfish opener neuromuscular preparation, regional differences in synaptic transmission are observed among the terminals of a single motoneuron. With a single stimulus, the high-output terminals of the proximal region of the muscle produce a larger excitatory postsynaptic potential than do the low-output terminals of the central region of the muscle. We tested the hypothesis that the low-output terminals exhibit more facilitation than do high-output terminals for twin-pulse, train, and continuous-stimulation paradigms. Previous studies have not employed several stimulation paradigms to induce facilitation among high- and low-output terminals of a single motoneuron. We found that the high-output terminals on the proximal fibers facilitate more than the low-output terminals on the central muscle fibers, in contrast with previous studies on similar muscles. The difference in measured facilitation is dependent on the stimulation paradigm. These results are important because ultrastructural differences between these high- and low-output terminals are known and can be used for correlation with physiological measurements. Short-term facilitation is a form of short-term memory at the synaptic level, and the processes understood at the crayfish neuromuscular junction may well be applicable to all chemical synapses.     

Fast-axon synapses of a crab leg muscle

Neuromuscular synapses of the "fast" excitatory axon supplying the main extensor muscle in the leg of the shore crab Pachygrapsus crassipes were studied with electrophysiological and electron-microscopic techniques. Electrical recording showed that many muscle fibers of the central region of the extensor muscle responded only to stimulation of the fast axon, and electron microscopy revealed many unitary subterminal axon branches. Maintained stimulation, even at a low frequency, resulted in depression of the excitatory junctional potentials (EJPs) set up by the fast axon but EJPs of different muscle fibers depressed at different rates, indicating some physiological heterogeneity among the fast-axon synapses. Focal recording at individual synaptic sites on the surfaces of the muscle fibers showed quantal contents ranging from 1.4 to 5.5 at different synapses; these values are relatively high in comparison with similar determinations made in the crayfish opener muscle. Synapse-bearing nerve terminals were generally relatively small in diameter and filiform, with many individual synaptic contact areas of uniform size averaging 0.6 micron2. All of the individual synapses had a presynaptic "dense body" at which synaptic vesicles clustered. If these structures represent release points for transmitter quanta, the initial high quantal content would have an ultrastructural basis. The mitochondial content of the nerve terminals, the synaptic vesicle population, and the specialized subsynaptic sarcoplasm were all much reduced in comparison with tonic axon synaptic regions in this and other crustaceans. The latter features may be correlated with the relatively infrequent use of this axon by the animal, and with rapid fatigue.     

Neurotransmitter release at ribbon synapses in the retina

The synapses of photoreceptors and bipolar cells in the retina are easily identified ultrastructurally by the presence of synaptic ribbons, electron-dense bars perpendicular to the plasma membrane at the active zones, extending about 0.5 microm into the cytoplasm. The neurotransmitter, glutamate, is released continuously (tonically) from these 'ribbon synapses' and the rate of release is modulated in response to graded changes in the membrane potential. This contrasts with action potential-driven bursts of release at conventional synapses. Similar to other synapses, neurotransmitter is released at ribbon synapses by the calcium-dependent exocytosis of synaptic vesicles. Most components of the molecular machinery governing transmitter release are conserved between ribbon and conventional synapses, but a few differences have been identified that may be important determinants of tonic transmitter release. For example, the presynaptic calcium channels of bipolar cells and photoreceptors are different from those elsewhere in the brain. Differences have also been found in the proteins involved in synaptic vesicle recruitment to the active zone and in synaptic vesicle fusion. These differences and others are discussed in terms of their implications for neurotransmitter release from photoreceptors and bipolar cells in the retina.     

Synaptic and Extrasynaptic Secretion of Serotonin

Serotonin is a major modulator of behavior in vertebrates and invertebrates and deficiencies in the serotonergic system account for several behavioral disorders in humans.The small numbers of serotonergic central neurons of vertebrates and invertebrates produce their effects by use of two modes of secretion: from synaptic terminals, acting locally in hard wired circuits, and from extrasynaptic axonal and somatodendritic release sites in the absence of postsynaptic targets, producing paracrine effects.In this paper, we review the evidence of synaptic and extrasynaptic release of serotonin and the mechanisms underlying each secretion mode by combining evidence from vertebrates and invertebrates. Particular emphasis is given to somatic secretion of serotonin by central neurons.Most of the mechanisms of serotonin release have been elucidated in cultured synapses made by Retzius neurons from the central nervous system of the leech. Serotonin release from synaptic terminals occurs from clear and dense core vesicles at active zones upon depolarization. In general, synaptic serotonin release is similar to release of acetylcholine in the neuromuscular junction.The soma of Retzius neurons releases serotonin from clusters of dense core vesicles in the absence of active zones. This type of secretion is dependent of the stimulation frequency, on L-type calcium channel activation and on calcium-induced calcium release.The characteristics of somatic secretion of serotonin in Retzius neurons are similar to those of somatic secretion of dopamine and peptides by other neuron types. In general, somatic secretion by neurons is different from transmitter release from clear vesicles at synapses and similar to secretion by excitable endocrine cells.     

Electrophysiological study of mediator secretion in the frog neuromuscular synapse under a colchicine block of axoplasmic transport

Two weeks after colchicine nerve treatment the evoked transmitter release was blocked in part of the frog sartorius synapses, with spontaneous activity being absent from some of them. In the synapses with evoked and spontaneous transmitter release preserved within this period of time, the magnitudes of the absolute refractory phase of nerve terminals were significantly higher than the control ones, while in part of synapses, the frequency of miniature end plate potentials (MEPP) was considerably increased. Nerve stimulation (5 imp.s-1) led to a rise of the amplitude of evoked potentials and of MEPP frequency followed by irreversible blockade of synaptic activity. It is concluded that substances transported by rapid axonal flow control the level of membrane potential of nerve terminals and are fairly important for presynaptic membrane integrity.     

Mobilization of the Readily Releasable Pool of Acetylcholine from a Sympathetic Ganglion by Tityustoxin in the Presence of Vesamicol

The present experiments tested whether preganglionic stimulation and direct depolarization of nerve terminals by tityustoxin could mobilize similar or different pools of acetylcholine (ACh) from the cat superior cervical ganglia in the presence of 2-(4-phenylpiperidino)cyclohexanol (vesamicol, AH5183), an inhibitor of ACh uptake into synaptic vesicles. In the absence of vesamicol, both nerve stimulation and tityustoxin increased ACh release. In the presence of vesamicol, the release of ACh induced by tityustoxin was inhibited, and just 16% of the initial tissue content could be released, a result similar to that obtained with electrical stimulation under the same condition. When the impulse-releasable pool of ACh had been depleted, tityustoxin still could release transmitter, amounting to some 10% of the ganglion's initial content. This pool of transmitter seemed to be preformed in the synaptic vesicles, rather than synthesized in response to stimuli, as tityustoxin could not release newly synthesized [3H]ACh formed in the presence of vesamicol, and hemicholinium-3 did not prevent the toxin-induced release. In contrast to the results with tityustoxin, preganglionic stimulation could not release transmitter when impulse-releasable or toxin-releasable compartments had been depleted. Our results confirm that vesamicol inhibits the mobilization of transmitter from a reserve to a more readily releasable pool, and they also suggest that, under these experimental conditions, there might be some futile transmitter mobilization, apparently to sites other than nerve terminal active zones.     

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     

Binding to Rab3A-interacting molecule RIM regulates the presynaptic recruitment of Munc13-1 and ubMunc13-2

Transmitter release at synapses between nerve cells is spatially restricted to active zones, where synaptic vesicle docking, priming, and Ca2+-dependent fusion take place in a temporally highly coordinated manner. Munc13s are essential for priming synaptic vesicles to a fusion competent state, and their specific active zone localization contributes to the active zone restriction of transmitter release and the speed of excitation-secretion coupling. However, the molecular mechanism of the active zone recruitment of Munc13s is not known. We show here that the active zone recruitment of Munc13 isoforms Munc13-1 and ubMunc13-2 is regulated by their binding to the Rab3A-interacting molecule RIM1alpha, a key determinant of long term potentiation of synaptic transmission at mossy fiber synapses in the hippocampus. We identify a single point mutation in Munc13-1 and ubMunc13-2 (I121N) that, depending on the type of assay used, strongly perturbs or abolishes RIM1alpha binding in vitro and in cultured fibroblasts, and we demonstrate that RIM1alpha binding-deficient ubMunc13-2(I121) is not efficiently recruited to synapses. Moreover, the levels of Munc13-1 and ubMunc13-2 levels are decreased in RIM1alpha-deficient brain, and Munc13-1 is not properly enriched at active zones of mossy fiber terminals of the mouse hippocampus if RIM1alpha is absent. We conclude that one function of the Munc13/RIM1alpha interaction is the active zone recruitment of Munc13-1 and ubMunc13-2.     

The actin cytoskeleton and neurotransmitter release: an overview

Here we review evidence that actin and its binding partners are involved in the release of neurotransmitters at synapses. The spatial and temporal characteristics of neurotransmitter release are determined by the distribution of synaptic vesicles at the active zones, presynaptic sites of secretion. Synaptic vesicles accumulate near active zones in a readily releasable pool that is docked at the plasma membrane and ready to fuse in response to calcium entry and a secondary, reserve pool that is in the interior of the presynaptic terminal. A network of actin filaments associated with synaptic vesicles might play an important role in maintaining synaptic vesicles within the reserve pool. Actin and myosin also have been implicated in the translocation of vesicles from the reserve pool to the presynaptic plasma membrane. Refilling of the readily releasable vesicle pool during intense stimulation of neurotransmitter release also implicates synapsins as reversible links between synaptic vesicles and actin filaments. The diversity of actin binding partners in nerve terminals suggests that actin might have presynaptic functions beyond synaptic vesicle tethering or movement. Because most of these actin-binding proteins are regulated by calcium, actin might be a pivotal participant in calcium signaling inside presynaptic nerve terminals. However, there is no evidence that actin participates in fusion of synaptic vesicles.     

Altered synaptic development and active zone spacing in endocytosis mutants

Many types of synapses have highly characteristic shapes and tightly regulated distributions of active zones, parameters that are important to the function of neuronal circuits. The development of terminal arborizations must therefore include mechanisms to regulate the spacing of terminals, the frequency of branching, and the distribution and density of release sites. At present, however, the mechanisms that control these features remain obscure. Here, we report the development of supernumerary or "satellite" boutons in a variety of endocytic mutants at the Drosophila neuromuscular junction. Mutants in endophilin, synaptojanin, dynamin, AP180, and synaptotagmin all show increases in supernumerary bouton structures. These satellite boutons contain releasable vesicles and normal complements of synaptic proteins that are correctly localized within terminals. Interestingly, however, synaptojanin terminals have more active zones per unit of surface area and more dense bodies (T-bars) within these active zones, which may in part compensate for reduced transmission per active zone. The altered structural development of the synapse is selectively encountered in endocytosis mutants and is not observed when synaptic transmission is reduced by mutations in glutamate receptors or when synaptic transmission is blocked by tetanus toxin. We propose that endocytosis plays a critical role in sculpting the structure of synapses, perhaps through the endocytosis of unknown regulatory signals that organize morphogenesis at synaptic terminals.