Differences in synaptic output between excitatory and inhibitory motoneurons in a crayfish muscle

A pair of antagonistic motoneurons, one excitatory and one inhibitory, innervates the distal accessory flexor muscle in the walking limb of the crayfish Procambarus clarkii. The number and size of synapses formed by these two axons on the muscle fibers (neuromuscular synapses) and on each other (axo-axonal synapses) were estimated using thin-section electron microscopy. Although profiles of nerve terminals of the two axons occur in roughly equal proportions, the frequency of occurrence of neuromuscular synapses differed markedly: 73% were excitatory and 27% were inhibitory. However, inhibitory synapses were 4–5 times larger than excitatory ones, and consequently, the total contact areas devoted to neuromuscular synapses were similar for both axons. Axo-axonal synapses were predominantly from the inhibitory axon to the excitatory axon (86%), and a few were from the excitatory axon to the inhibitory axon (14%). The role of the inhibitory axo-axonal synapse is presynaptic inhibition, but that of the excitatory axo-axonal synapse is not known. The differences in size of neuromuscular synapses between the two axons may reflect intrinsic determinants of the neuron, while the similarity in total synaptic area may reflect retrograde influences from the muscle for regulating synapse number.     

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.     

Neuromuscular synapses on the dactyl opener muscle of the lobster Homarus americanus

The crustacean dactyl opener neuromuscular system has been studied extensively as a model system that exhibits several forms of synaptic plasticity. We report the ultrastructural features of the synapses on dactyl opener of the lobster (Homarus americanus) as determined by examination of serial thin sections. Several innervation sites supplied by an inhibitory motoneuron have been observed without nearby excitatory innervation, indicating that excitatory and inhibitory inputs to the muscle are not always closely matched. The ultrastructural features of the lobster synapses are generally similar to those described previously for the homologous crayfish muscle, with one major distinction: few dense bars are seen at the presynaptic membranes of these lobster synapses. The majority of the lobster neuromuscular synapses lack dense bars altogether, and the mean number of dense bars per synapse is relatively low. In view of the finding that the physiology of the lobster dactyl opener synapses is similar to that reported for crayfish, these ultrastructural observations suggest that the structural complexity of the synapses may not be a critical factor determining synaptic plasticity.This work was supported by funds from the University of Virginia Research and Development Committee.     

Structural definition of the neuromuscular system in the swimming-paddle opener muscle of blue crabs

Blue crabs are excellent swimmers, using their highly modified last pereiopods as sculling paddles. Hence, the hypertrophied paddle opener muscle was examined for adaptations of its motor innervation by an excitor and a specific inhibitor axon. The muscle has a uniform composition of slow fibers with long (6-12 microm) sarcomere lengths. Individual fibers are richly innervated with approximately two-thirds excitatory and one-third inhibitory innervation. The profuse excitatory innervation reflects the high activity levels of this motoneuron in swimming. Adaptation to sustained activity associated with swimming is also reflected in the motor nerve terminals by a high concentration of energy source, which is equally divided between glycogen granules and mitochondria, the former providing a more rapid source of energy. The excitor axon makes predominantly neuromuscular synapses, but also a few synapses onto the inhibitor axon. The location of these excitatory axoaxonal synapses suggests regional modulation of the inhibitor axon. The specific inhibitor axon makes less than two-thirds of its synapses with the muscle fiber, regulating contraction via postsynaptic inhibition. The remaining inhibitory synapses are onto the excitor axon, signaling very strong presynaptic inhibition. Such presynaptic inhibition will effectively decouple the opener muscle from the stretcher muscle even though both are innervated by a single excitor axon.     

Regenerated synaptic terminals on a crayfish slow muscle identify with transplanted phasic or tonic axons

Phasic or tonic nerves transplanted onto a denervated slow superficial flexor muscle in adult crayfish regenerated synaptic connections that displayed large or small excitatory postsynaptic potentials (EPSPs), respectively, suggesting that the neuron specifies the type of synapse that forms (Krause et al., J Neurophysiol 80:994-997, 1998). To test the hypothesis that such neuronal specification would extend to the synaptic structure as well, we examined the regenerated synaptic terminals with thin serial section electron microscopy. There are distinct differences in structure between regenerated phasic and tonic innervation. The phasic nerve provides more profuse innervation because innervation sites occurred more frequently and contained larger numbers of synaptic terminals than the tonic nerve. Preterminal axons of the phasic nerve also had many more sprouts than those of the tonic nerve. Phasic terminals were thinner and had a lower mitochondrial volume than their tonic counterparts. Phasic synapses were half the size of tonic ones, although their active zone-dense bars were similar in length. The density of active zones was higher in the phasic compared with the tonic innervation, based on estimates of the number of dense bars per synapse, per synaptic area, and per nerve terminal volume. Because these differences mirror those seen between phasic and tonic axons in crayfish muscle in situ, we conclude that the structure of the regenerated synaptic terminals identify with their transplanted axons rather than with their target muscle. Therefore, during neuromuscular regeneration in adult crayfish, the motoneuron appears to specify the identity of synaptic connections.     

Growth of inhibitory innervation in a lobster muscle

The fine structure of inhibitory innervation to a limb muscle was examined in larval, juvenile, and adult lobsters. The innervation is essentially similar in qualitative features among these different stages, although there are some marked quantitative changes associated with growth. From being localized to discrete regions in the larval muscle, the inhibitory innervation spreads to groups of muscle fibers in the early juvenile muscle and to single fibers in the late juvenile and adult muscles. Concurrently, its neuromuscular synapses enlarge in area, become perforated, and acquire more active sites of transmitter release. Inhibitory nerve terminals occur in close proximity to their excitatory counterparts in the muscles of larval and early juvenile stages, although in later stages this juxtaposition occurs preferentially in some muscle fibers but not others. The inhibitory innervation is, nevertheless, much more restricted in occurrence than is the excitatory innervation.     

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.     

Proliferation and relocation of developing lobster neuromuscular synapses

The development of multiterminal innervation from a single identifiable excitatory motoneuron to the lobster distal accessory flexor muscle (DAFM) was studied by serial section electron microscopy. The number, size, and location of neuromuscular synapses and presynaptic dense bars within the peripheral branching pattern of the axon was determined in cross sections of the DAFM in 1st (24-hr-old)-, 4th (2-week-old)-, and 12th (1-year-old)-stage lobsters. The mean size of synapses remains fairly constant in these three stages but synaptic density, i.e., the number of synapses per unit length of fiber, increased more than 20-fold between the 1st and 4th stages and more than 5-fold between the 4th and 12th stages. Synaptic surface area per fiber length showed a parallel increase. Consequently there is a proliferation of synapses along the length of individual muscle fibers during primary development. Furthermore from the 1st stage where only a few fibers are innervated, synapses proliferate to many more fibers in the 4th and to all fibers in the 12th stage. The neuromuscular synapses are distributed in different proportions within the axonal branching pattern in the three stages. Based on the number and size of synapses and presynaptic dense bars, the main axon and primary branches provide almost equal amounts of innervation in the 1st stage. With further branching in the 4th stage, the main axon accounts for only 20–25% of the innervation; the primary branches for 45% and other finer branches the remainder. By the 12th-stage synapses are found only on branches other than the main axon and its primary offshoots. There is therefore a shift in innervation from the main axon to the primary branches and then to the finer branches during primary development. This shift in innervation involves the formation of new synaptic terminals and the restructuring of existing ones into axonal areas. In this way the multiterminal innervation arising from an identifiable motoneuron is remodeled.     

Synaptic differentiation between two phasic motoneurons to a crayfish fast muscle

Synaptic differentiation among crustacean phasic motoneurons was investigated by characterizing the synaptic output and nerve terminal morphology of the two axons to the adductor exopodite muscle in the crayfish uropod. The muscle is of the fast type with short sarcomeres (2–3 μm) and a low thin to thick filament number (6:1). On single muscle fibers, excitatory postsynaptic potentials generated by the large-diameter axon are significantly larger than those by the small-diameter axon suggesting a presynaptic origin for these differences. Nerve terminals arising from these two axons have typical phasic features, filiform shape and a low (6–8%) mitochondrial density. Synaptic contacts are similar in size between the two axons as is the length and number of active zone dense bars at these synapses. The large-diameter axon, however, exhibits a twofold larger area of nerve terminal than the small-diameter axon resulting in a higher density of synapses per muscle fiber. Hence, differences in synaptic density may in part account for differences in synaptic output between these paired phasic axons. Electronic Publication     

Nonhomogeneous excitatory synapses of a crab stomach muscle

Neuromuscular synapses of pyloric muscle P1 in the blue crab Callinectes sapidus were examined using electrophysiological and electron microscopic methods. The muscle is innervated by a single excitatory axon of the stomatogastric ganglion. Excitatory postsynaptic potentials show striking facilitation at very low frequencies of stimulation, indicating very slow decay of the facilitation process after a single nerve impulse. Quantal content of transmitter release at a low frequency of stimulation averaged 1.5. Evidence was obtained that not all synapses on a muscle fiber are equivalent. This was particularly evident at the morphological level in serially sectioned nerve terminals. On each nerve terminal examined, a wide range of synapse sizes was found. Synaptic contact areas ranged from less than 0.5 micron2 to almost 10 micron2; the latter value is large compared with those obtained for other crustacean neuromuscular synapses. Most of the smaller synapses lacked the presynaptic dense bodies which are putative release sites for the transmitter substance. The larger synapses all had presynaptic dense bodies, and some showed evidence of splitting apart into smaller subunits. It is postulated that about half the morphologically identified synapses are relatively inactive.     

The innervation pattern of crustacean skeletal muscle

Summary The innervation pattern of distal muscle fibers of the opener muscle of walking legs of crayfish (Astacus leptodactylus) was investigated using methylene-blue staining, cobalt infiltration, and electron microscopy. A quantitative analysis of the entire innervation of single muscle fibers was attempted.It was found that instead of the generally assumed parallel array of numerous excitatory and inhibitory terminals, innervation consists of only a few branched terminals. The branches of excitatory and inhibitory terminals lie side-by-side. Both types are characterized by numerous varicosities (see Fig. 9B). The aggregate length of excitatory as well as inhibitory terminals on one muscle fiber is, on the average, about 1,500 m with a total of 152 varicosities spaced about 10 m apart. The average diameter of the varicosities is 4.26 m, that of the connecting thin segments about 0.5 m. Total terminal surface of motor or inhibitory terminals amounts to about 10,000 m² per muscle fiber. There are approximately 2,000 motor synapses on each muscle fiber, but their average total area is only about 6% of the terminal membrane area, or 0.06% of the (idealized) muscle fiber surface.There are conspicuous differences in the postsynaptic specializations associated with excitatory and inhibitory terminals; these are described in detail.The results are discussed in a functional context and with regard to design and results of electrophysiological experiments.Supported by Sonderforschungsbereich 138 of the Deutsche Forschungsgemeinschaft     

GABA and neuroligin signaling: linking synaptic activity and adhesion in inhibitory synapse development

GABA-mediated synaptic inhibition is crucial in neural circuit operations. In mammalian brains, the development of inhibitory synapses and innervation patterns is often a prolonged postnatal process, regulated by neural activity. Emerging evidence indicates that gamma-aminobutyric acid (GABA) acts beyond inhibitory transmission and regulates inhibitory synapse development. Indeed, GABA(A) receptors not only function as chloride channels that regulate membrane voltage and conductance but also play structural roles in synapse maturation and stabilization. The link from GABA(A) receptors to postsynaptic and presynaptic adhesion is probably mediated, partly by neuroligin-reurexin interactions, which are potent in promoting GABAergic synapse formation. Therefore, similar to glutamate signaling at excitatory synapse, GABA signaling may coordinate maturation of presynaptic and postsynaptic sites at inhibitory synapses. Defining the many steps from GABA signaling to receptor trafficking/stability and neuroligin function will provide further mechanistic insights into activity-dependent development and possibly plasticity of inhibitory synapses.     

Synaptic restructuring during long-term facilitation at the crayfish neuromuscular junction

Long-term facilitation was induced by 20-Hz stimulation of the motor axon innervating the opener muscle of the crayfish, Procambarus clarkii. Excitatory postsynaptic potentials remained potentiated for several hours after stimulation. Structural correlates of potentiation were sought. Nerve terminals of the motor axon were fixed for electron microscopy in unstimulated preparations (controls), and during and after 20-Hz stimulation. Synapses were reconstructed from micrographs obtained from serial sections. Synaptic contact area and the number of vesicles at the presynaptic membrane did not change after 20-Hz stimulation, but the latter decreased during stimulation. Presynaptic dense bars ("active zones") decreased in number during and increased after stimulation, while perforated synapses increased after stimulation. Modification of presynaptic structures occurs rapidly and may be linked to long-lasting changes in quantal content of transmission.     

Triple innervation of the crayfish opener muscle: the astacuran common inhibitor

Part of the much-studied crayfish opener muscle receives a second inhibitory input in addition to its well known specific excitatory and inhibitory innervation. This second inhibitor, formerly thought to innervate only four of the seven peripheral leg muscles, is in fact a common inhibitor of all seven. This has significance both for previous findings in this muscle and for the role of the common inhibitor in decapods.     

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     

Postmetamorphic development of neuromuscular junctions and muscle fibers in the frog cutaneous pectoris

Synaptic size, synaptic remodelling, polyneuronal innervation, and synaptic efficacy of neuromuscular junctions were studied as a function of growth in cutaneous pectoris muscles of postmetamorphic Rana pipiens. Recently metamorphosed frogs grew rapidly, and this growth was accompanied by hypertrophy of muscle fibers, myogenesis, and increases in the size and complexity of neuromuscular junctions. There were pronounced gradients in pre- and postsynaptic size across the width of the muscle, with neuromuscular junctions and muscle fibers near the medial edge being smaller than in more lateral regions. The incidence of polyneuronal innervation, measured physiologically and histologically, was also higher near the medial edge. Growth-associated declines in all measures of polyneuronal innervation indicated that synapse elimination occurs throughout life. Electrophysiology also demonstrated regional differences in synaptic efficacy and showed that doubly innervated junctions have lower synaptic efficacy than singly innervated junctions. Repeated, in vivo observations revealed extensive growth and remodelling of motor nerve terminals and confirmed that synapse elimination is a slow process. It was concluded that some processes normally associated with embryonic development persist long into adulthood in frog muscles.     

Synaptic plasticity at the crayfish opener neuromuscular preparation

The crayfish opener neuromuscular preparation exhibits most of the plasticities yet described for any synapse, including facilitation, long-term potentiation, presynaptic inhibition, and modulation. Since the presynaptic terminals and postsynaptic muscle fibers can both be intracellularly penetrated, one can now more easily examine the cellular/molecular bases for these plasticities. Data from such studies suggest that facilitation may be influenced by something other than residual free calcium and that presynaptic inhibition is produced by a conductance increase to chloride in the terminals of the excitor axon. Several drugs (ethanol, pentobarbital) have significant effects on these synaptic plasticities over concentration ranges which produce obvious behavioral effects in crayfish and mammals. Hence, this preparation should be a useful model system to determine cellular/molecular bases for various synaptic plasticities and the effects of drugs on these plasticities.     

Synaptic destabilization by neuronal Nogo-A

Formation and maintenance of a neuronal network is based on a balance between plasticity and stability of synaptic connections. Several molecules have been found to regulate the maintenance of excitatory synapses but nothing is known about the molecular mechanisms involved in synaptic stabilization versus disassembly at inhibitory synapses. Here, we demonstrate that Nogo-A, which is well known to be present in myelin and inhibit growth in the adult CNS, is present in inhibitory presynaptic terminals in cerebellar Purkinje cells at the time of Purkinje cell-Deep Cerebellar Nuclei (DCN) inhibitory synapse formation and is then downregulated during synapse maturation. We addressed the role of neuronal Nogo-A in synapse maturation by generating several mouse lines overexpressing Nogo-A, starting at postnatal ages and throughout adult life, specifically in cerebellar Purkinje cells and their terminals. The overexpression of Nogo-A induced a progressive disassembly, retraction and loss of the inhibitory Purkinje cell terminals. This led to deficits in motor learning and coordination in the transgenic mice. Prior to synapse disassembly, the overexpression of neuronal Nogo-A led to the downregulation of the synaptic scaffold proteins spectrin, spectrin-E and β-catenin in the postsynaptic neurons. Our data suggest that neuronal Nogo-A might play a role in the maintenance of inhibitory synapses by modulating the expression of synaptic anchoring molecules. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Pumping up the synapse

Synchronized control of excitatory and inhibitory synapse maturation is crucial for normal brain wiring, while its dysfunction leads to neurodevelopmental disorders, including autism. A paper in this issue of Neuron identified a novel role for the KCC2 pump, also responsible for the GABAergic synapse developmental switch, in regulating spiny excitatory synapse maturation, implicating it in the coordinated maturation of inhibitory and excitatory synapses.     

The ultrastructure of neuromuscular and neural-neural relationships in in vitro maintained Dirofilaria Immitis

Relationships of neuromuscular junctions of the somatic musculature and associated neural-neural synapses in the ventral nerve trunk of the canine adult heartworm, Dirofilaria immitis, were studied by transmission electron microscopy. The heartworms were maintained in vitro prior to study. Nerve fibres in the trunk were highly invaginated into the cytoplasm of hypodermal cells and connected through the intercellular spaces via mesaxons. The nerve fibres contained neurotubules, neurofilaments and ribosomes. The nerve trunk and the muscle arms were separated by an epineurium averaging 250 nm in width. At the junctional site, a marked reduction in width of the epineurium was noted at the synaptic cleft. Often when two adjacent nerve fibres had adjacent neuromuscular junctions, an axo-axonal synapse and common mesaxon between the adjacent fibres were present. Varicosities were evident on some cross-sections through nerve fibres and ranged from a simple outward swelling against the muscle arm mass to exaggerated outgrowths measuring several micrometers in length.