Allotransplanted nerves regenerate inhibitory synapses on a crayfish muscle: Possible postsynaptic specification

Donor nerves of different origins, when transplanted onto a previously denervated adult crayfish abdominal superficial flexor muscle (SFM), regenerate excitatory synaptic connections. Here we report that an inhibitory axon in these nerves also regenerates synaptic connections based on observation of nerve terminals with irregular to elliptically shaped synaptic vesicles characteristic of the inhibitory axon in aldehyde fixed tissue. Inhibitory terminals were found at reinnervated sites in all 12 allotransplanted-SFMs, underscoring the fact that the inhibitory axon regenerates just as reliably as the excitatory axons. At sites with degenerating nerve terminals and at sparsely reinnervated sites, we observe densely stained membranes, reminiscent of postsynaptic membranes, but occurring as paired, opposing membranes, extending between extracellular channels of the subsynaptic reticulum. These structures are not found at richly innervated sites in allotransplanted SFMs, in control SFMs, or at several other crustacean muscles. Although their identity is unknown, they are likely to be remnant postsynaptic membranes that become paired with collapse of degenerated nerve terminals of excitatory and inhibitory axons. Because these two axons have uniquely different receptor channels and intramembrane structure, their remnant postsynaptic membranes may therefore attract regenerating nerve terminals to form synaptic contacts selectively by excitatory or inhibitory axons, resulting in postsynaptic specification.     

The closer muscle is a second target for the stretcher inhibitor motoneuron of the crayfish's thoracic limbs

Summary The stretcher inhibitor motoneuron of each thoracic limb of a crayfish (Pacifastacus leniusculus) was consistently found to innervate parts of the closer muscle, in addition to the stretcher muscle; it is thus not a specific inhibitor as previously thought. The common inhibitory motoneuron also innervates parts of both muscles. Some individual closer muscle fibers are inhibited more strongly by one inhibitor, some by the other, and some fairly equally by both; no general rule governing the inhibitors' closer muscle outputs became evident. In the claw, the distal closer fibres with the longest membrane time constants are all strongly inhibited by the stretcher inhibitor, and some by the common inhibitor as well.No other thoracic limb muscles were found to receive the stretcher inhibitor. The opener inhibitor's effects could be detected only in the opener muscle. The common inhibitor inhibits all walking leg muscles effectively. In the cheliped, it consistently inhibits all except the opener muscle, where its output may be vestigial. Its axon emerges through the ganglion's first root, whereas the opener and stretcher inhibitors' axons pass through the second root. The fast and slow excitatory axons to the extensor muscle also exit separately through the first and second roots, as in locusts.Abbreviations CI common inhibitor - EJP excitatory junctional potential - IJP inhibitory junctional potential - OI opener inhibitor - SI stretcher inhibitor     

Identification of common excitatory motoneurons in Drosophila melanogaster larvae

In insects, four types of motoneurons have long been known, including fast motoneurons, slow motoneurons, common inhibitory motoneurons, and DUM neurons. They innervate the same muscle and control its contraction together. Recent studies in Drosophila have suggested the existence of another type of motoneuron, the common excitatory motoneuron. Here, we found that shakB-GAL4 produced by labels this type of motoneuron in Drosophila larvae. We found that Drosophila larvae have two common excitatory motoneurons in each abdominal segment, RP2 for dorsal muscles and MNSNb/d-Is for ventral muscles. They innervate most of the internal longitudinal or oblique muscles on the dorsal or ventral body wall with type-Is terminals and use glutamate as a transmitter. Electrophysiological recording indicated that stimulation of the RP2 axon evoked excitatory junctional potential in a dorsal muscle.     

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.     

An Attempt to Account for the Diversity of Crustacean Muscles

Crustacean muscles are known to contain muscle fibers of variableproperties and to be innervated by phasic and/or tonic motoneuronswhich may possess synapses of diverse physiological properties.Frequently, phasic motor axons innervate short-sarcomere phasicmuscle fibers and tonic motor axons innervate long-sarcomeretonic muscle fibers, but some muscles receiving a single (tonic)motor axon contain both phasic and tonic muscle fibers. Althoughit is not known whether neural trophic influences are involvedin muscle differentiation, some neural trophic effects havebeen found in crustaceans, and it is reasonable to assume thatsuch influences may be involved in establishing the definitiveproperties of the muscle. Several other postulates must be made:(1) Phasic and tonic motor axons differ in their trophic effectiveness:(2) muscle fibers innervated relatively early in developmentby a tonic motor axon acquire the properties of tonic musclefibers, while those innervated later become intermediate orphasic muscle fibers; (3) the developmental stage of a growingor regenerating axon terminal plays a role in determinationof synaptic properties. Studies on regenerating limb buds supportthe hypothesis, which can account for the genesis of all observedtypes of crustacean neuromuscular system. Further experimentalwork is necessary to test the hypothesis.     

The oculomotor system of Daphnia magna

Summary The highly mobile cyclopic compound eye of Daphnia magna is rotated by six muscles arranged as three bilateral pairs. The three muscles on each side of the head share a common origin on the carapace and insert dorsally, laterally and ventrally on the eye. The dorsal and ventral muscles are each composed of two muscle fibers and the lateral muscle is composed of from two to five fibers, with three the most common number. Individual muscle fibers are spindle-shaped mononucleated cells with organized bundles of myofilaments. Lateral eye-muscle fibers are thinner than those of the other muscles but are otherwise similar in ultrastructure. Two motor neurons innervate each dorsal and each ventral muscle and one motor neuron innervates each lateral muscle. The cell bodies of the motor neurons are situated dorsally in the supraesophageal ganglion (SEG) and are ipsilateral to the muscles they innervate. The dendritic fields of the dorsal-muscle motor neurons are ipsilateral to their cell bodies; those of the ventral-muscle motor neurons are bilateral though predominantly contralateral. The central projections of the lateral-muscle motor neurons are unknown. In the dorsal and ventral muscles one motor axon synapses principally with one muscle fiber; in each lateral muscle the single motor axon branches to, and forms synapses with, all the fibers. The neuromuscular junctions, characterized by pre- and postsynaptic densities and clear vesicles, are similar in all the eye muscles.     

Synaptic development in the crayfish opener muscle

Nerve terminal regions in walking leg opener muscles of several crayfish of different ages (0 to 245 days after hatching) were examined by means of electron microscopy. This muscle is innervated by two axons (excitatory and inhibitory) and at maturity contains three classes of synapse: excitatory and inhibitory neuromuscular synapses, and inhibitory axo-axonal synapses. The muscle itself is initially a syncytium, which gradually becomes subdivided into distinct “muscle fibers” as the animal matures. Innervation was not found in the opener muscle just before or just after hatching, but was present in restricted locations on the inner side of the muscle within a few days of hatching. As the muscle enlarged and became subdivided, innervation appeared in various other locations. Synaptic contacts were located in young stages soon after hatching, and in later stages. Morphological differences characteristic of excitatory and inhibitory nerve terminals could be found even at the earliest stages of innervation. Both excitatory and inhibitory synapses, but particularly the former, showed evidence of progressive enlargement to a final size within the first two months, and no evidence for further enlargement of existing synapses thereafter. Synaptic maturation also involved the appearance of presynaptic “dense bodies” thought to be regions at which transmitter substance is preferentially released. Nerve terminals at different levels of maturation were observed in opener muscles of young crayfish. Clear evidence for differential maturation of the three types of synapse present in this muscle was obtained. The inhibitory neuromuscular synapses attained their final average size and developed their dense bodies sooner than the excitatory neuromuscular synapses. The inhibitory axo-axonal synapses were the last to appear and to mature.     

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.     

Innervation pattern of the deep extensor abdominal muscle in the crayfish species Astacus astacus

The deep extensor abdominal muscle consisting of one medial and two lateral muscle bundles together with the nerve innervating the muscles of crayfish species Astacus astacus, was prepared. Light microscopic investigations of methylene blue stained preparations showed that the nerve innervating the deep extensor abdominal muscle consists of five distinct axons. The five axons were stained separately with lucifer yellow and the innervation pattern of the axons was determined. To confirm the histological results the axons were also stimulated with a suction electrode to elicit excitatory postsynaptic currents on the muscle membrane which were detected using a macro patch electrode. The muscle is innervated by a common excitatory and a common inhibitory axon branching over all three muscle bundles and sending additionally a branch to the L1-bundle of the next posterior segment, and by two axons specific for the two lateral muscle bundles. The axon specific for the innervation of the L1-bundle sends also a branch to the L1-bundle of the next posterior segment. In addition there is one excitatory axon which directly innervates the medial muscle bundle of the next posterior segment branching in most of the cases also to the medial bundle of the segment where it originates.Abbreviations DEAM deep extensor abdominal muscle - EPSC excitatory postsynaptic current - IPSC inhibitory postsynaptic current - L lateral - M medial - GABA -aminobutyric acid     

Independent development of sensory and motor innervation patterns in embryonic chick hindlimbs

Previous studies suggest that sensory axon outgrowth is guided by motoneurons, which are specified to innervate particular target muscles. Here we present evidence that questions this conclusion. We have used a new approach to assess the pathfinding abilities of bona fide sensory neurons, first by eliminating motoneurons after neural crest cells have coalesced into dorsal root ganglia (DRG) and second by challenging sensory neurons to innervate muscles in a novel environment created by shifting a limb bud rostrally. The resulting sensory innervation patterns mapped with the lipophilic dyes DiI and DiA showed that sensory axons projected robustly to muscles in the absence of motoneurons, if motoneurons were eliminated after DRG formation. Moreover, sensory neurons projected appropriately to their usual target muscles under these conditions. In contrast, following limb shifts, muscle sensory innervation was often derived from inappropriate segments. In this novel environment, sensory neurons tended to make more "mistakes" than motoneurons. Whereas motoneurons tended to innervate their embryologically correct muscles, sensory innervation was more widespread and was generally from more rostral segments than normal. Similar results were obtained when motoneurons were eliminated in embryos with limb shifts. These findings show that sensory neurons are capable of navigating through their usual terrain without guidance from motor axons. However, unlike motor axons, sensory axons do not appear to actively seek out appropriate target muscles when confronted with a novel terrain. These findings suggest that sensory neuron identity with regard to pathway and target choice may be unspecified or quite plastic at the time of initial axon outgrowth.     

Neuromuscular relationships in the abdomen of the Californian shore crab Pachygrapsus crassipes

There are two pairs of muscles in each abdominal segment of the crab; one pair of flexors and one pair of extensors. In the early larval stages the muscles have short sarcomeres--a property of fast fibers--and high thin to thick filament ratios--a property of slow fibers. In the adult the abdominal muscles are intermediate and slow, since they have fibers with intermediate and long sarcomeres, high thin to thick filament ratios, low myofibrillar ATPase activity, and high NADH diaphorase activity. The different fiber types are regionally distributed within the flexor muscle. Microelectrode recordings from single flexor muscle fibers in the adult showed that most fibers are supplied by three excitatory motor axons, although some are supplied by as many as five efferents. One axon supplies all of the flexor muscle fibers in its own hemisegment, and the evoked junctional potentials exhibit depression. This feature together with the innervation patterns of the fibers are similar to those reported for the deep flexor muscles of crayfish and lobsters. Therefore, in the adult crab, the abdominal flexor muscles have some features in common with the slow superficial flexors of crayfish and other features in common with the fast deep flexor muscles.     

Local innervation patterns of the metathoracic flexor and extensor tibiae motor neurons in the cricket Gryllus bimaculatus

To elucidate neural mechanisms underlying walking and jumping in insects, motor neurons supplying femoral muscles have been identified mainly in locusts and katydids, but not in crickets. In this study, the motor innervation patterns of the metathoracic flexor and extensor tibiae muscles in the cricket, Gryllus bimaculatus were investigated by differential back-fills and nerve recordings. Whereas the extensor tibiae muscle has an innervation pattern similar to that of other orthopterans, the flexor has an innervation unique to this species. The main body of the flexor muscle is divided into the proximal, middle and distal regions, which receive morphologically unique terminations from almost non-overlapping sets of motor neurons. The proximal region is innervated by about 12 moderate-sized excitatory motor neurons and two inhibitory neurons while the middle and distal regions are innervated by three and four large excitatory motor neurons, respectively. The most-distally located accessory flexor muscle, inserting on a common flexor apodeme with the main muscle, is innervated by at least four small excitatory (slow-type) and two common inhibitory motor neurons. The two excitatory and two inhibitory motor neurons that innervate the accessory flexor muscle also innervate the proximal bundles of the main flexor muscle. This suggests that the most proximal and distal parts of the flexor muscle participate synergistically in fine motor control while the rest participates in powerful drive of tibial flexion movement.     

The metabolism of gamma aminobutyric acid in the lobster nervous system. Enzymes in single excitatory and inhibitory axons

γ-aminobutyric acid (GABA) is the inhibitory transmitter compound at the lobster neuromuscular junction. This paper presents a comparison of the enzymes of GABA metabolism in single identified inhibitory and excitatory axons from lobster walking legs. Inhibitory axons contain more than 100 times as much glutamic decarboxylase activity as do excitatory axons. GABA-glutamic transaminase is found in both excitatory and inhibitory axons, but about 50% more enzyme is present in inhibitory axons. The kinetic and electrophoretic behavior of the transaminase activity in excitatory and inhibitory axons is similar. Succinic semialdehyde dehydrogenase is found in both axon types, as is an unknown enzyme which converts a contaminant in radioactive glutamic acid to GABA. In lobster inhibitory neurons, therefore, the ability to accumulate GABA ultimately rests on the ability of the neuron to accumulate the enzyme glutamic decarboxylase.     

Roles for ephrins in positionally selective synaptogenesis between motor neurons and muscle fibers

Motor axons form topographic maps on muscles: rostral motor pools innervate rostral muscles, and rostral portions of motor pools innervate rostral fibers within their targets. Here, we implicate A subfamily ephrins in this topographic mapping. First, developing muscles express all five of the ephrin-A genes. Second, rostrally and caudally derived motor axons differ in sensitivity to outgrowth inhibition by ephrin-A5. Third, the topographic map of motor axons on the gluteus muscle is degraded in transgenic mice that overexpress ephrin-A5 in muscles. Fourth, topographic mapping is impaired in muscles of mutant mice lacking ephrin-A2 plus ephrin-A5. Thus, ephrins mediate or modulate positionally selective synapse formation. In addition, the rostrocaudal position of at least one motor pool is altered in ephrin-A5 mutant mice, indicating that ephrins affect nerve-muscle matching by intraspinal as well as intramuscular mechanisms.     

The specificity of motor innervation of the chick wing does not depend upon the segmental origin of muscles

In vertebrate embryos, motor axons originating from a particular craniocaudal position in the neural tube innervate limb muscles derived from myoblasts of the same segmental level. We have investigated whether this relationship is important for the formation of specific nerve-muscle connections, by altering the segmental origin of muscles and examining their resulting innervation. First, by grafting quail wing somites to a new craniocaudal position opposite the chick wing, we established that the segmental origin of a muscle can be altered: presumptive muscle cells migrated according to their new, rather than their original, somitic level, colonizing a different subset of muscles. However, after reversal of a length of brachial somitic mesoderm along the craniocaudal axis, or exchange or shift of brachial somites, the craniocaudal position of wing muscle motoneurone pools within the spinal cord was undisturbed, despite the new segmental origin of the muscles themselves. While not excluding the possibility that muscles and their motor nerves are labelled segmentally, we conclude that specific motor axon guidance in the wing does not depend upon the existence of such labels.     

Levels of free amino acids in excitatory, inhibitory and sensory axons of the walking limbs of the lobster

Using a gas chromatography procedure, the levels of several amino acids were determined in individual excitatory and inhibitory axons, in bundles of sensory fibers, and in muscle tissue from the walking limb of the lobster, $\text{Homarus}$$\text{americanus}$. In addition, the levels of amino acids in the hemolymph were also determined. Of the amino acids assayed in the excitatory and inhibitory axons and in the sensory fibers the level of aspartate was highest whereas in hemolymph and muscle, aspartate had one of the lowest values. The levels of glutamate, glycine and proline were significantly higher in the excitatory axons than in the inhibitory axons. GABA was present in inhibitor axons and in the muscle tissue which these axons innervate and was not detected in the other axons assayed nor in the hemolymph. β-Alanine was present at low levels in hemolymph and in muscle but was not detected in the excitatory nor in the inhibitory axons.     

Regeneration of motorneurones to a cricket muscle after partial or complete denervation

The metathoracic extensor tibiae muscle of the cricket Teleogryllus oceanicus is innervated by two excitatory axons; one of which leaves the metathoracic ganglion through nerve 5, the other through nerve 3. Axons in nerve 5 frequently regenerate to reinnervate the extensor tibiae if the nerve is sectioned in a late nymphal stage; functional reinnervation is rare if the nerve is sectioned in young adults. The muscle may become reinnervated by several axons regenerating through nerve 5, and individual muscle fibres may receive inputs from two regenerated axons. Axons regrowing through nerve 5 to a partially-denervated extensor tibiae preferentially innervate fibres in the central portion of the muscle, which is the normal innervation field of nerve 5. If the muscle is totally denervated by transection of both nerve 5 and nerve 3b, reinnervation is less specific and fibres throughout the muscle may be reinnervated by axons in either nerve. Reinnervation by regenerating axons is progressive. The proportion of muscles which are functionally reinnervated by regenerated axons increases with survival time as does the proportion of fibres within a muscle with reinnervation. The amplitude of excitatory junctional potentials and of muscle contraction evoked by regenerated axons both increase with survival time.     

The tonotopic organization of the bullfrog amphibian papilla,an auditory organ lacking a basilar membrane

Summary Intracellular dye-injection studies have revealed tonotopic organization of the bullfrog (Rana catesbeiana) amphibian papilla, an auditory organ lacking a basilar membrane or its equivalent. The best excitatory frequency (BEF) for auditory stimuli was identified in each of twenty-nine VIIIth-nerve afferent axons that subsequently were traced to their peripheral terminations at the sensory surface. Among those axons, the five with BEFs greater than 550 Hz all terminated in the caudalmost region of the papilla, the ten with the BEFs greater than 300 Hz and less than or equal to 550 Hz all terminated in the central region of the papilla, and the fourteen with BEFs equal to or less than 300 Hz all terminated in the rostralmost region of the papilla (Fig. 4). The tectorium is very much larger and presumably more massive under the low-frequency region of the papilla than it is under the high-frequency region (Fig. 1). Higher-frequency axons tended to innervate few (one to four) receptor cells, and low-frequency axons tended to innervate many (six or more). Higher-frequency axons often terminated in large claw-like structures that engulfed the basal portions of individual hair cells and in this way were morphologically similar to type I terminals in the inner ears of higher vertebrates.Abbreviations BEF best excitatory frequency - HRP horseradish peroxidase     

Neocortical Axon Arbors Trade-off Material and Conduction Delay Conservation

The brain contains a complex network of axons rapidly communicating information between billions of synaptically connected neurons. The morphology of individual axons, therefore, defines the course of information flow within the brain. More than a century ago, Ramón y Cajal proposed that conservation laws to save material (wire) length and limit conduction delay regulate the design of individual axon arbors in cerebral cortex. Yet the spatial and temporal communication costs of single neocortical axons remain undefined. Here, using reconstructions of in vivo labelled excitatory spiny cell and inhibitory basket cell intracortical axons combined with a variety of graph optimization algorithms, we empirically investigated Cajal''s conservation laws in cerebral cortex for whole three-dimensional (3D) axon arbors, to our knowledge the first study of its kind. We found intracortical axons were significantly longer than optimal. The temporal cost of cortical axons was also suboptimal though far superior to wire-minimized arbors. We discovered that cortical axon branching appears to promote a low temporal dispersion of axonal latencies and a tight relationship between cortical distance and axonal latency. In addition, inhibitory basket cell axonal latencies may occur within a much narrower temporal window than excitatory spiny cell axons, which may help boost signal detection. Thus, to optimize neuronal network communication we find that a modest excess of axonal wire is traded-off to enhance arbor temporal economy and precision. Our results offer insight into the principles of brain organization and communication in and development of grey matter, where temporal precision is a crucial prerequisite for coincidence detection, synchronization and rapid network oscillations.     

The distribution of the common inhibitory neuron in brachyuran limb musculature

Summary In the walking legs of two common crabs, antidromic stimulation of the common inhibitory axon (CI) from either opener or closer nerve produces inhibitory potentials in certain fibers of every muscle distal to the ischiopodite. In particular, CI inhibits the flexor and accessory flexor muscles of the meropodite and abolishes or reduces contractile force in the flexor. The specific opener inhibitor, OI, formerly believed to innervate the flexor, has no electrical or mechanical effect on this muscle. The brachyuran inhibitory limb innervation thus appears to be the same as that accepted for the anomurans, comprising one universally distributed common inhibitor and two truly specific inhibitors serving the opener and stretcher muscles.Abbreviations CI common inhibitor - OI opener inhibitor - SI stretcher inhibitor - FI flexor inhibitor - FE flexor excitor(s)