Regeneration of an identifiable motoneuron in the crayfish. I. Patterns of reconnection and synaptic strength established in normal and altered target areas

The superficial flexor muscles of the crayfish are innervated in a position-dependent connectivity pattern, which can be reestablished when the nerve to the muscle is cut. This article deals with the regeneration of the largest excitor motoneuron under three different target scenarios: (1) a normal target with all the muscle fibers present, (2) a reduced target lacking the medial or the lateral muscle fiber population, and (3) when the nerve enters the target in the middle of the muscle field. In scenario 1 the neuron is able to regenerate the normal connectivity pattern within 10 weeks after surgery: all the lateral fibers become innervated, with a linear decline in the probability of connections over the medial fibers. The medial fibers become transiently hyperinnervated before the normal pattern of connections is established. In scenario 2 the normal pattern of connections is established only when the lateral fibers were present; with only medial cells as a target, the transient hyperinnervation stage is stable and no decline in connections was observed. Analysis of regenerated junction potential sizes during the stable hyperinnervation stage show abnormal patterns, suggesting that some aspects of the regeneration program of this neuron can be affected when signals from its prime target cells are missing. In scenario 3 growth begins in both directions until the entire muscle becomes innervated. The normal pattern of connectivity finally emerges after continued lateral growth and diminished medial growth, suggesting that the position of the muscle fibers influences connectivity patterns during the final stages of regeneration.     

Regeneration studies on a crayfish neuromuscular system. II. Effect of changing the nerve entry point into the muscle field on the gradient of innervation

The superficial flexor muscle of the crayfish is a neuromuscular system in which the neurons form position-dependent connectivity patterns with the muscle fibers. This system could be formed with the help of a single medial-to-lateral gradient during development that embodies positional information. To test this gradient hypothesis we changed the nerve's normal medial entry point into the muscle by transplanting it to the middle of the muscle sheet. When all the muscle fibers were present in the target area, most of the neurons studied passed through a stage during regeneration in which they showed preference for either medial or lateral synapse formation. Those neurons that in normal animals innervated preferentially the medial fibers showed a medial preference for new contacts; the neuron that normally innervated the lateral fibers showed a lateral preference for new contacts; the neuron that normally innervated everywhere regenerated equally well into both medial and lateral fibers. Therefore, these neurons are able to detect information regarding their position within the muscle mass and respond to it by preferential synapse formation. The effect of a positional gradient could not be detected when half of the target field was removed prior to regeneration. In this instance, the neuron that innervated the missing target area now regenerated to almost all the available fibers. It is suggested that the interplay of positional cues with other factors at different points in time could determine the final connectivity patterns formed by these cells.     

Regeneration of neuromuscular connections in crayfish allotransplanted neurons

Transplantation of whole ganglia was used to study the regeneration of four of the neurons that innervate the superficial flexor muscles of the crayfish Procambarus clarkii. The isolated ganglia containing the somas of these neurons were successfully transplanted from one crayfish to another. Reinnervation proceeded across the muscle surface and by 8 to 10 weeks connections were detected across the entire target field. At different time periods after the transplant, junction potentials (JPs) produced in phase with spontaneous neuronal spikes were recorded. The distribution of JP sizes and their decay times were examined. JPs from transplanted preparations were smaller than JPs from control or normal regeneration animals. These JPs also failed to facilitate when stimulated at 1 and 10 Hz. These are normal characteristics of immature terminals, but in the transplant preparations, once established, they remained stable for the duration of the study. Thus, synaptogenesis appears to be arrested at a stage before synaptic efficacy is established in the allotransplants. In addition, connectivity maps were plotted for each axon over the muscle surface. Some muscle fibers did not receive any contacts, and overall innervation leveled off at around 60% of the muscle fibers, remaining stable for the duration of this study. Despite the incomplete physiological innervation, however, three of the four neurons showed the same medial/lateral preferences observed in control animals, regenerating their original patterns of connectivity across the muscle surface. © 1995 John Wiley & Sons, Inc.     

Regeneration of an identifiable motoneuron in the crayfish. II. Patterns of reconnection and synaptic strength established in the presence of an extra nerve

The regeneration of neuromuscular connections to the superficial flexor muscle system in the crayfish has been studied under a variety of experimental manipulations. These have provided insight into the factors that can influence the regeneration program of neurons. In this work the regeneration of the largest excitor motoneuron was studied under two different conditions: (1) when the original neuron and a transplanted neuron were growing simultaneously into a denervated target, and (2) when a transplanted neuron was growing into a target that had its original nerve supply intact. In condition 1 both the transplanted and the original neuron formed normal patterns of connectivity and synaptic strength in comparable periods of time. In condition 2 the rate of growth of the transplanted neuron is significantly reduced and does not extend into the lateral fibers of the muscle. It is concluded that the regeneration program of this neuron is not affected by the presence of other neurons growing at the same time into a denervated muscle. Since regeneration is seriously affected if growth occurs into a fully innervated target area, it is suggested that lack of growth stimuli from the target or competitive interactions between established and growing synaptic terminals could influence the regeneration program of this neuron.     

The structure and function of serially homologous leg motor neurons in the locust. II. Physiology

Simultaneous intracellular recordings were made from pairs of motor neurons in the pro- or mesothoracic ganglion of the locust. Though central connections were sought between pairs of motor neurons, none were found. This is in sharp contrast to the findings that flexor and extensor tibiae neurons in the metathoracic ganglion make certain connections between themselves (Hoyle and Burrows, 1973; Heitler and Burrows, 1977a). As the previously mentioned authors believed that the metathoracic flexor-extensor connections were used as part of the motor program for jumping and kicking, the present results strongly support their hypothesis. Common PSPs have been found in a variety of pairs of motor neurons. Of note are common PSPs of the same sign to antagonists. Different innervation patterns have been found for the flexor and extensor muscles. It is proposed that serially homologous motor neurons serving similar functions are, to a first approximation, similar in the locust. Serially homologous motor neurons serving different functions will, in most cases, have altered structures and/or functions.     

Plasticity in the cockroach neuromuscular system

The retrograde transport of wheat germ agglutinin-conjugated horseradish peroxidase extracellularly injected into a leg muscle was used to identify the regenerating cockroach motor neurons that have grown an axonal branch into that muscle. At least 66% of the animals with crushed nerve roots eventually reform the original innervation pattern of this muscle with no mistakes. In spite of this apparent specificity the cockroach neuromuscular system can express plasticity as evidenced by the correction of mistakes made at early stages of regeneration. These mistakes are corrected through elimination during the time interval between 40 and 60 days after nerve crush. In addition, when the distal segments of the leg are removed, thus depriving some motor neurons of their normal target muscles, many of them form stable inappropriate axonal branches in denervated as well as fully innervated muscles. These observations are discussed in terms of possible mechanisms responsible for the specificity of the cellular interactions and in terms of their relevance to understanding the development of vertebrate neuromuscular systems.     

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.     

Thenar and hypothenar muscles and their innervation by the ulnar and median nerves in the human hand.

The thenar and hypothenar muscles as well as their supplying nerves were analyzed with an improved dissecting method. Among the four thenar muscles, the m. abductor pollicis brevis (AbPB) has a separate muscle belly, whereas the m. opponens pollicis (OP), the superficial and deep heads of the flexor pollicis brevis (sFPB and dFPB), and the adductor pollicis (AdP) are fused with each other to make a single mass (deep thenar muscle group). These muscles are innervated by branches of the recurrent nerve and the accessory recurrent nerve from the median nerve as well as by terminal branches of the deep branch (ramus profundus) of the ulnar nerve. These three nerves frequently form a loop within the deep thenar muscle group (thenar loop), and a branch to the OP and one to deep parts of the sFPB often make a smaller loop (intrathenar loop), whereas the AbPB receives a separate nerve branch. Among the hypothenar muscle, the m. abductor digiti minimi and the m. flexor digiti minimi brevis are fused with each other, and their supplying nerves frequently form a loop in these muscles (intrahypothenar loop), whereas the m. opponens digiti minimi is separated from the others and receives a separate nerve branch. In the distribution pattern of supplying nerves to the thenar and hypothenar muscles, we find regularities in that they branch off in a regular manner from the ulnar and the median nerve, and that nerve branches to those muscles with fused bellies frequently communicate with each other to make loops.     

Location and connectivity of abdominal motoneurons in the embryo and larva of Drosophila melanogaster

The soma location and peripheral connectivity of motoneurons in abdominal segments of the embryo and larva of the fruitfly, Drosophila melanogaster are described as an initial step in determining the mechanisms by which motoneurons make connections with their target muscles in a genetically accessible organism. Embryonic motoneuron somata were retrogradely labelled by application of the fluorescent dye, DiI, to the whole peripheral nerve or to its separate anterior or posterior fascicles in segments A5-A7 of late stage 15/early stage 16 embryos. This technique reveals a stereotyped, segmentally repeated population of 34 motoneurons per hemisegment, several of which can be individually identified from their soma position. The same set of motoneurons was revealed in third instar larvae of D. melanogaster by cobalt backfilling of abdominal peripheral nerves, although the positions of some of these neurons change during larval development. The peripheral connectivity and axon morphology of several of the abdominal motoneurons was determined by intracellular injection with Lucifer Yellow in stage 16 embryos. For the motoneurons with axons in the anterior fascicle there is no clear relationship between somata groupings and the muscle targets innervated: contrary to earlier claims, these motoneurons arborize over both ventral and dorsal muscles. Individual motoneurons possess a stereotyped pattern of terminal arborization.     

Intersegmental variation of afferent pathways to giant interneurons of the earthworm,Lumbricus terrestris L.

Summary We have investigated the connectivity of four classes of mechanosensory afferents to giant interneurons in the earthwormLumbricus. Three of these classes of afferents change their specification for connection to medial giant (MGF) and lateral giant (LGF) fibers along the length of the animal. Near the caudal end, stimulation of touch, pressure and small tactile fibers generates excitatory post-synaptic potentials, epsp's, in the two LGF's but not in the MGF. Near the rostral end these afferents produce much smaller epsp's in the LGFs but produce large epsp's in the MGF. In the middle region of the animal an overlap region exists where both giant fibers receive approximately equal inputs from these afferents. The amplitude of these inputs are reduced compared to the maxima seen at either end. The fourth class of sensory afferents investigated, the stretch neurons, have no synaptic effect on the giant fibers anywhere in the nerve cord.These results explain at least part of the basis, in neuronal connectivity, for the differences in response to tactile stimulation of the head and tail segments previously characterized in terms of behavior and giant fiber impulse activity. In this system developmental mechanisms generating synaptic connectivity patterns have coded certain classes of homologous afferent neurons and interneurons to make different connections in different segments.Abbreviations MGF medial giant fiber - LGF lateral giant fiber - SN1 first segmental root - SN2 second segmental root - SN3 third segmental root - RIN giant interneuron     

Anatomical partitioning of three multiarticular human muscles.

To examine neuromuscular partitioning within human muscles, the innervation patterns and muscle fiber architecture of the flexor carpi radialis (FCR), extensor carpi radialis longus (ECRL) and lateral gastrocnemius (LG) muscles were examined. Consistent patterns of innervation between specimens were found within each of the three muscles. The nerve to the FCR clearly innervates three major architectural divisions of the muscle. The ECRL is innervated by two different muscle nerves. Branches of these nerves innervate at least two distinct anatomical subvolumes. However, the subvolumes of the ECRL defined by muscle architecture are not totally congruent with those defined by the innervation pattern. In the LG, the single muscle nerve branches into two main divisions, and these subsequently divide into branches which supply the three heads. However, each head does not receive a completely private nerve. These results indicate that human muscles are partitioned in a manner roughly similar to the divisions of the same muscles in cats and rats, but with less congruency of architecture and innervation.     

Absence of competitive interactions among axon terminals of regenerating motor neurons

Competition among axon terminals is usually considered to contribute to the formation of patterned synaptic connections. During axonal regeneration of motor neurons in the cockroach, leg muscles initially become innervated by appropriate and inappropriate motor neurons. All axon terminals from inappropriate neurons eventually are eliminated, resulting in the reformation of the original innervation pattern. Destruction of an identified motor neuron by the intracellular injection of pronase did not prevent the elimination of inappropriate axon terminals in the muscle normally innervated by that motor neuron. Therefore, competition does not play a role in the reinnervation of the leg muscles. This indicates a major role for specific cell-cell recognition.     

Structure of allotransplanted ganglia and regenerated neuromuscular connections in crayfish

In adult crayfish, Procambarus clarkii, motoneurons to a denervated abdominal superficial flexor muscle regenerate long-lasting and highly specific synaptic connections as seen from recordings of excitatory postsynaptic potentials, even when they arise from the ganglion of another crayfish. To confirm the morphological origins of these physiological connections we examined the fine structure of the allotransplanted tissue that consisted of the third abdominal ganglion and the nerve to the superficial flexor muscle (the fourth ganglion and the connecting ventral nerve cord were also included). Although there is considerable degeneration, the allotransplanted ganglia display intact areas of axon tracts, neuropil, and somata. Thus in both short (6–8 weeks) and long (24–30 weeks) term transplants approximately 20 healthy somata are present and this is more than the five axons regenerated to the host muscle. The principal neurite and dendrites of these somata receive both excitatory and inhibitory synaptic inputs, and these types of synaptic contacts also occur among the dendritic profiles of the neuropil. Axon tracts in the allotransplanted ganglia and ventral nerve cord consist largely of small diameter axons; most of the large axons including the medial and lateral giant axons are lost. The transplanted ganglia have many blood vessels and blood lacunae ensuring long-term survival. The transplanted superficial flexor nerve regenerates from the ventral to the dorsal surface of the muscle where it has five axons, each consisting of many profiles rather than a single profile. This indicates sprouting of the individual axons and accounts for the enlarged size of the regenerated nerve. The regenerated axons give rise to normal-looking synaptic terminals with well-defined synaptic contacts and presynaptic dense bars or active zones. Some of these synaptic terminals lie in close proximity to degenerating terminals, suggesting that they may inhabit old sites and in this way ensure target specificity. The presence of intact somata, neuropil, and axon tracts are factors that would contribute to the spontaneous firing of the transplanted motoneurons. © 1996 John Wiley & Sons, Inc.     

Synaptic repression at crayfish neuromuscular junctions. I. Generation after partial target area removal

Synaptic repression, the inability of synaptic junctions to generate normal-sized postsynaptic potentials under normal physiological conditions, is reported here for crayfish neuromuscular synapses. The synapses in the superficial flexor muscle system of the crayfish change their efficiency in generating a postsynaptic response as a result of a specific alteration in their immediate environment. When the superficial flexor nerve is cut halfway into the target muscle field and the lateral muscle fibers are removed, the intact medial synapses do not generate normal-sized junction potentials (JP) at the 17° –19°C temperature of the Ringers solution. JPs cannot be recorded in 83% of the muscle fibers at 2 weeks after the operation and of the few JPs that can be detected, 80% are smaller than 1 mV in size. By 8 weeks after the operation, JPs were detected in 55% of the muscle fibers, and now only 46% of these are smaller than 1 mV. When the lateral muscle fibers are left in place during the original operation, providing a target area for the cut nerve to grow into, JPs were then detected in 60%–80% of all medial fibers at all time periods after the operation; their size profile, with 10%–25% of the muscle fibers having JP's less than 1 mV, was similar to control values. These results suggest that the efficiency of these synaptic contacts become affected as a result of partial axotomy and removal of the target area of the cut branches of the axons. © 1993 John Wiley & Sons, Inc.     

Homology of the muscles within the uropatagium membrane in Leschenault’s rousette (Rousettus leschenaultii)

Pteropodidae possess unique muscles in the uropatagium called Musculus uropatagialis and M. depressor ossis styliformis. The homology of these muscles is important for the phylogenetic analysis of bats because the wing membrane is a characteristic feature for them. Here, I discuss the homology of M. uropatagialis and M. depressor ossis styliformis in Rousettus leschenaultii by tracing their innervations. I found that the dominant nerve for the M. uropatagialis contains the components of the sciatic nerve associated with the dominant nerve of the flexor muscles of the thigh. This result shows that M. uropatagialis is homologous to the flexor muscles of the thigh. The dominant nerve of M. depressor ossis styliformis is the lateral plantar nerve derived from the tibial nerve. Thus, this innervation pattern proposes the hypothesis that M. depressor ossis styliformis is homologous to one of the muscles of the foot sole.     

The effects of 5-HT on sensory, central and motor neurons driving the abdominal superficial flexor muscles in the crayfish

Serotonin (5-HT) induces a variety of physiological and behavioral effects in crustaceans. However, the mechanisms employed by 5-HT to effect behavorial changes are not fully understood. Among the mechanisms by which these changes might occur are alterations in synaptic drive and efficacy of sensory, interneurons and motor neurons, as well as direct effects on muscles. We investigated these aspects with the use of a defined sensory-motor system, which is entirely contained within a single abdominal segment and consists of a ‘cuticular sensory neurons–segmental ganglia–abdominal superficial flexor motor neurons–muscles’ circuit. Our studies address the role of 5-HT in altering (1) the activity of motor neurons induced by sensory stimulation; (2) the inherent excitability of superficial flexor motor neurons; (3) transmitter release properties of the motor nerve terminal and (4) input resistance of the muscle. Using en passant recordings from the motor nerve, with and without sensory stimulation, and intracellular recordings from the muscle, we show that 5-HT enhances sensory drive and output from the ventral nerve cord resulting in an increase in the firing frequency of the motor neurons. Also, 5-HT increases transmitter release at the neuromuscular junction, and alters input resistance of the muscle fibers     

Muscle development in the grasshopper embryo. I. Muscles, nerves, and apodemes in the metathoracic leg

Much is known about the development of nerve pathways in the metathoracic limb bud of the grasshopper embryo. In this series of three papers, we report on the development of muscles in the same embryonic appendage. In a fourth paper (E. E. Ball, R. K. Ho, and C. S. Goodman, 1985, J. Neurosci, in press) we examine the development of specific neuromuscular connections for one of these muscles (coxal muscle 133a). In this first paper, we present an overview of the development of muscles, nerves, and apodemes (tendons). We previously reported on a class of large mesodermal cells, called muscle pioneers (MPs), that arises early in development and appears to act as a scaffold for developing muscles and guidance cue for motoneuron growth cones (R. K. Ho, E. E. Ball, and C. S. Goodman, 1983, Nature (London) 301, 66-69). We have used the I-5 monoclonal antibody (which specifically labels the MPs as well as the nerve pathways), HRP immunocytochemistry, and Normarski optics to visualize muscle, nerve, and apodeme development in the embryonic metathoracic limb bud from 27.5% (before the appearance of the MPs) to 55% (after the muscles have attained their basic adult pattern). Cell fusions, cell migration, and cell death all appear to play important roles in the development of MPs. The patterns of muscle development vary greatly, ranging from (i) single MPs for simple muscles (which in the adult have only one bundle of muscle fibers, e.g., coxal muscle 133a), to (ii) arrays of MPs for complex muscles [which in the adult have many bundles of muscle fibers each with separate sites of insertion, e.g., the extensor tibiae (ETi) and flexor tibiae (FlTi) muscles in the femur].     

Innervation and motor patterns of the abdominal superficial flexor muscles in larval lobsters

The pattern of innervation and motor program of the abdominal superficial flexor muscle was investigated electrophysiologically in larval lobsters (Homarus americanus). The muscle receives both excitatory and inhibitory innervation in the larval as well as in the embryonic stages. Individual muscle fibers receive a single inhibitory neuron (f5) and a maximum of three excitors. Based on spike heights these axons belong to either the small (f1 or f2) or large (f3, f4) motoneurons. While the small axons preferentially innervate the medial muscle fibers the large axons innervate medial as well as lateral fibers. This larval pattern of innervation resembles the pattern in the adult lobster. The resemblance extends to the firing patterns as well with both large and small excitors firing spontaneously. Furthermore, evoked activity in the larvae produces reciprocal (and occasionally cyclical) bursts of excitor and inhibitor neurons denoting abdominal extension and flexion and resembling the firing patterns in adults. Consequently motor programs employed in steering the pelagic larvae are reminiscent of the programs for maintaining posture in the benthic adult lobsters.     

Musculature,nervous system and glands of metasomal abdominal segments of the male of Nomia melanderi Ckll. (Hymenoptera,Apoidea)

Each muscle of the third metasomal segment of the male of Nomia melanderi Ckll. is described in detail. The points of attachment of each muscle are compared with their homologs in other pregenital segments and with their homologs in the female. The function desgnated for each muscle describes its action alone or in conjunction with other muscle(s). New names are given to genital muscles by referring in the name to their points of attachment. Each intratergal muscle has homologous points of attachment in the pregenital segments of both sexes. The median tergo-dorsoplical muscle of the seventh segment and the oblique tergo-dorsoplical muscle of the eighth segment have changed their points of attachment. The intrasternal muscles are modified to suit the needs of courtship and mating, thus they are different from their homologs in the female. The spiracular muscles are well developed in all segments except the eighth, where the sterno-spiracular muscle is absent. The extrinsic genital muscles are derived from the intrasternal muscles of the eighth and ninth segments. The parameral and volsellar muscles are reduced in number. The aedeagal muscles, except the aedeago-phallic, have retained similar points of attachment to those found in primitive Hymenoptera. The topography of the metasomal nervous system is reported in detail by following each nerve and nervule to its termination. The study shows that (at least in Nomia) the criterion of nerve-concentration should not be used alone to indicate evolutionary levels. To accommodate the morphological changes in the terminal segments the Anterior and Posterior Lateral Nerves have migrated to new locations. The pattern of nerve topography (even at the nervule level) is homologous both in the different pregenital segments and between the sexes. The fact that homology does not exist between the external genitalia of the male and the modified ovipositor of the female supports the thesis that the male genital capsule is of phallic rather than prephallic origin. A pair of intersegmental membrane glands located between the seventh and eighth sterna is described. These glands may be the source of a pheromon responsible for gregariousness among “sleeping” males.