Neuromuscular Connections Research Articles

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.

The Drosophila abrupt gene encodes a BTB-zinc finger regulatory protein that controls the specificity of neuromuscular connections.

The Drosophila abrupt gene encodes a BTB-zinc finger regulatory protein that controls the specificity of neuromuscular connections.

Neuromuscular contacts of expanded motor units in rat soleus muscles are rescued by leupeptin

In the soleus muscle of the rat following section of the L5 ventral ramus (partial denervation) the remaining motor axons increase their territory by sprouting. Nerve sprouts are first seen two to three days after the operation, their number peaks at 10–14 days and subsequently remains at this level. The time course of the initial sprouting in partially denervated muscles is not altered by paralysing the muscles with α-bungarotoxin, and the initial extent of the sprouting is, if anything, greater in the paralysed muscles. However, unlike in controls, this level of sprouting is not maintained and neuromuscular contacts are lost when muscles recover from the paralysis. The loss of these contacts can be prevented by treatment of these partially denervated paralysed muscles with leupeptin, an inhibitor of calcium-activated neutral protease. Interestingly, more contacts are rescued when leupeptin is applied 10 days after α-bungarotoxin treatment, when sprouting has reached high levels, than at three days, when sprouting has just begun. The neuromuscular connections rescued by leupeptin are functional. Maximum tetanic tension produced by untreated soleus muscles two to five months after partial denervation is66 ± 9% of contralateral control muscles, but only39 ± 8% when the muscles were paralysed with α-bungarotoxin for 12–14 days after partial denervation. However, when partially denervated paralysed muscles were treated with leupeptin three and 10 days after α-bungarotoxin treatment their tension output is74 ± 3% and81± 8%, respectively. After partial denervation alone, motor units are twice their normal size. Short-term paralysis with α-bungarotoxin prevents this increase in motor unit territory. However, the application of leupeptin to the paralysed muscles rescues neuromuscular contacts, allowing motor unit size to remain expanded, at around 2–2.5-fold. Thus, following recovery from temporary paralysis with α-bungarotoxin, there is a sudden withdrawal of neuromuscular contacts and these can be rescued by treatment with leupeptin.

Neuromuscular regeneration by buccal motoneuron B15 after peripheral nerve crush in Aplysia californica.

1. We studied regeneration of neuromuscular connections by identified buccal motoneuron B15 after axotomy produced by crushing nerve 4; the intact contralateral nerve 4 served as control. Electrophysiological recordings, intracellular dye injections, and light and electron microscopy were used to characterize the nature and time course of neuromuscular reinnervation as well as the fate of the isolated distal stump of the motor axon. 2. Axonal outgrowth or sprouting in the form of numerous "regenerites" occurred from the proximal stump of the transected B15 axon, and these regenerites projected through the crush site along the length of the nerve to innervate target muscles at the periphery. 3. Reinnervation of one of the target muscles, the accessory radula closer (I5), was first detected 3 wk after nerve crush. Neuromuscular excitatory postsynaptic potentials measured in individual I5 muscle fibers were initially small and approached control amplitudes by 8 wk postlesion. Newly regenerated neuromuscular synapses displayed facilitation and depression to repeated B15 stimulation with properties similar to those of control synapses, even at early times postlesion. 4. Reinnervation of other buccal muscles by B15, such as I4, appeared slightly delayed relative to that observed for I5. No evidence of abnormal or enlarged fields of innervation were observed, and as in control preparations, regenerated neuromuscular connections were strictly limited to muscles ipsilateral to the B15 cell body. 5. Physiological evidence suggested that the distal axon stumps of B15, although isolated from their cell bodies, survive for several weeks after axotomy. In addition, several large axon profiles indicative of motor axons were seen in cross-sections of nerve 4 taken close to the muscle and distal to the crush site, indicating survival of distal axon stumps. 6. When B15 was selectively stimulated, the newly formed regenerites failed to fire the distal axon stump of B15, demonstrating that the regenerites do not reinnervate the distal stump. 7. Degeneration of axons in nerve 4 distal to the crush site was observed in cross-sections of the nerve at 8 wk postlesion; using ultrathin sections we found cellular debris in individual axon profiles as well as large acellular masses within nerve 4, the latter likely representing the concretion of many axons. Additional evidence for such degenerative changes appeared in the form of autofluorescing spherical bodies or "spheroids" both in individual axons and the nerve distal to the crush site.(ABSTRACT TRUNCATED AT 400 WORDS)

Persistence of functional neuromuscular junctions formed in a denervated skeletal muscle of the adult rat by axons that have regrown from the injured spinal cord through a peripheral nerve autograft

In previous 'short-term' (2 to 7 months) experiments, we had demonstrated, in the adult rat, that motoneurons of the injured cervical spinal cord could extend lengthy axons into an autologous peripheral nerve segment which was connected to a nearby denervated skeletal muscle. In addition, we had shown that new functional motor endplates were formed by these axons both at the original sites of innervation and at ectopic locations of the denervated muscle. This substitution motor system, although quite functional, was anatomically very different from the original model of innervation in the intact animal, relating to its motoneuronal pool, the course of its motor axons and the sites of terminal innervation. The present 'long-term' (11 to 21 months) experiments demonstrate the anatomical and functional permanency of the new motor circuitry, despite a lack of strict specificity in the new neuromuscular connections. However, some minor modifications or adjustments were observed with time: (i) the maintenance of functional ectopic endplates could be consistently demonstrated, while functional reinnervated endplates at the initial sites of innervation were rare or even lacking; (ii) there was a definitive withdrawal of all non target-specific regenerated axons from the vicinity of the muscle. It is now necessary to address the question to what extent this substitution motor system is actually controlled by central and/or peripheral inputs.

The cell adhesion molecule, connectin, and the development of the Drosophila neuromuscular system.

The connectin gene of Drosophila has been identified as a candidate direct target of homeotic gene control and has also been implicated in the formation of specific neuromuscular connections. The gene product, connectin, is a member of the leucine-rich repeat protein family and we show that it is attached to the cell surface via a glycosylphosphatidylinositol linkage and that it can mediate homotypic cell-cell adhesion in vitro. The expression of connectin protein during Drosophila embryogenesis provides support for a role in adhesion in vivo. In the central nervous system, it is initially expressed on longitudinal glia and on a few identified neurons. These cells extend processes and connect up to form a continuous scaffold of connectin-expressing cells, presaging the development of axonal pathways. Later, connectin is expressed on specific axons as they track along the connectin scaffold. Glial expression then declines and connectin appears on axons that fasciculate with pre-existing connectin-positive bundles. Thus scaffold formation, axon pathfinding and fasciculation involve specific contacts between connectin-positive cells. The timing and pattern of connectin expression suggest that it may play an important role in mediating specific interactions through homotypic cell adhesion.

Methods for ablating neurons

This past year, laser ablation has been applied to investigations of neuromuscular connectivity in Drosophila, neuronal function in nematode, and mammalian central nervous system development. Ablation by targeted gene expression has been refined and applied to questions of neural development. Chromophore-assisted laser inactivation has been used to demonstrate distinct functions for two proteins during grasshopper neural development.

Cellular mechanisms governing synaptic development in Drosophila melanogaster

The neuromuscular connections of Drosophila are ideally suited for studying synaptic function and development. Hypotheses about cell recognition can be tested in a simple array of pre- and postsynaptic elements. Drosophila muscle fibers are multiply innervated by individually identifiable motoneurons. The neurons express several synaptic cotransmitters, including glutamate, proctolin, and octopamine, and are specialized by their synaptic morphology, neurotransmitters, and connectivity. During larval development the initial motoneuron endings grow extensively over the surface of the muscle fibers, and differentiate synaptic boutons of characteristic morphology. While considerable growth occurs postembryonically, the initial wiring of motoneurons to muscle fibers is accomplished during mid-to-late embryogenesis (stages 15-17). Efferent growth cones sample multiple muscle fibers with rapidly moving filopodia. Upon reaching their target muscle fibers, the growth cones rapidly differentiate into synaptic contacts whose morphology prefigures that of the larval junction. Mismatch experiments show that growth cones recognize specific muscle fibers, and can do so when the surrounding musculature is radically altered. However, when denied their normal targets, motoneurons can establish functional synapses on alternate muscle fibers. Blocking synaptic activity with either injected toxins or ion channel mutants does not derange synaptogenesis, but may influence the number of motor ending processes. The molecular mechanisms governing cellular recognition during synaptogenesis remain to be identified. However, several cell surface glycoproteins known to mediate cellular adhesion events in vitro are expressed by the developing synapses. Furthermore, enhancer detector lines have identified genes with expression restricted to small subsets of muscle fibers and/or motoneurons during the period of synaptogenesis. These observations suggest that in Drosophila a mechanism of target chemoaffinity may be involved in the genesis of stereotypic synaptic wiring.

Genes that control neuromuscular specificity in Drosophila

In each abdominal hemisegment of the Drosophila embryo, an array of 30 muscle fibers is innervated by about 34 motoneurons in a highly stereotyped and cell-specific fashion. To begin to elucidate the molecular basis of neural specificity in this system, we conducted a genetic screen for mutations affecting neuromuscular connectivity. We focus on 5 genes required for specific aspects of pathway ( beaten path, stranded, and short stop) and target ( walkabout and clueless) recognition. The different classes of mutant phenotypes suggest that neural specificity is controlled by a hierarchy of molecular mechanisms: motoneurons are guided toward the correct region of mesoderm, in many cases navigating a series of choice points along the way; they then display an affinity for a particular domain of neighboring muscles; and finally, they recognize their specific muscle target from within this domain.

Presynaptic and postsynaptic competition in models for the development of neuromuscular connections.

In the establishment of connections between nerve and muscle there is an initial stage when each muscle fibre is innervated by several different motor axons. Withdrawal of connections then takes place until each fibre has contact from just a single axon. The evidence suggests that the withdrawal process involves competition between nerve terminals. We examine in formal models several types of competitive mechanism that have been proposed for this phenomenon. We show that a model which combines competition for a presynaptic resource with competition for a postsynaptic resource is superior to others. This model accounts for many anatomical and physiological findings and has a biologically plausible implementation. Intrinsic withdrawal appears to be a side effect of the competitive mechanism rather than a separate non-competitive feature. The model's capabilities are confirmed by theoretical analysis and full scale computer simulations.

Testosterone fails to rescue motoneurons from axotomy-induced death in young rats.

This study was undertaken to determine whether testosterone acting on muscle alone could enhance motoneuron survival after axotomy. Facial nerves were transected unilaterally in 10-day old rats in which androgen receptors were already present in skeletal muscle but not yet developed in motoneurons. Testosterone treatment lessened chromatolysis but failed to reduce neuronal loss which reached more than 50% 2 weeks postaxotomy. The inefficacy of testosterone was attributed to the death of motoneurons before they could re-establish synaptic contact with targets, thereby rendering target-derived trophic substances stimulated by testosterone unable to rescue motoneurons in a timely manner. The present finding is consistent with our hypothesis that functional neuromuscular connectivity is a prerequisite for testosterone to promote neuronal survival.

Expression of the myogenic gene MRF4 during Xenopus development

In a search for myogenic genes in Xenopus, I have cloned homology of the mammalian myogenic genes MRF4 and myogenin. The myogenin clone is a genomic fragment encoding an amino acid sequence with 62% identity to the N-terminal region of rat myogenin. No myogenin transcript has been detected and no cDNA has been isolated, suggesting that Xenopus myogenin, if it is expressed at all, is likely to be expressed at low levels or transiently during development. A Xenopus MRF4 cDNA has been isolated and encodes an amino acid sequence with 72% identity to rat MRF4. In adult frogs, MRF4 RNA is detectable only in skeletal muscle (whereas MyoD, unexpectedly, is also expressed at low levels in the heart). During embryonic development, MRF4 RNA appears later than MyoD, at a time when the embryonic musculature already shows many differentiated features. This implies that MRF4 is not involved in the commitment or early differentiation of muscle cells. The accumulation of Xenopus MRF4 RNA overlaps with the formation of neuromuscular connections, suggesting that it may be induced by innervation. Consistent with this possibility, the level of Xenopus MRF4, but not MyoD, RNA is reduced in response to denervation of adult frog muscle.

Induction of synaptic development in cultured muscle cells by basic fibroblast growth factor

The role of basic fibroblast growth factor (bFGF) in signaling the development of the neuromuscular junction was examined. Beads coated with bFGF induced the formation of acetylcholine receptor (AChR) clusters in cultured Xenopus myotomal muscle cells. Tyrphostin, a tyrosine kinase inhibitor, abolished AChR clustering induced by bFGF beads, suggesting a role of tyrosine kinase activation in AChR clustering. Using specific antibodies, we demonstrated the presence of both bFG F and its receptor in the myotomal muscle in vivo during the period of neuromuscular connection. However, similar tissue from older animals with mature neuromuscular junctions showed an apparently truncated form of the bFGF receptor. These data suggest that bFG F may play a role in signaling synaptogenesis in skeletal muscle.

Serotonin-immunoreactive varicosities in the cell body region and neural sheath of the snail, Helix pomatia, ganglia: An electron microscopic immunocytochemical study

The distribution and connections of serotonin-immunoreactive fibers in the cell body region and neural sheath of the central ganglia of the snail, Helix pomatia, have been examined. The cell body region of the ganglia is supplied by an extremely dense network of varicose serotonin-immunoreactive fibers which surround neuronal perikarya in the ganglia. Immunoreactive processes also run to the neural sheath of both the ganglia and the peripheral nerve roots, forming a dense network. Electron microscopy revealed five different connections of serotonin-immunoreactive varicosities, according to their target: (i) non-specialized contacts with neuronal perikarya; (ii) non-specialized contacts with axon processes on the surface of the peripheral nerve roots; (iii) non-specialized neuromuscular connections with smooth muscle fibers in the neural sheath; (iv) varicosities engulfed by glial processes in both the cell body region and neural sheath; (v) varicosities embedded in the connective tissue elements of the sheath either partly or completely free of glial processes. In all cases of appositions no membrane specializations could be observed on either site of the contacts. These observations provide morphological evidence for non-synaptic regulatory actions of serotonin-containing neurons in Helix central nervous system: (i) modulation of the activity of neuronal perikarya; (ii) involvement in neuromuscular regulation; (iii) neurohormonal modulation of peripheral processes by release through the neural sheath.

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.

Differential loss of neuromuscular connections according to activity level and spinal position of neonatal rabbit soleus motor neurons

We have tested whether the ability of synapses to compete for occupancy of endplates during neuromuscular synapse elimination is affected by differences in the spinal position or in the activity level of the parent motor neuron. To test the role of spinal position, the relative sizes of motor units for motor neurons from middle and extreme (rostral/caudal) positions in the rabbit soleus motor pool were determined at 3 postnatal ages: 4-5 d ("early" ages, when the soleus is heavily polyinnervated), 8-9 d ("intermediate"), and 11-15 d ("late," when the soleus has just reached singly innervated state). Average motor unit sizes from extreme ventral roots were similar to those from middle ventral roots in early-aged soleus muscles but were significantly smaller (by 18-27%) for both intermediate and late muscles. Thus, motor neurons from extreme positions evidently compete less effectively for retention of synapses than those from middle positions. To test the role of differential activity, inactive and active synapses were pitted directly against one another by implanting Silastic plugs laden with tetrodotoxin (TTX) into one of the spinal nerves containing a minority of the soleus motor axons. Differential activity was maintained during a period of extensive synapse loss, from the time of the implant at day 4 or 5 until the intermediate age (day 8-9). Motor unit twitch tensions were subsequently measured to determine the relative number of synapses retained by individual active and inactive motor neurons. The inactivated motor units were on average significantly larger (by more than 50%) than the corresponding group from normal and control-implanted animals. The abnormally large size of inactivated motor units persisted in animals allowed to recover from the TTX block and examined after multiple innervation had disappeared. Hence, the effect of the TTX block cannot be attributed to a simple slowing of synapse elimination specifically among the inactive motor neurons. We conclude that complete presynaptic inactivity improves the chances of survival relative to that for normal activity during synapse elimination in the neonatal rabbit soleus muscle. This difference in competitive ability may contribute to the development of an important characteristic of adult muscles, the correlation between motor unit size and recruitment threshold.

The role of gonadal hormones in neuromuscular synapse elimination in rats. I. Androgen delays the loss of multiple innervation in the levator ani muscle

The normal period of synapse elimination in the androgen-sensitive levator ani (LA) muscle occurs between 2 and 4 weeks after birth, well after the period of synapse elimination for most other rat muscles. To evaluate whether gonadal androgen might be involved in the delayed development of single innervation in the LA, we compared the time course of synapse elimination in LA muscles that lacked endogenous gonadal androgen or were exposed to exogenous androgen. Tetranitroblue tetrazolium was used to stain neuromuscular connections. Our results suggest that both endogenous and exogenous androgen delay the normal process of synapse elimination. Removing endogenous androgen resulted in lower levels of multiple innervation in the LA, suggesting that androgen may normally influence synapse elimination. Moreover, androgen treatment prevented much of the normal loss of multiple innervation in the LA. Androgen treatment during the normal period of synapse elimination also increased the diameter of LA muscle fibers, enhanced the development of preterminal branching, and increased the number of junctional sites on some LA fibers. Because androgen did not appear to induce the formation of new synapses through sprouting, we conclude that androgen maintained multiple innervation in the LA by preventing the normal ontogenetic process of synapse elimination.

Reformation of specific neuromuscular connections during axolotl limb regeneration: evidence that the first contacts are correct.

Retrograde neuronal tracing with horseradish peroxidase was used to determine the position in the spinal cord of the motor neurone pools of a proximal (biceps) and a distal (extensor digitorum) limb muscle at various times during axolotl limb regeneration. It was found that from the earliest stages of muscle redifferentiation (as judged by light and electron microscopic analysis) the vast majority of axons innervating the regenerating muscles came from cells within the bounds of the normal motor neurone pool for each muscle. A few incorrect projections were noted in that the regenerating proximal muscle was sometimes innervated by some cells caudal to its normal motor neurone pool. The results are discussed in terms of mechanisms that may be operating in the regenerating limb to ensure that specific neuromuscular connections are made.

The role of synapse elimination in the establishment of neuromuscular compartments

To investigate the specificity of development of initial neuromuscular connections, we examined the compartmental distribution of synapses in neonatal rat lateral gastrocnemius (LG) muscle. Initial neuromuscular connections might be restricted to the compartmental territories present in adults; alternatively, synapse elimination could establish the compartments from a less precise pattern of innervation. We examined 46 pups of ages 0 to 14 postnatal days using a variety of techniques. The principle method was evoked electromyographic (EMG) activity in response to nerve stimulation. The nerve branch to one neuromuscular compartment was cut and the remainder of the nerve was stimulated. The presence of EMG activity was used to identify the areas of muscle contracting in response to nerve stimulation. After cutting a particular branch, EMG activity generally could not be recorded from the denervated compartment. These results indicate that the pattern of innervation at birth is essentially compartment-specific, and that neuromuscular compartments are not shaped from some less precise pattern by postnatal synapse elimination. The factors which operate prenatally to determine this high degree of specificity in neuromuscular connectivity seen at the time of birth, however, remain unknown.

Substrate interactions affecting motor growth cone guidance during development and regeneration.

Most serious injuries of spinal nerves or roots in man and other higher vertebrates lead to permanent loss of control of skeletal muscles. In some cases this may be due to a failure of motor axons to regenerate, although even when functional neuromuscular connections are re-established, coordinated use of body and limb muscles may be absent. In both mammals and lower vertebrates, damaged motor axons usually regrow and reform functional connections with muscles, although these connections are often inappropriate. The selectivity of reinnervation is improved by maintaining alignment of the severed ends of the nerve. Thus, factors operating near the lesion site may direct regenerating motor axons into fascicles in the distal nerve stump that lead to inappropriate muscles. The identity of some of these factors is suggested by recent studies of developing systems which have shown that motor axons are directed in their growth. (a) The filopodia of their growth cones sample a limited region of the periphery. If motor growth cones extend too far from their normal pathways they establish connections with inappropriate muscles. (b) Motor growth cones normally extend into regions of embryos rich in the extracellular matrix molecule laminin, and avoid regions containing fibronectin. Moreover, motor growth cones extend on laminin but not on fibronectin substrates in vitro. In peripheral nerves, these two molecules are differentially distributed; laminin is expressed by Schwann cells in the endoneurium whereas fibronectin is expressed by fibroblasts primarily in the perineurium. These studies suggest that regenerating motor growth cones may be directed to appropriate muscles if their original fascicles within the distal nerve stump are within filopodial reach but may not be able to escape the fibronectin-rich perineurial sheath once directed into an inappropriate fascicle.