Nerve sprouting and endplate growth induced in normal muscle by contralateral partial denervation of rat plantaris

The incidence of motor nerve and terminal sprouting was quantitatively analyzed in normal unoperated muscles, in homologous muscles contralateral to muscles which have been partially denervated, in partially denervated muscles, and in sham-operated muscles. Muscles were studied by light microscopy after staining motor endplates by a combined silver-cholinesterase stain. In addition, the incidence of endplates containing terminal sprouts, the number of terminal branch points per endplate, and endplate size were also assessed in the various groups examined. We observed that following section of the L₄ spinal nerve, the incidence of sprouting (preterminal and intranodal) in the contralateral muscle exhibited a 2-fold increase over sham-operated controls. We also found a correlation between nerve terminal sprouting, terminal branch point number and endplate size. All of these parameters were significantly increased in the contralateral muscles as compared to the sham-operated control muscles. These findings suggest that normal muscles undergo sprouting which can be enhanced by contraleteral partial denervation. The possible underlying mechanism may be the transneuronal induction of sprouting.     

Neuromuscular relationships in a muscle having segregated motor endplate zones. II. The response to partial denervation

The cranial belly of the anterior gracilis muscle of the rat has two discrete motor endplate zones. A proximal zone is innervated by short branches of the obturator nerve, and a distal zone is innervated by (usually) two longer branches. Each muscle fiber is innervated at a single motor endplate although a substantial number lie within both endplate zones. In addition, motor units are divided between the two zones. In order to dissociate the role of the denervated endplate from that of the denervated muscle fiber in the promotion of motoneuron sprouting, the distal endplate zone in this model was denervated and the response at the proximal zone was studied. Comparisons were made with partial denervation of the muscle by division of the L4 ventral ramus and with partial denervation of the distal endplate zone. Denervation of the distal endplate zone produced profuse terminal sprouting at the proximal zone whereas division of L4 predominantly produced nodal sprouting at both zones. Partial denervation of the distal zone resulted in nodal sprouts in that zone and again mainly terminal sprouts at the proximal zone. The repeated association of terminal sprouting with division of the motor axons supplying the distal zone together with the knowledge that motor units are distributed between the two zones led to the conclusion that the terminal sprouting was stimulated by the reduction in size of motor units rather than by the presence of denervated muscle fibers in the vicinity of the endplates.     

Spinal irradiation does not inhibit distal axonal sprouting

In an attempt to determine the relative importance of the nerve cell body and of the axon in initiating and controlling axonal regeneration, nerve cell bodies were irradiated and the ability of the distal axon to sprout was examined. Mice were subjected to either 25 or 50 Gray (Gy) of x-irradiation localized to the lumbar spinal cord. After times varying from 1 day to 6 months after irradiation, a sublethal dose of botulinum toxin (BoTx) was injected into the calf muscles of one leg. The soleus muscle was examined histologically after times varying from 1 week to 6 months after injection, and BoTx-induced ultraterminal axonal sprouting was assessed by the number of motor endplates showing sprouts, the length of the sprouts, and the long term endplate morphology. Apart from some irradiated subgroups having slightly shorter sprout lengths, no significant differences were found between irradiated and nonirradiated groups. The results suggest either that the processes in the nerve cell body responsible for initiating and supporting axonal growth are resistant to large doses of irradiation, or that growth regulatory mechanisms in the distal axon are under local control.     

Neuromuscular transmission at terminals of sprouted mammalian motor neurones

Motor nerve sprouting was induced in the tensor fasciae latae muscle of mice by partial denervation produced either by cutting (to prevent reinnervation) or crushing (to allow subsequent reinnervation) spinal nerve L4 unilaterally. The quantum content (m) of endplate potentials recorded intracellularly in vitro in the presence of high-Mg²⁺ and low-Ca²⁺ ion concentrations was determined up to 400 days later in non-reinnervated, reinnervated and contralateral control muscles. The muscles were then either fixed and stained with silver and cholinesterase for light microscopy, or fixed and examined in the electron microscope. The average value of m in control muscles increased by 4–5-fold as the animals matured in the 4 months following the operations. The average value of m at terminals of sprouted motor neurones in the absence of reinnervation also increased with time after partial denervation but was always less than the value in the corresponding control muscle. In electron micrographs of muscles following L4 section the nerve terminals closely apposed on average only two-thirds of the proportion of junctional folds apposed by terminals in control muscles. When muscles were reinnervated following L4 crush the average value of m at terminals of sprouted and reinnervating motor neurones equalled and sometimes exceeded m in contralateral control muscles. A proportion of muscle fibres had endplate potentials from reinnervating and sprouted axons, and the silver stain showed that these muscle fibres were innervated at the site of the original endplate. At these endplates the fraction of the total quantum content contributed by presumed sprout terminals fell significantly in the 4 months following L4 crush. It is concluded that: (i) in the absence of reinnervation, sprout terminals grow in size but a significant number never occupy all endplate site available to them; and (ii) in the presence of reinnervation, reinnervating axon terminals share some endplates with sprout terminals and grow at the expense of the sprout terminals which are eventually withdrawn from some shared endplates.     

Muscle fiber orientation in muscles commonly injected with botulinum toxin: An anatomical pilot study

The endplate zone is assumed to be at about the midpoint of a muscle fiber. This study was designed to locate the middle of the muscle fibers of commonly injected muscles, thus identifying the endplate zone of these muscles. The proximal and distal musculotendinous junctions in muscles of the upper and lower extremities were identified. Orientation of muscle fibers was determined. Measurements using common surface landmarks were used to determine the relationship of these muscles with the landmarks (e.g., biceps muscle bulk extends from the upper fourth to the lower fourth of the humerus). Figures were developed using these measurements so as to be able to extrapolate these measurements to other patients of varying sizes. Illustrations of muscle fiber orientation were done and the assumed location of motor endplate bands marked. Color illustrations will be shown. With the thought that the endplate zone is at the middle of the muscle fiber, this detailed study of muscle fibers helps identify assumed location of motor endplates of specific muscles, thereby improving technique and efficacy of botulinum toxin injections.     

Neuromuscular relationships in a muscle having segregated motor endplate zones. I. Anatomical and physiological considerations

The cranial portion of the rat anterior gracilis muscle is innervated by the obturator nerve at two discrete motor endplate zones of which the distal is supplied by well-defined branches of the nerve. Prior to the use of this muscle in a study of motoneuron sprouting, further morphological and physiological studies have shown that its fibers vary in length but a number traverse the whole muscle or are long enough to extend through both endplate zones. The distribution of muscle fiber types is typical of a rat fast-twitch muscle, and each fiber is innervated at a single endplate. Myography showed that the spinal cord segments, which may contribute to the muscle's innervation, are L2, 3, and 4, of which L3 is constant and predominant, and that denervation of the distal endplate zone leads to a 50% reduction of the maximum isometric tension developed by the muscle. Antidromic stimulation of nerves supplying the distal endplate zone produced contraction of the proximal part of the muscle and, following similar antidromic stimulation, intracellular recordings made at proximal zone endplates showed the presence of endplate potentials. It was concluded from these data that endplates at both the proximal and distal zones can form part of the same motor unit.     

Motor nerve terminal outgrowth and acetylcholine receptors: inhibition of terminal outgrowth by alpha-bungarotoxin and anti-acetylcholine receptor antibody

Motor nerves undergo extensive terminal outgrowth when the muscles they supply are "functionally denervated." In this study, we have investigated the role of the acetylcholine receptors (AChRs), newly appearing in such muscles, in promoting nerve terminal outgrowth. The amount of outgrowth was determined by morphometric measurement of nerve terminal branching, endplate length, and ultraterminal sprouts, in cholinesterase-silver-stained neuromuscular junctions. Presynaptic neuromuscular blockade with botulinum toxin induced pronounced nerve terminal outgrowth in both the rat and mouse soleus muscles, although ultraterminal sprouts did not occur in the rat soleus. By contrast, postsynaptic neuromuscular blockade with alpha-bungarotoxin (alpha-BuTx) induced little or no terminal outgrowth, although it caused "functional denervation." Moreover, alpha-BuTx and anti-AChR antibody inhibited the terminal outgrowth otherwise induced by botulinum toxin. Other types of motor nerve growth, such as nerve regeneration, were unaffected by these agents. Our results are consistent with the concept that extrajunctional AChRs in skeletal muscle play an important role in the control of motor nerve terminal outgrowth at neuromuscular junctions.     

Changes in terminal sprout formation in rat sternocostalis muscle during chronic intoxication with 2,5 hexanedione

Qualitative and quantitative morphological studies of the sternocostalis muscle innervation were made on rats chronically intoxicated with 2,5 hexanedione (2,5 HD) using the zinc iodide-osmium (ZIO) technique. Two distinct phases were seen in the events at the motor endplate. First, the number of motor endplates forming spontaneous terminal sprouts was found to increase linearly with time and, from the third week onward, the sprouts appeared to become progressively elongated. This latter change was associated with the appearance of swollen axons within intramuscular nerve bundles. Second, from the sixth week onward, wallerian degeneration of nerve fibers was seen and terminal sprouts began to make new arborizations on muscle fibers. By the eighth week, this occurred in as many as 66% of the rats, and collateral sprouting was also observed at this time. The occurrence of increased spontaneous terminal sprouting due to altered neuromuscular function is discussed in the light of axonal changes resulting from neurofilament accumulation following 2,5 HD intoxication.     

Motor endplate position of rat gastrocnemius muscle

In this study, the relative endplate position of fibers of rat gastrocnemius caput mediale (GM) muscle was determined by counting numbers of sarcomeres. Isolated fibers were teased from the proximal, intermediate, and distal regions of the muscle. Endplates of distal fibers were located on the proximal third of their lengths. Endplates of intermediate fibers were located at half fiber length, and for proximal fibers, a variable endplate position was obtained: in half of the muscles studied, endplates occurred around the proximal one-third and in the other half near the midpoint of the fiber. Endplate position relative to fiber length was thus found to be dependent on the region of the muscle. Changes in the orientation of endplate zone relative to the muscle belly is likely to take place with changes in muscle length, as shown by a planimetric muscle model. It is argued that architecture of pennate muscles may highly affect characteristics of motor unit potentials.     

Terminal sprouting of mouse motor nerves when the post-synaptic membrane degenerates

Injection of ecothiopate, 4-aminopyridine and caffeine into the mouse calf produced necrosis in the endplate region of approximately 40% of soleus muscle fibres. Within two days terminal sprouting, as seen by zinc iodide/osmium tetroxide staining, had occurred at nearly a quarter of such endplates, but not at neighbouring intact ones. Almost half of these sprouts were greater than 50 μm in length. Terminal sprouting at degenerating endplates was also seen in identically treated silver-stained gluteus maximus muscles. Muscle degeneration caused by mechanical damage produced similar effects. In transverse sections of the gluteus maximus preparation, the terminals could be found within the necrotic muscle fibres, having penetrated the synaptic basal lamina. It is concluded that motor nerve terminals have an intrinsic tendency to grow, and are normally prevented from doing so by their formation of synapses with muscle fibres. Destruction of this relationship alone can cause terminal sprouting.     

Motor nerve terminal sprouting in formamide-treated inactive amphibian skeletal muscle

Motor axons can form sprouts from their terminal arborizations in response to partial denervation, and when exposed to pharmacological blocking agents like TTX, botulinum toxins alpha-bungarotoxin, or curare. Each of these experimental procedures has cessation of muscle contractile activity as a common feature. We tested the specific role of muscle fiber inactivity in regulating nerve terminal sprouting by chronically treating adult frog (Rana pipiens) cutaneous pectoris muscles with formamide. Exposure to formamide, unlike the other compounds used to study sprouting, selectively inhibits muscle contractions without blocking pre- or postsynaptic transmission or muscle fiber action potentials. Repeated formamide applications were used to achieve chronic block of muscle contractile activity in vivo for up to 6 weeks. Motor axons in formamide-treated inactive muscle sprouted only from their terminal arborizations, but not from nodes of Ranvier. The onset of this sprouting was protracted compared with that seen in pharmacologically blocked mammalian muscles, and sprouts in formamide-treated muscles were more complex and ornate. The frequency of sprouting terminals was less in these formamide-treated muscles than that seen after alternate methods of contractile block, and this suggests that contractile inactivity alone serves as only a moderate cue for sprouting. The possibility is discussed that the prolific sprouting seen following neurotoxin administration may, in fact, be due to perturbations in synaptic transmission or muscle electrical activity rather than muscle fiber inactivity.     

Transneuronal and peripheral mechanisms for the induction of motor neuron sprouting

After injury to the nerve to one cutaneous pectoris muscle of the frog, the intact nerve to the contralateral muscle sprouts and forms additional synaptic connections with already innervated muscle fibers. It has been suggested (Rotshenker, S. (1979) J. Physiol. (Lond.) 292: 535-547; Rotshenker, S., and F. Reichert (1980) J. Comp. Neurol. 193: 413-422) that axotomy initiates a signal for sprouting in the injured neurons that is transferred transneuronally across the spinal cord to intact motor neurons. The present study was designed to test the hypothesis that axotomy initiates the signal for sprouting by interfering with some trophic signaling between the injured neurons and denervated muscle. Colchicine therefore was applied to the nerve to the left muscles to inhibit axonal transport on whose integrity trophic interactions depend. Consequently, supernumerary innervation developed in contralateral right intact muscles much the same as after axotom. Surprisingly, axons that were exposed to the drug also sprouted and formed synapses. Furthermore, the sprouting response of axons that were exposed to the drug also was produced in nerve fibers that were separated from their cell bodies. These results suggest two ways in which colchicine may produce sprouting and synapse formation and thereby suggest two mechanisms by which motor neurons may be induced to sprout: (a) transneuronally, by presenting growth stimuli to their cell bodies and central processes in the central nervous system and (b) by presenting growth stimuli to their peripheral extensions.     

Morphological and electrophysiological study of distal motor nerve fiber degeneration and sprouting after irreversible cholinesterase inhibition

A single subcutaneous injection of a sublethal dose of the irreversible organophosphate sarin (0.08 mg/kg) in rats induced a non-Wallerian-type axonal degeneration of the neuromuscular synapse in the slow twitch, soleus muscle. These alterations of the endplate region were more obvious in the soleus than in the fast extensor digitorum longus muscle and were slowly reversible, complete recovery requiring about 10 days. Silver-cholinesterase staining and electrophysiological techniques were used to define the spatiotemporal evolution of prejunctional abnormalities. The non-Wallerian-type axonal degeneration of the neuromuscular synapse was characterized by bead or balloon-like varicosities of the focal, distal, and terminal nerve fibers and a retraction of terminal axons. Axonal degeneration was accompanied by junctional and extrajunctional membrane depolarization and was followed by nerve sprouting at focal, distal, and terminal nerve fibers. Transients similar to miniature endplate potentials were recorded along the muscle fiber at distances of 800-2500 microns away from the parent endplate. New ectopic endings, originating from the same endplate, were discovered adjacent to the terminal axon and also distant from the parent endplate. Very elaborate terminal arborization and occasional multibranching arose from a progressive growth sprout. The new sprouting may have served to compensate for the loss of synaptic contact caused by sarin. Thus the present study demonstrates a direct cytotoxic effect of sarin and indicates that this organophosphate agent may be an important neurotoxicological tool to understand the mechanisms involved in nerve sprouting.     

Influence of spinal cord stimulation on the innervation pattern of muscle fibers in vivo.

In chick embryo, chronic stimulation of the brachial spinal cord at a fast rhythm from days 7 to 18 of development induced an increase in AChE activity sites and ACh receptor (AChR) clusters in slow anterior latissimus dorsi (ALD) muscle. Most AChR clusters and AChE spots were contacted by nerve endings. A previous study showed that such spinal cord stimulation causes changes in ALD muscle properties, especially the appearance of a high proportion of fast type II fibers (Fournier Le Ray et al., 1989). Analysis of the synaptic pattern in different fiber types of experimental ALD muscle indicated a decrease in the distance between successive AChE spots in slow type III fibers compared to controls, whereas the intersynaptic distance in fast type II fibers was very similar to that in the rare fast fibers developing in control ALD. Fast fibers of experimental muscles exhibited less AChR than did slow fibers. The increased number of neuromuscular junctions in ALD muscle after spinal cord stimulation appeared to be preferentially located in slow fibers. Electron microscopy showed no change in the number of axons in ALD nerve after spinal cord stimulation. The activity imposed on brachial motoneurons apparently caused terminal sprouting of ALD nerve in target muscle, thus accounting for the increase in neuromuscular contacts in ALD muscle fibers. Differences in the distribution of nerve contacts indicate that the type of muscle fiber innervated may play a critical role in the synaptic pattern during chick embryogenesis.     

Distinct patterns of motor nerve terminal sprouting induced by ciliary neurotrophic factor vs. botulinum toxin

Both diffusible and surface-bound molecules are thought to induce sprouting of motor nerve terminals in response to paralysis. Here we report that the sprouting induced by ciliary neurotrophic factor (CNTF) is qualitatively different from the sprouting induced by botulinum toxin (BoTX). We show first that subcutaneous application of CNTF to levator auris longus muscles of adult mice evokes sprouting from nearly all nerve terminals. Surprisingly, however, most terminal sprouts remain within the boundaries of the endplate region and rarely grow extrasynaptically even if CNTF is administered chronically. In contrast, terminal sprouts induced by BoTX extend vigorously along the extrasynaptic muscle surface. The different patterns of sprout elongation are attributable in part to different patterns of initiation: whereas CNTF-induced sprouts emerge randomly from the surface of terminal branches, BoTX-induced sprouts emerge exclusively along the perimeter of terminal branches in direct apposition to muscle fiber membranes. Combined treatment with CNTF and BoTX produces exceptionally robust extraterminal sprouting with little if any intrasynaptic growth of terminal sprouts. We interpret these results as showing that paralysis induces sprouting primarily by muscle-associated, surface-bound molecules rather than by diffusible factors. Our findings may be useful in defining the physiological role of the numerous candidate sprouting-inducers and in promoting compensatory sprouting after nerve injury for therapeutic benefit.     

Na+ currents near and away from endplates on human fast and slow twitch muscle fibers

Fast and slow twitch muscle fibers have distinct contractile properties. Here we determined that membrane excitability also varies with fiber type. Na⁺ currents (I_NA) were studied with the loose-patch voltage clamp technique on 29 histochemically classified human intercostal skeletal muscle fibers at the endplate border and <200 μm from the endplate (extrajunctional). Fast and slow twitch fibers showed slow inactivation of endplate border and extrajunctional I_NA and had increased I_NA at the endplate border compared to extrajunctional membrane. The voltage dependencies of I_NA were similar on the endplate border and extrajunctional membrane, which suggests thatboth regions have physiclogically similar channels. Fast twitch fibers had larger I_NA on the endplate border and extrajunctional membrane and manifest fast and slow inactivation of I_NA at more negative potentials than slow twitch fibers. For normal muscle, the differences between I_NA on fast and slow twitch fibers might: (1) enable fast twitch fibers to operate at high firing frequencies for brief periods; and (2) enable slow twitch fibers to operate at low firing frequencies for prolonged times. Disorders of skeletal membrane excitability, such as the periodic paralyses and myotonias, may impact fast and slow twitch fibers differently due to the distinctive Na⁺ channel properties of each fiber type. © 1993 John Wiley & Sons, Inc.     

Motoneurons of twitch and nontwitch extraocular muscle fibers in the abducens, trochlear, and oculomotor nuclei of monkeys

Eye muscle fibers can be divided into two categories: nontwitch, multiply innervated muscle fibers (MIFs), and twitch, singly innervated muscle fibers (SIFs). We investigated the location of motoneurons supplying SIFs and MIFs in the six extraocular muscles of monkeys. Injections of retrograde tracers into eye muscles were placed either centrally, within the central SIF endplate zone; in an intermediate zone, outside the SIF endplate zone, targeting MIF endplates along the length of muscle fiber; or distally, into the myotendinous junction containing palisade endings. Central injections labeled large motoneurons within the abducens, trochlear or oculomotor nucleus, and smaller motoneurons lying mainly around the periphery of the motor nuclei. Intermediate injections labeled some large motoneurons within the motor nuclei but also labeled many peripheral motoneurons. Distal injections labeled small and medium-large peripheral neurons strongly and almost exclusively. The peripheral neurons labeled from the lateral rectus muscle surround the medial half of the abducens nucleus: from superior oblique, they form a cap over the dorsal trochlear nucleus; from inferior oblique and superior rectus, they are scattered bilaterally around the midline, between the oculomotor nucleus; from both medial and inferior rectus, they lie mainly in the C-group, on the dorsomedial border of oculomotor nucleus. In the medial rectus distal injections, a "C-group extension" extended up to the Edinger-Westphal nucleus and labeled dendrites within the supraoculomotor area. We conclude that large motoneurons within the motor nuclei innervate twitch fibers, whereas smaller motoneurons around the periphery innervate nontwitch, MIF fibers. The peripheral subgroups also contain medium-large neurons which may be associated with the palisade endings of global MIFs. The role of MIFs in eye movements is unclear, but the concept of a final common pathway must now be reconsidered.     

Histochemical changes in innervated and denervated skeletal muscle fibers following treatment with bupivacaine (marcain).

Widespread degenerative and regenerative changes were produced in the fast tibialis anterior and slow soleus muscles of rats by intramuscular injection of bupivacaine combined with hyaluronidase. The redifferentiation of muscle fiber types during the period of regeneration was followed in innervated and denervated muscles using histochemical methods. Evidence of differentiation was present in fast and slow innervated muscles 2 weeks after treatment and three fiber types could be recognized in the tibialis anterior at three weeks. By four weeks tibialis anterior and soleus showed a normal distribution of fiber types and associated differences in fiber caliber. Following denervation some enzyme activity was seen to appear in both muscles, but after four weeks fiber caliber remained markedly reduced and no differentiation of the enzyme activity of fibers was seen. It was concluded that a complete dedifferentiation of fibers had followed treatment with bupivacaine and that innervation was essential for the redifferentiation of fiber types.     

Initiation of differentiation into skeletal muscle fiber types

A monoclonal antibody directed against a slow isoform of a 100-K myofibrillar protein has been used to study the differentiation of muscle fibers in the fast and slow muscles of the rat. Immunohistochemical studies have shown that initiation of differentiation into fast and slow muscle fibers occurs very early during development. The distribution pattern of cell types in different muscles is unique and is also determined very early during fetal muscle development. It is concluded that motor innervation is probably not essential to initiate differentiation into distinct muscle fiber types.     

In vivo visualization of pre- and postsynaptic changes during synapse elimination in reinnervated mouse muscle

Using a vital nerve terminal dye (4-Di-2-ASP) and fluorescently tagged alpha-bungarotoxin to stain postsynaptic acetylcholine (ACh) receptors, we viewed the same muscle fibers at multiple times in the sternomastoid muscle of living mice during the process of reinnervation following nerve crush. Soon after axons reenter the muscle, they precisely reoccupy the original endplate sites. However, in contrast to normal adult muscle, during the first several weeks of reinnervation, anatomical and physiological measures show that many of the endplate sites are innervated by more than one axon. Typically, one axon reinnervates the original endplate site by growing up the old Schwann cell tube while another originates as a sprout from a nearby endplate. Within 2 weeks after reinnervation nerve terminal staining shows that most of the sprouts have regressed and physiological evidence of multiple innervation has returned to the normal low level. By repeatedly observing the same endplates during the period of synapse elimination, we could directly view this phenomenon. At some endplates, nerve terminal boutons in one region of the endplate were eliminated at the same time a sprout entering that area regressed. These unoccupied sites seemed permanently eliminated as they are not subsequently occupied by sprouts from the axon remaining at the endplate. We were surprised to find that there is a corresponding permanent loss of ACh receptors within the muscle fiber membrane precisely underneath the eliminated nerve terminals. The decrease in receptors at sites of synapse elimination is due to both a selective loss of ACh receptors already incorporated into these sites and to a lack of insertion of new receptors at the same regions. These sites of pre- and postsynaptic loss, however, maintain cholinesterase staining in the basal lamina for long periods. Control experiments showed that endplates that were permanently denervated, incompletely reoccupied by reinnervating axons, or stained and viewed multiple times in normal muscle do not lose postsynaptic receptor regions. Interestingly, receptors appear to be eliminated before there is any obvious change in the staining of the overlying nerve terminal. Because of the lag between receptor and nerve terminal loss, we could predict which synaptic boutons would be eliminated by looking for lightly stained receptor regions. One interpretation of these data is that the removal or redistribution of relevant postsynaptic molecules by one innervating axon may instigate the elimination of competing terminals.