Neuromuscular activity impairs axonal sprouting in partially denervated muscles by inhibiting bridge formation of perisynaptic Schwann cells

Following partial denervation of rat hindlimb muscle, terminal Schwann cells extend processes from denervated endplates to induce and guide sprouting from the remaining intact axons. Increased neuromuscular activity significantly reduces motor unit enlargement and sprouting during the acute phase of sprouting. These findings led to the hypothesis that increased neuromuscular activity perturbs formation of Schwann cell bridges and thereby reduces sprouting. Adult rat tibialis anterior (TA) muscles were extensively denervated by avulsion of L4 spinal root and were immediately subjected to normal caged activity or running exercise (8 h daily) for 3, 7, 14, 21, and 28 days. Combined silver/cholinesterase histochemical staining revealed that the progressive reinnervation of denervated endplates by sprouts over a 1 month period in the extensively partially denervated TA muscles was completely abolished by increased neuromuscular activity. Immunohistochemical staining and triple immunofluorescence revealed that the increased neuromuscular activity did not perturb the production of Schwann cell processes, but prevented bridging between Schwann cell processes at innervated and denervated endplates. Our findings suggest that failure of Schwann cell processes to bridge between endplates accounts, at least in part, for the inhibitory effect of increased neuromuscular activity on sprouting.     

Terminal Schwann cells guide the reinnervation of muscle after nerve injury

Schwann cells and axons labeled by transgene-encoded, fluorescent proteins can be repeatedly imaged in living mice to observe the reinnervation of neuromuscular junctions. Axons typically return to denervated junctions by growing along Schwann cells contained in the old nerve sheaths or “Schwann cell tubes”. These axons then commonly “escape” the synaptic sites by growing along the Schwann cell processes extended during the period of denervation. These “escaped fibers” grow to innervate adjacent synaptic sites along Schwann cells bridging these sites. Within the synaptic site, Schwann cells, originally positioned above the synaptic site continue to cover the acetylcholine receptors (AChRs) immediately following denervation, but gradually vacate portions of this site. When regenerating axons return, they first deploy along the Schwann cells and ignore sites of AChRs vacated by Schwann cells. In many cases these vacated sites are never reinnervated and are ultimately lost. Following partial denervation, Schwann cells grow in an apparently tropic fashion from denervated to nearby innervated synaptic sites and serve as the substrates for nerve sprouting. These experiments show that Schwann cells provide pathways that stimulate axon growth and insure the rapid reinnervation of denervated or partially denervated muscles.     

In vivo observations of terminal Schwann cells at normal, denervated, and reinnervated mouse neuromuscular junctions

We found a low-molecular-mass, fluorescent dye, Calcein blue am ester (CB), that labels terminal Schwann cells at neuromuscular junctions in vivo without damaging them. This dye was used to follow terminal Schwann cells at neuromuscular junctions in the mouse sternomastoid muscle over periods of days to months. Terminal Schwann cell bodies and processes were stable in their spatial distribution over these intervals, with processes that in most junctions were precisely aligned with motor nerve terminal branches. Three days after nerve cut, the extensive processes elaborated by terminal Schwann cells in denervated muscle were labeled by CB. The number and length of CB-labeled terminal Schwann cell processes decreased between 3 days and 1 month after denervation, suggesting that terminal Schwann cell processes are only transiently maintained in the absence of innervation. During reinnervation after nerve crush, however, terminal Schwann cell processes were extended in advance of axon sprouts, and these processes persisted until reinnervation was completed. By viewing the same junctions twice during reinnervation, we directly observed that axon sprouts used existing Schwann cell processes and chains of cell bodies as substrates for outgrowth. Thus, CB can be used to monitor the dynamic behavior of terminal Schwann cells, whose interactions with motor axons and their terminals are important for junction homeostasis and repair.     

Mechanisms controlling axonal sprouting at the neuromuscular junction

This review considers the relative roles of sprouting stimuli, perisynaptic Schwann cells and neuromuscular activity in axonal sprouting at the neuromuscular junction in partially denervated muscles. A number of sprouting stimuli, including insulin-like growth factor II, which are generated from inactive muscle fibers in partially denervated and paralyzed skeletal muscles, has been considered. There is also evidence that perisynaptic Schwann cells induce and guide axonal sprouting in adult partially denervated muscles. Excessive neuromuscular activity significantly reduces bridging of perisynaptic Schwann cell processes between innervated and denervated endplates and thereby inhibits axonal sprouting in partially denervated adult muscles. Elimination of neuromuscular activity is also detrimental to sprouting in these muscles, suggesting that calcium influx into the nerve is crucial for axonal sprouting. The role of neuromuscular activity in axonal sprouting will be considered critically in the context of the roles of sprouting stimuli and perisynaptic Schwann cells in the process of axonal sprouting.     

Schwann cell dynamics with respect to newly formed motor—nerve terminal branches on mature (Bufo marinus) muscle fibers

A study has been made of the formation of synaptic terminals from long processes formed at the end of motor nerve branches of endplates in mature amphibian (Bufo marinus) muscle. Injection of fluorescent dyes into individual motor axons showed the full extent of their branches at single endplates. Synaptic vesicle clusters at these branches were identified with styryl dyes. Some terminal branches consisted of well separated varicosities, each possessing a cluster of functioning synaptic vesicles whilst others formed by the same axon consisted of closely spaced clusters of vesicles in a branch of approximately uniform diameter. All the varicosities gave rise to calcium transients on stimulation of their parent axon. Both types of branches sometimes possessed short processes (<5 μm long) or very long thin processes (>10 μm long) which ended in a bulb that possessed a functional synaptic vesicle cluster. These thin processes could move and form a varicosity along their length in less than 30 min. Injection of a fluorescent dye into terminal Schwann cells (TSCs) at an endplate showed that they also possessed very long thin processes (>10 μm long) which could move over relatively short times (<30 min). Injecting fluorescent dyes into both axons and their associated TSCs showed that on some occasions long TSC processes were accompanied by a long nerve terminal process and at other times they were not. It is suggested that the mature motor-nerve terminal is a dynamic structure in which the formation of processes by TSCs guides nerve terminal sprouting.     

Terminal sprouting in the sternocostalis muscle of the rat

Summary Partial denervation of the sternocostalis muscle was achieved by sectioning two out of five of its intercostal nerves. The terminal sprouting response was markedly increased compared to that found following section of only one nerve. The increase in the response was greater for B type than for C type end plates, although B type end plates appear unable to produce terminal sprouts longer than 20 m after partial denervation.Double nerve section allowed terminal sprouts from C type end plates to increase in length up to three days, after which time they appeared to retract. It is postulated that the onset of collateral sprouting resulted in reinnervation of empty end plate sites and hence removed the target for terminal sprouts.     

Nerve sprouting in innervated adult skeletal muscle induced by exposure to elevated levels of insulin-like growth factors

Partial denervation or paralysis of adult skeletal muscle is followed by nerve sprouting, probably due to release of diffusible sprout-inducing activity by inactive muscle. Insulin-like growth factors (IGF1 and IFG2) are candidates for muscle-derived sprouting activity, because (a) they induce neurite growth from peripheral neurons in vitro; and (b) their mRNA levels in adult skeletal muscle increase severalfold after denervation or paralysis. We sought to determine whether the presence of elevated levels of IGFs in innervated adult skeletal muscle was sufficient to produce intramuscular nerve growth. Low concentrations of IGFs induced massive neurite growth from enriched embryonic chick motoneurons in vitro. Half-maximal responses required 0.2 nM IGF2 or IGF1, or 20 nM insulin. Similar hormone binding properties of motoneuron processes in vitro were observed. Exposure of adult rat or mouse gluteus muscle in vivo to low quantities of exogenous IGF2 or IGF1 led to intramuscular nerve sprouting. Numbers of sprouts in IGF-exposed muscles were 10-fold higher than in vehicle-exposed or untreated muscles, and 12.2% of the end plates in IGF-exposed muscle (control: 2.7%) had sprouts growing from them. The nerve growth reaction was accompanied by elevated levels of intramuscular nerve-specific growth-associated protein GAP43. Additional properties of IGF-exposed muscle included modest proliferation of interstitial cells and elevated interstitial J1 immunoreactivity. These results suggest that elevated levels of IGFs in denervated or paralyzed muscle might trigger coordinate regenerative reactions, including nerve sprouting and expression of nerve growth-supporting substrate molecules by activated interstitial cells.     

Transient inability of neonatal rat motoneurons to reinnervate muscle

We studied the reinnervation of internal intercostal muscles of newborn rats. The distal halves were denervated by nerve section at various ages between birth and 6 weeks. Regardless of the age at denervation, neither evoked nor spontaneous nerve-muscle transmission reappeared until the animals were at least 3 weeks old. Older rats recovered a substantial degree of function within 7 days of nerve section. Normally the motor units in this muscle are narrowly distributed, so most axotomized motoneurons lost their entire synaptic periphery. Reinnervation was by axons which had been sectioned, and regenerated motor units were of normal size and number. There was no collateral sprouting from end plates left intact. Motoneurons axotomized at birth did regenerate axons the full length of the muscle within 7 days of operation. Their failure to reinnervate the muscle was due to delay in forming functional end plates. Nerve section in animals aged 1 month or older resulted in an abnormal pattern of reinnervation; reinnervated motor units were diffusely spread through large portions of the muscle, although they still did not overlap with the region left intact. This indicates that thoracic motoneurons respond to axotomy differently in neonatal rats than they do in adults.     

DEVELOPMENT OF THE NEUROMUSCULAR JUNCTION : III. Degeneration of Motor End Plates After Denervation and Maintenance In Vitro by Nerve Explants

To determine the effects of nerve explants on the integrity of motor end plates in vitro, cholinesterase activity and structure of end plates were compared in newt muscle denervated in vivo, cultured in the absence of nerve explants, and cultured in the presence of sensory ganglia. In neuromuscular junctions denervated in vivo or in vitro, the synaptic vesicles become clumped and fragmented. A few intact vesicles escape into the synaptic cleft. Axon terminals degenerate until they are left as residual bodies within the Schwann cell cytoplasm. Junctional folds on the muscle surface are reduced in height and are no longer evident once traces of axoplasm within the Schwann cell disappear. End plate cholinesterase activity is reduced as junctional folds are lost. When muscle is cultured in the presence of a sensory ganglion, the terminal axoplasm degenerates in the same manner but junctional folds persist on the muscle surface. Moderately intense cholinesterase activity remains in association with the junctional folds, so that normal motor end plates are maintained in the absence of innervation. These results show that degenerative changes in the structure of the motor end plate and loss of cholinesterase activity occurring in organ culture as a result of denervation can be retarded by nerve explants that do not directly innervate the muscle.     

Perisynaptic Schwann cells at neuromuscular junctions revealed by a novel monoclonal antibody

This study aimed to generate a probe for perisynaptic Schwann cells (PSCs) to investigate the emerging role of these synapse-associated glial cells in the formation and maintenance of the neuromuscular junction (NMJ). We have obtained a novel monoclonal antibody, 2A12, which labels the external surface of PSC membranes at the frog NMJ. The antibody reveals PSC fine processes or “fingers” that are interposed between nerve terminal and muscle membrane, interdigitating with bands of acetylcholine receptors. This antibody also labels PSCs at the avian neuromuscular junction and recognizes a 200 kDa protein in Torpedo electric organs. In frog muscles, axotomy induces sprouting of PSC processes beyond clusters of acetylcholine receptors and acetylcholinesterase at denervated junctional branches. PSC branches often extend across several muscle fibers. At some junctions, PSC sprouts join the tips of neighboring branches. The average length of PSC sprouts is approximately 156 µ at 3-week denervated NMJs. PSC sprouting is accompanied by a significant increase in the number of Schwann cell bodies per NMJ. Following nerve regeneration, nerve terminals reinnervate the junction along the PSC processes. In vivo observations of normal frog muscles also show PSC processes longer than nerve terminals at some junctional branches. The results suggest that nerve injury induces profuse PSC sprouting that may play a role in guiding nerve terminal regeneration at frog NMJs. In addition, antibody 2A12 reveals the fine morphology of PSCs in relation to other synaptic elements and is a useful probe in elucidating the function of these synapse-associated glial cells in vivo.     

Reciprocal interactions between perisynaptic Schwann cells and regenerating nerve terminals at the frog neuromuscular junction

The perisynaptic Schwann cell (PSC) has gained recent attention with respect to its roles in synaptic function, remodeling, and regeneration at the vertebrate neuromuscular junction (NMJ). Here we test the hypothesis that, following nerve injury, processes extended by PSCs guide regenerating nerve terminals (NTs) in vivo, and that the extension of sprouts by PSCs is triggered by the arrival of regenerating NTs. Frog NMJs were double-stained with a fluorescent dye, FM4-64, for NTs, and fluorescein isothiocyanate (FITC)-tagged peanut agglutinin (PNA) for PSCs. Identified NMJs were imaged in vivo repeatedly for several months after nerve injury. PSCs sprouted profusely beginning 3-4 weeks after nerve transection and, as reinnervation progressed, regenerating NTs closely followed the preceding PSC sprouts, which could extend tens to hundreds of microns beyond the original synaptic site. The pattern of reinnervation was dictated by PSC sprouts, which could form novel routes joining neighboring junctions or develop into new myelinated axonal pathways. In contrast to mammals, profuse PSC sprouting in frog muscles was not seen in response to axotomy alone, and did not occur at chronically denervated NMJs. Instead, sprouting coincided with the arrival of regenerating NTs. Immunofluorescent staining revealed that in muscle undergoing reinnervation 4 weeks after axotomy, 91% of NMJs bore PSC sprouts, compared to only 6% of NMJs in muscle that was chronically denervated for 4 weeks. These results suggest that reciprocal interactions between regenerating NTs and PSCs govern the process of reinnervation at frog NMJs: regenerating NTs induce PSCs to sprout, and PSC sprouts, in turn, lead and guide the elaboration of NTs.     

Metabolic stabilization of acetylcholine receptors in vertebrate neuromuscular junction by muscle activity

The effects of muscle activity on the growth of synaptic acetylcholine receptor (AChR) accumulations and on the metabolic AChR stability were investigated in rat skeletal muscle. Ectopic end plates induced surgically in adult soleus muscle were denervated early during development when junctional AChR number and stability were still low and, subsequently, muscles were either left inactive or they were kept active by chronic exogenous stimulation. AChR numbers per ectopic AChR cluster and AChR stabilities were estimated from the radioactivity and its decay with time, respectively, of end plate sites whose AChRs had been labeled with 125I-alpha-bungarotoxin (alpha-butx). The results show that the metabolic stability of the AChRs in ectopic clusters is reversibly increased by muscle activity even when innervation is eliminated very early in development. 1 d of stimulation is sufficient to stabilize the AChRs in ectopic AChR clusters. Muscle stimulation also produced an increase in the number of AChRs at early denervated end plates. Activity-induced cluster growth occurs mainly by an increase in area rather than in AChR density, and for at least 10 d after denervation is comparable to that in normally developing ectopic end plates. The possible involvement of AChR stabilization in end plate growth is discussed.     

Terminal sprouting in rat sternocostalis muscle following partial denervation

Summary The sternocostalis muscle of the rat was examined at one to five days after partial denervation and levels of terminal sprouting were assessed.The removal of one intercostal nerve caused localised degeneration which did not extend more than a few muscle fibres deep into the field of distribution of the adjacent nerve. Terminal sprouting was clearly seen at 24 h after operation and did not appear to develop further up to five days.There was no difference in the sprouting responses to section of either intercostal nerve 2, 4 or 5. There was, however, a decrease in the response with increasing distance from the cut nerve. No sprouting response was observed in the contralateral muscle.Comparison of sprouting levels of B and C type end plates revealed a greater percentage of C type end plates with sprouts. However, the response of B type end plates, considered in relation to the levels of spontaneous sprouting, was greater than that of C type end plates.     

Galectin-1 expression in innervated and denervated skeletal muscle

Galectin-1 is a soluble carbohydrate-binding protein with a particularly high expression in skeletal muscle. Galectin-1 has been implicated in skeletal muscle development and in adult muscle regeneration, but also in the degeneration of neuronal processes and/or in peripheral nerve regeneration. Exogenously supplied oxidized galectin-1, which lacks carbohydrate-binding properties, has been shown to promote neurite outgrowth after sciatic nerve sectioning. In this study, we compared the expression of galectin-1 mRNA and immunoreactivity in innervated and denervated mouse and rat hind-limb and hemidiaphragm muscles. The results show that galectin-1 mRNA expression and immunoreactivity are up-regulated following denervation. The galectin-1 mRNA is expressed in the extrasynaptic and perisynaptic regions of the muscle, and its immunoreactivity can be detected in both regions by Western blot analysis. The results are compatible with a role for galectin-1 in facilitating reinnervation of denervated skeletal muscle.     

Cell accumulation in the junctional region of denervated muscle

If skeletal muscles are denervated, the number of mononucleated cells in the connective tissue between muscle fibers increases. Since interstitial cells might remodel extracellular matrix, and since extracellular matrix in nerve and muscle plays a direct role in reinnervation of the sites of the original neuromuscular junctions, we sought to determine whether interstitial cell accumulation differs between junctional and extrajunctional regions of denervated muscle. We found in muscles from frog and rat that the increase in interstitial cell number was severalfold (14-fold for frog, sevenfold for rat) greater in the vicinity of junctional sites than in extrajunctional regions. Characteristics of the response at the junctional sites of frog muscles are as follows. During chronic denervation, the accumulation of interstitial cells begins within 1 wk and it is maximal by 3 wk. Reinnervation 1-2 wk after nerve damage prevents the maximal accumulation. Processes of the cells form a multilayered veil around muscle fibers but make little, if any, contact with the muscle cell or its basal lamina sheath. The results of additional experiments indicate that the accumulated cells do not originate from terminal Schwann cells or from muscle satellite cells. Most likely the cells are derived from fibroblasts that normally occupy the space between muscle fibers and are known to make and degrade extracellular matrix components.     

Effect of reinnervation on collagen synthesis in rat skeletal muscle.

The effect of reinnervation on the activities of prolyl 4-hydroxylase (PH) and galactosylhydroxylysyl glucosyltransferase (GGT), both enzymes of collagen biosynthesis, and on the concentration of hydroxyproline (Hyp) was studied in gastrocnemius, soleus, and tibialis anterior muscles of rat 19, 26, 40, and 61 days after crush denervation of the sciatic nerve. The GGT activity was elevated in denervated gastrocnemius and soleus muscles and the PH activity in gastrocnemius. Muscular Hyp concentration was increased in denervated tibialis anterior muscle. Both the PH and GGT activities and the Hyp concentration returned to the control level during the reinnervation period (19-61 days from the start of denervation). It seems that denervation atrophy of skeletal muscle is associated with an increased rate of muscular collagen biosynthesis and that during reinnervation collagen synthesis rate decreases despite accelerated muscular growth. The results thus suggest that innervation is a powerful suppressive regulator of muscular collagen biosynthesis.     

Sustained cell proliferation in denervated skeletal muscle of mice

Summary Cellular proliferation in skeletal muscle was measured throughout the first 4 weeks after denervation. Twenty four mice had one leg denervated, and 4 groups of 6 of these mice were injected with tritiated thymidine once daily for 7 days, either during the first, second, third or fourth week after denervation. Autoradiographic labelling of muscle and connective tissue nuclei in denervated muscles was compared with innervated muscles from the opposite innervated legs of the same mice. Labelling of connective tissue and muscle (myonuclear and satellite cell) nuclei was significantly higher in denervated muscles, compared with innervated muscles on the unoperated side. There were no significant differences among labelling of nuclei in muscles denervated for 1, 2, 3 or 4 weeks. However, connective tissue labelling after 1 week of denervation was significantly higher than at later times. This study shows that nuclei of muscle and connective tissue cells proliferate and turnover at high levels for at least one month after denervation.     

Impulse Activity Evokes Precocious Sprouting of Nociceptive Nerves into Denervated Skin

We have studied the sprouting of intact high-threshold mechanosensory nerves into adjacent denervated trunk skin in adult rats behaviorally, histologically, and electrophysiologically. In the anesthetized animal, stimulation of high-threshold endings in back skin by localized pinching elicits a bilateral reflex excitation of the underlying skeletal muscle, the cutaneous trunci muscle (CTM), visible as a twitch-like puckering of the skin. The reflex was also evoked by electrical excitation of Aδ and of C fibers in the dorsal cutaneous nerves (DCNs), with characteristic latencies of 7–20 msec and 40–60 msec, respectively; excitation of low-threshold (Aα) fibers was ineffective. After cutting selected DCNs, the deprived skin became insensible, but pinch responsiveness gradually recovered over the following 2 weeks. Regeneration of cut axons was not responsible for this recovery; when neighboring intact DCNs were cut, however, all responses were abolished in the recovered skin that had been initially denervated. By 3–5 days after denervation, axons in the dermis were all histologically absent or degenerating; when pinch sensitivity was restored to such skin, silver-stainable axons reappeared in the formerly empty Schwann tubes. During the work we noticed that the periodic examination by pinching, used to follow the time course of recovery of function in individual animals, led to an earlier development of this recovery than in animals that were examined only once at a specified time after denervation, This apparent acceleration in the redevelopment of pinch sensitivity was correlated with the appearance of axons in the recovered skin, and was shown to be due to the impulse activity evoked in the remaining nerves by the periodic pinching; it did not occur when the nerves were blocked by tetrodotoxin (TTX), and it was mimicked by a brief (10-min) period of electrical excitation of the Aδ fibers in a remaining nerve carried out at the time when the denervation of skin was done. The time course of the phenomenon suggested that the principal effect of the impulses was to shorten the latency to the onset of sprouting in the activated Aδ axons; that is, they induced precocious sprouting. The impulses needed to be conducted centrally for the effect to occur, and precocious sprouting failed to occur if the impulses were allowed to proceed only distally toward the skin.     

Denervated muscle fibers explain the deficit in specific force following reinnervation of the rat extensor digitorum longus muscle

The authors tested the hypothesis that, after denervation and reinnervation of skeletal muscle, observed deficits in specific force can be completely attributed to the presence of denervated muscle fibers. The peroneal nerve innervating the extensor digitorum longus muscle in rats was sectioned and the distal stump was coapted to the proximal stump, allowing either a large number of motor axons (nonreduced, n = 12) or a drastically reduced number of axons access to the distal nerve stump (drastically reduced, n = 18). A control group of rats underwent exposure of the peroneal nerve, without transection, followed by wound closure (control, n = 9). Four months after the operation, the maximum tetanic isometric force (Fo) of the extensor digitorum longus muscle was measured in situ and the specific force (sFo) was calculated. Cross-sections of the muscles were labeled for neural cell adhesion molecule (NCAM) protein to distinguish between innervated and denervated muscle fibers. Compared with extensor digitorum longus muscles from rats in the control (295 +/- 11 kN/m2) and nonreduced (276 +/- 12 kN/m2) groups, sFo of the extensor digitorum longus muscles from animals in the drastically reduced group was decreased (227 +/- 15 kN/m2, p < 0.05). The percentage of denervated muscle fibers in the extensor digitorum longus muscles from animals in the drastically reduced group (18 +/- 3 percent) was significantly higher than in the control (3 +/- 1 percent) group, but not compared with the nonreduced (9 +/- 2 percent) group. After exclusion of the denervated fibers, sFo did not differ between extensor digitorum longus muscles from animals in the drastically reduced (270 +/- 20 kN/m2), nonreduced (301 +/- 13 kN/m2), or control (303 +/- 10 kN/m2) groups. The authors conclude that, under circumstances of denervation and rapid reinnervation, the decrease in sFo of muscle can be attributed to the presence of denervated muscle fibers.     

Histological evidences of reparative and regenerative effects of beta-adrenoceptor agonists, clenbuterol and isoproterenol, in denervated rat skeletal muscle

The aim of this study was to determine the contribution of beta-adrenoceptor activation in the reconstruction of the structural and functional organization of denervated skeletal muscle. beta-agonists, clenbuterol (1.2 mg/kg body weight) and isoproterenol (2 mg/kg body weight), administration (daily oral administration; maximum 7 days) to normal innervated rats as well as denervated animals caused muscle hypertrophy. An increase in mean fiber diameter confirmed this stimulated growth both in normal innervated and denervated rat gastrocnemius muscle. Examination of muscle nuclei from treated but normal innervated rat gastrocnemius exhibited features like large size, active nucleoplasm and an increase in their number per fiber cross section and per mm mean fiber length indicating towards an elevated biosynthetic activity in tissue in the presence of beta adrenoceptor agonists. Administration of drugs to normal innervated animals resulted in an emergence of central muscle nuclei. The hyperactive and enlarged muscle nuclei ultimately organized themselves into unusually elongated nuclear streaks. beta agonist treatment to denervated rats resulted in amelioration of atrophic state of tissue characterized by hypertrophy of muscle fibers thus lending to a restoration of structural organization of tissue. Bizarre shapes of nuclei in denervated muscle tend to recover to that characteristic to normal innervated muscle in presence of clenbuterol and isoproterenol hydrochloride. All observations were confirmed by administering butoxamine, a beta-adrenoceptor antagonist along with beta-agonists. The results suggests that both clenbuterol and isoproterenol hydrochloride are capable of mimicking normal innervation functions in skeletal muscle and thus play important role in the structural and functional reorganization of tissue. Amelioration of denervation atrophy in rat gastrocnemius in the presence of beta-agonists supports this.