Age changes of neuromuscular junctions in the extensor digitorum longus muscle of spontaneous thymoma BUF/Mna rats

BUF/Mna rats spontaneously develop thymomas and cause muscle weakness of hind legs at an advanced age. This rat strain has been recognized as a suitable animal model for human myasthenia gravis or related myopathies. To characterize the structural changes of neuromuscular junctions (NMJs) in BUF/Mna rats, subneural apparatuses (SNAs) of extensor digitorum longus muscles of young-adult (4-month-old) and aged (22- to 25-month-old) male rats were examined using scanning and transmission electron microscopy. The SNAs of NMJs in young rats consist of complex labyrinthine gutters with numerous slit-like junctional folds. SNAs in aged BUF/Mna rats, however, are characterized by: (1) a group of cup-like depressions with very wide slit-like junctional folds in relatively large muscle fibers (the major type), (2) the presence of slit-like folds on the flat sarcoplasm outside the cup-like depressions or on the protruded sarcoplasm, and (3) winding gutters or a small number of round depressions with poorly developed synaptic folds in small and medium-sized muscle fibers (the minor type). Since similar structural changes have been reported in dystrophic mice or normally aged rats, it is suggested that both the slowly progressing muscle atrophy and age-dependent turnover of muscle fibers may occur in the aged BUF/Mna rats. Received: 19 October 1999 / Accepted: 16 March 2000     

The morphological variability of neuromuscular junctions in the rat extraocular muscles: a scanning electron microscopical study

Six morphologically distinct types of neuromuscular junctions were identified by scanning electron microscopy in the rat extraocular muscles: two diffuse and four focal types. The diffuse junctions, spreading out extensively over the muscle fiber surface, were characterized by two types of varicose swellings (or terminal varicosities) of nerve endings. One type consisted of several ramifying nerve endings and shallow postsynaptic depressions with poorly-developed junctional folds. The other type consisted of a single axon and formed many synaptic contacts along the long axis of a muscle fiber. Round synaptic depressions facing the varicosities contained several junctional folds. The focal junctions, confined to an oval area on the muscle fiber surface, were characterized by the complexity and variability of their subneural apparatuses. Four different types of apparatuses, i.e., focal junctions, were found: 1) an apparatus consisting of labyrinthine gutters with numerous slit-like junctional folds, 2) apparatuses consisting of a large number (more than 20) of cup-like depressions with either a small number of pit-like junctional folds or 3) numerous slit-like ones, and 4) an apparatus consisting of a small number (about 10) of cup-like depressions with a few junctional folds. The findings indicate that the rat extraocular muscles contain six different types of muscle fibers.     

Postnatal morphodifferentiation of the subneural apparatuses of the posterior cricoarytenoid muscle in rats: a scanning electron microscopy study

The subneural apparatus, i. e., the post-synaptic component of the neuromuscular junction, in the posterior cricoarytenoid muscle of the rat was studied by scanning electron microscopy, with special attention given to its postnatal differentiation along with the functional development of the muscle. Primitive synaptic troughs observed in the first postnatal week consisted of single cup-like depressions 5-6 microm in diameter. On the 7th day, low sarcoplasmic ridges appeared in the trough. In the second postnatal week, muscle fibers could be classified into two groups: large (10-15 microm in diameter) and small (less than 10 microm in diameter). In the large muscle fibers, many low ridges became circular and protruded to transform the single trough into numerous cup-like depressions (2-5 microm in diameter). In contrast, the subneural apparatus in the small muscle fibers consisted of a small number of cup-like depressions. The two types of subneural apparatus differentiated into adult forms by the 28th postnatal day, although they remained smaller in size than those of adults. In the large muscle fibers, the number of pit-like or elongated invaginations increased and gradually transformed into slit-like junctional folds by the 28th postnatal day, while the small muscle fibers still possessed a few pit-like or elongated junctional folds at this point in time. The two types of morphodifferentiation of the subneural apparatus are thought to reflect the two types of muscle fibers in the rat posterior cricoarytenoid muscle.     

Alterations in neuromuscular junction morphology during fast-to-slow transformation of rabbit skeletal muscles

Summary Chronic low frequency stimulation of motor nerves results in transformation of muscle fibre phenotype from fast- to slow-twitch. We examined the light and electron microscopic structure of neuromuscular junctions in normally fast twitch muscles, tibialis anterior and extensor digitorum longus of rabbit after 3 weeks of stimulation to determine whether synaptic structure is also modified during fibre type transformation. Neuromuscular junctions of stimulated and unstimulated (control) tibialis anterior and extensor digitorum longus muscles and unstimulated slow twitch soleus muscle were visualized with rhodamine-conjugated -bungarotoxin. Video light microscopic images of neuromuscular junctions were digitized to allow quantification of their surface areas, perimeters, lengths and widths. Three weeks of stimulation resulted in a decrease in the maximal velocity of muscle fibre shortening and augmentation of mitochondrial volume in fast muscles, demonstrating the efficacy of the stimulation protocol employed in altering muscle fibre phenotype. Neuromuscular junctions of control tibialis anterior and extensor digitorum longus are thin, compact, and continuous, with complex branching patterns. In contrast, those of slow-twitch soleus are thicker and discontinuous. Neuromuscular junctions in control tibialis anterior and extensor digitorum longus are larger than those in soleus. Three weeks of stimulation causes a marked decrease in the size of neuromuscular junctions in tibialis anterior and extensor digitorum longus, as reflected in the significant reduction in neuromuscular junction surface area, length and width. Electron microscopy of these junctions suggests that secondary postsynaptic folds in stimulated muscles are more closely spaced. Also, axon terminals of stimulated muscles appear to contain more densely packed synaptic vesicles and mitochondria than controls. Decreases in neuromuscular junction dimensions can be partly explained by muscle fibre atrophy. However, the decrease in neuromuscular junction size is proportionately greater than that of muscle fibre diameter in both muscles, indicating that factors other than fibre atrophy may contribute to the reduced neuromuscular junction size in stimulated muscles. Neuromuscular junctions of stimulated tibialis anterior and extensor digitorum longus muscles exhibit some features characteristic of normal soleus neuromuscular junctions, indicating structural adaptations consistent with the altered muscle fibre phenotype. On the other hand, neuromuscular junctions of 3 week stimulated tibialis anterior and extensor digitorum longus and their synaptic branches remain as thin and continuous as those of unstimulated controls, suggesting that the transformation of neuromuscular junctions towards a morphology characteristic of slow muscle, is only partial. These results demonstrate that an altered pattern of impulse activity causes significant synaptic remodelling in adult rabbit skeletal muscles.     

Topographic comparison of neuromuscular junctions in mouse slow and fast twitch muscles

The neuromuscular junctions of mammalian slow and fast twitch muscles are activated differently in vivo and show corresponding physiological differences in vitro, but the structural basis or consequences of these differences are relatively unexplored. Therefore, neuromuscular junctions of mouse fast (extensor digitorum longus) and slow (soleus) twitch muscles were compared by use of new scanning and light microscopy techniques. In both muscles, the endplate appeared as an elliptical area raised to a variable extent above the surrounding sarcolemma and containing the primary clefts. In most soleus endplates, this raised surface area was considerably higher and wider and about three times larger than in extensor digitorum longus. In addition, the primary cleft area was about two-fold greater in soleus than in extensor digitorum longus, even though cleft length was the same. The primary clefts formed either an elliptical shape along the outer margin of the endplate with inward-directed branches or a group of relatively rectilinear dendritic branches orthogonally oriented to one another. The latter type was most frequent in soleus and the elliptical type in extensor digitorum longus. Corresponding patterns of nerve terminal arborizations were seen by light microscopy. Although nerve terminal areas were the same in fast and slow muscles, in the former, numerous diverticulae significantly increased the length of the nerve terminal outline. The possible physiological significance of the different synaptic structure of slow and fast muscle is discussed.     

Degeneration and regeneration of neuromuscular junction architecture in rat skeletal muscle fibers damaged by bupivacaine hydrochloride

We evaluated the degeneration and regeneration of neuromuscular junctions (NMJs) on the extensor digitorum longus muscle of Fischer 344 rats between 4 h and 3 weeks after bupivacaine hydrochloride (BPVC) injection, which induces muscle fiber necrosis, using histochemical staining by acetylcholine esterase (AchE)-silver and electron microscopy. Degeneration of muscle fibers and NMJs was observed 4 h after BPVC injection. One week after BPVC injection, some terminal axons were almost completely retracted, and the level of basal lamina-associated AchE in some NMJ regions had gradually disappeared. At that time, the depression contained a few, mostly pit-like or elongated oval invaginations: the incipient junctional folds and some NMJs did not have any secondary junctional fold. By 2 weeks after the BPVC injection, secondary junctional folds began to develop: however, the number of secondary junctional folds was clearly less than that in normal NMJs. At 3 weeks when regeneration of muscle fibers was well advanced, the staining for AchE at the end-plates became stronger and better-defined. The volume density of mitochondria in the terminal area of the terminal significantly decreased upon BPVC-induced destruction of the NMJ, and the density reached the lowest value 24 h after BPVC injection. Significant changes in the ultrastructural features of the architecture of NMJs occurred in skeletal muscle fibers damaged by BPVC during both the degeneration and regeneration processes. The changes in the ultrastructural and morphological features of the NMJ architecture during the regeneration of degenerated muscle fibers resembled those that occur during the differentiation of normal muscle fibers.     

Effects of age on the number and histochemical properties of muscle fibers and motoneurons in the rat extensor digitorum longus muscle

The number and histochemical properties of muscle fibers and motoneurons were studied in the extensor digitorum longus muscle in female albino rats at the ages of 10, 60 and 120 weeks. The number of fast twitch glycolytic (FG) fibers was decreased at 60 weeks, while that of fast twitch oxidative glycolytic (FOG) fibers was decreased at 120 weeks. The number and oxidative enzyme activity of motoneurons were decreased later at 120 weeks. Thus, it is suggested that the decrease in FG fibers at 60 weeks was due to selective muscle fiber atrophy or to degeneration of neuromuscular junctions, while on the other hand, the decrease in FOG fibers at 120 weeks was due mainly to both a decrease in motoneurons and a type shift of fibers from FOG to FG.     

The overall morphology of neuromuscular junctions as revealed by scanning electron microscopy

Summary Skeletal neuromuscular junctions (NMJs) of vertebrates were examined by scanning electron microscopy after removal of connective tissue components by HCl hydrolysis. In addition to the surface texture of NMJs, the subsynaptic organization of the sarcolemma was visualized in specimens in which nerve endings were detached from the muscle surface.A remarkable morphological variability between animal species was observed. The NMJs in the frog sartorius muscle consisted of longitudinal ribbon-like endings which fitted into a shallow synaptic gutter containing highly ordered cross-bands of junctional folds. The NMJs of the posterior latissimus dorsi muscle of the zebra finch were characterized by varicose swellings of the nerve endings which fitted into a round pit of the sarcolemma. NMJs in the sternothyroid muscle of the Chinese hamster consisted of thin ramified endings which were confined to an oval area on the muscle surface. The labyrinthine synaptic groove contained well-developed junctional folds without preferential spatial arrangement.The procedure used for the present study illustrates in great detail the terminal arborization of the motor nerve ending and the surface features of the subsynaptic sarcolemma. It may also allow quantitative study of the synaptic morphology of NMJs.     

Targeting functional subtypes of spinal motoneurons and skeletal muscle fibers in vivo by intramuscular injection of adenoviral and adeno-associated viral vectors

We report that functional subtypes of spinal motoneurons and skeletal muscle fibers can be selectively transduced using replication-defective adenoviral (ADV) or adeno-associated (AAV) viral vectors. After intramuscular injection in adult rodents, ADV vectors transduced both fast-twitch and slow-twitch skeletal muscle fibers. Intramuscular injection of ADV vectors also caused transduction of spinal motoneurons and dorsal root ganglion cells. However, only neurons innervating the injected muscle were transduced, as shown by co-injection of a retrograde axonal tracer. In adult male rats it is therefore possible to transduce fast or slow spinal motoneurons and muscle fibers selectively since in these animals, the extensor digitorum longus and soleus muscles contain almost exclusively fast or slow motor units, respectively. In rats, AAV vectors transduced muscle fibers in the predominantly fast extensor digitorum longus but not in the predominantly slow soleus muscle. We did not observe any transduction of spinal motoneurons following intramuscular injection of AAV vectors. These results show that physiologically and clinically important subpopulations of cells in the neuromuscular system can be selectively transduced by viral vectors.     

Infraterminal spreading and extrajunctional expression of nicotinic acetylcholine receptors in denervated rat skeletal muscle

 Through the use of biotinylated-bungarotoxin and monoclonal antibodies, the nicotinic acetylcholine receptor (nAChR) was localized in the subneural apparatus of mammalian motor end plates of the flexor digitorum brevis muscle of the adult rat at the light and electron microscopic levels. Under normal conditions, nAChR was located in the primary post-synaptic membrane of the neuromuscular junction, and the depths of the junctional folds constituting the secondary post-synaptic membrane did not contain any nAChR. Up to 75 days after repeated transection of the related motor nerve (sciatic), there was no major alteration in the light-microscopic localization of junctional nAChR in the subneural apparatus, except for a moderate shrinkage and increased immunocytochemical reactivity of the subneural apparatus. At the electron microscopic level, however, immunocytochemical reactivity gradually occupied the entire extent of the secondary post-synaptic membrane, including the depths of the junctional folds, which exhibited extensive branching. In non-innervated portions of the muscle fibers, nAChR receptor appeared in a linear localization on the surfaces of denervated muscle fibers. This linear reaction was not continuous with the nAChR reaction of the motor end plates. It is concluded that denervation supersensitivity might not be due to spreading of junctional nAChR from the end-plate area, but rather to expression of nAChR in non-innervated portions of the muscle fiber and to the infraterminal (subsynaptic) spreading of nAChR into the depths of junctional folds. Received: 29 May 1998 / Accepted: 8 December 1998     

Ultrastructural studies of young and old mouse neuromuscular junctions

Summary The ultrastructure of the neuromuscular junction of young and old male CBF-1 mice was analysed both qualitatively and quantitatively. The age-related findings were similar in both the phasic extensor digitorum longus muscle and the tonic soleus muscle but more pronounced in the latter. Presynaptic terminals of old mice compared to young showed decreases in nerve terminal area, mitochondria and synaptic vesicles, but increases in smooth endoplasmic reticulum, coated vesicles, cisternae, microtubules and probably neurofilaments. On the postsynaptic side there were increases in complexity of junctional folds and subsarcolemmal vesicles, and the appearance of lipofuscin deposits. Occasional denervated postsynaptic regions were encountered in old neuromuscular junctions, but the predominant characteristics of aging changes were not those of denervation. Rather, a unique and uniform process involving most of the population of nerve terminals, possibly of physiologically adaptive significance, appears to occur with age in both phasic and tonic limb muscles.     

Three-dimensional structure of the synaptic contact of the neuromuscular junction in the rat lumbrical muscle.

This study examined the three-dimensional structures of the synaptic contact in rat lumbrical muscles by scanning electron microscopy using three different methods: the aldehyde prefix-osmium-dimethyl sulfoxide-osmium method (A-O-D-O method), the cell-extraction method, and the NaOH-digestion method. These three methods visualized the motor nerve endings, subneural basal lamina and postsynaptic sarcolemma, respectively. The motor nerve endings were composed of a cluster of spherical and cylindrical terminals. Pores on the presynaptic membrane were considered openings of exocytotic vesicles. The postsynaptic side of the subneural basal lamina showed numerous ridges, corresponding to junctional folds. Most of the ridges rose vertically from their base. The ridges showed widening, narrowing, and branching. The subneural basal lamina appeared to be composed of small granular substances. The basal lamina of the primary synaptic clefts had pores 25-30 nm in diameter, which may facilitate the transport of acetylcholine (ACh) without being hydrolyzed by ACh esterase in the lamina. On the outer surface of the postsynaptic sarcolemma in a sole plate, the primary synaptic clefts were composed of a mixture of depressions and gutters; so far as we know, this represents the only example of such a phenomenon. These depressions and gutters seem to fit respectively into the spherical and cylindrical terminals of the motor nerve endings. The openings of the junctional folds consisted of a mixture of many slits and a few pits in the primary synaptic clefts.     

Factors causing different properties at neuromuscular junctions in fast and slow rat skeletal muscles

Neuromuscular junctions on fast and slow skeletal muscle fibers have different properties. Possible reasons for these differences were examined in adult rat soleus (SOL) muscle fibers reinnervated at new ectopic or old denervated sites by fast fibular (FIB) or slow SOL motoneurons. FIB motoneurons formed large ectopic junctions with a high density of nerve terminal varicosities (fast appearance), whereas SOL motoneurons formed small ectopic junctions with a low density of varicosities (slow appearance). Both FIB and SOL motoneurons formed small junctions with a slow appearance when reinnervating old SOL endplates. FIB nerves innervating ectopic sites and SOL nerves reinnervating old sites had the same appearance whether they contacted the SOL fibers alone (single innervation) or together (dual innervation). Continuous stimulation of the FIB nerve at 10 Hz for 3–4 months reduced the size of ectopic FIB and intact extensor digitorum longus (EDL) junctions and caused a modest reduction in density of terminal varicosities in EDL. Junction size and muscle fiber diameter were positively correlated, but the slope describing this relation was steeper for FIB junctions than for SOL junctions. It is concluded that in the present system (1) motoneuron type and not muscle fiber type determines the fast or slow character of the neuromuscular junction, (2) denervated endplates of one type place stable and severe constraints on the termination pattern of reinnervating axons of another type, (3) the appearance of fast EDL junctions undergoes a modest fast to slow transformation when exposed to long-term slow pattern stimulation, and (4) not only the size of the muscle fibers, but also the type and firing pattern of the motoneurons and the spatial constraints at preformed endplates influence the relation between junction size and muscle fiber diameter.     

Transformation of contraction speed in muscle following cross-reinnervation; dependence on muscle size

Summary The characteristics of isometric contractions and the force-velocity relation were studied in flexor digitorum longus, flexor hallucis longus and soleus muscles of the cat,in situ, at 37° C and with nerve stimulation. The two flexors were identified as typical fast twitch muscles and the soleus as a typical slow twitch muscle. Following self-reinnervation, both fast and slow muscles retained, to a large extent, their basic contraction characteristics. The soleus muscle, when cross-reinnervated with the nerve of either flexor hallucis longus muscle or extensor digitorum longus muscle exhibited a more complete slow-to-fast transformation than when cross-reinnervated with the nerve of flexor digitorum longus muscle. The flexor digitorum longus muscle underwent a greater degree of fast-to-slow transformation than the flexor hallucis longus muscle, when each was cross-reinnervated with the soleus nerve. The data previously reported for sarcomere shortening velocities of the cross-reinnervated muscles in the rat, the rabbit and the cat are reviewed in the light of present findings. It is found that the discrepancies obtained between species and between different muscles in the same species, with respect to the degree of muscle-speed transformation following cross-reinnervation, are correlated with the differences in the size-ratio of the muscles used in the cross-reinnervation procedure.     

家兔趾长伸肌肌内神经、运动终板和肌梭的分布

目的：探究家兔趾长伸肌肌内神经、运动终板与肌梭的分布。方法：改良Sihler’s染色法、乙酰胆碱酯酶染色法及HE染色法。结果：趾长伸肌是短肌束构成的长肌,肌束起点高的,止点相应亦较高。连于第2趾的肌纤维占据了肌腹上1／3,止于其余3趾的肌纤维位于下2／3。趾长伸肌的神经来自2条肌外神经干,上支及其分支支配第2趾的肌纤维,下支司其余3趾。运动终板分布除冠状切面上为弥漫的黑色颗粒外,其余各切面均为一条连续的运动终板带。各部的肌梭密度分别是：起端33.95个／g、肌腹中部44.76个／g、止端为零。结论：家兔趾长伸肌内,肌梭分布不均匀。肌内神经分支密集的部位,运动终板集聚、肌梭密度亦高。家兔趾长伸肌具有划分亚部的特征。     

Postnatal development of rat motor nerve terminals

Summary The nerve terminals of neuromuscular junctions in the rat diaphragm, extensor digitorum longus muscle and soleus muscle have been studied in animals between 3 weeks and 2.5 years of age using methylene blue stain and light microscopy. Dimensions, structure and organization of the nerve terminals were shown to change during life at various rates in different muscles and postnatal periods. The area and length of the terminals increase in all three muscles until young adult age. Later these dimensions continue to increase in the extensor digitorum longus and soleus muscles. In the diaphragm only the length increases, and this occurs late in adult life. The area also increases in relation to the diameter of the corresponding muscle fiber. Adult soleus terminals are more elongated than terminals in the diaphragm and extensor digitorum longus muscle. During adult life the extension of nerve terminals in relation to muscle fiber length increases in the extensor digitorum longus and soleus muscles, but is almost unchanged in the diaphragm. The nerve terminal branches are mainly coarse and irregular in young animals, but possess varying numbers of varicosities in adult animals. The number of varicosities is high in the extensor digitorum longus muscle and low in the diaphragm. In old animals the number of varicosities tends to be reduced. With increasing age the nerve terminal branches become organized in distinct groups with increasing distance between the groups. This is prominent in the soleus.     

Demonstration of glutamate-like immunoreactivity at rat neuromuscular junctions by quantitative electron microscopic immunocytochemistry

Motor nerve terminals and adjacent structures in the extensor digitorum longus and soleus muscles of young adult rats were examined for their content of glutamate by means of quantitative, electron microscopic immunocytochemistry employing colloidal gold particles as markers. The level of glutamate immunoreactivity was stronger in the extensor digitorum longus terminals than in the soleus terminals. In both muscles the glutamate immunolabelling was stronger in the nerve terminals than in the synaptic clefts and the postsynaptic tissue separating the secondary clefts, but the differences were larger in the extensor digitorum longus than in the soleus muscle. The myofibrils of the soleus muscle were more densely labelled than those in the extensor digitorum longus muscle: The level of immunoreactivity was high in the Schwann cells of both muscles. By comparing the labelling intensity of motor nerve terminals with that of muscle fibres and hippocampal mossy fibres (compartments that have been analysed previously with respect to their glutamate content), the mean concentration of fixed glutamate in the extensor digitorum terminals was estimated to be in the range of 10–20 mmol/l. An association of glutamate immunoreactivity with synaptic vesicles was demonstrated in the most strongly labelled terminals. Whether these epitopes were localized in the interior of the vesicles or at their external surface could not be resolved with the present technique. These data indicate that motor nerve terminals contain glutamate, and that the enrichment of this amino acid is more pronounced in the terminals of the extensor digitorum longus muscle (a fast muscle) than in those of the soleus muscle (a slow muscle). A possible modulatory or trophic role of glutamate in the mammalian neuromuscular junction should be considered.     

Different effects of physical training on the morphology of motor nerve terminals in the rat extensor digitorum longus and soleus muscles

Summary The morphology of nerve terminals in the rat extensor digitorum longus and soleus muscles was studied with light microscopy in 13-week-old male animals after 6 weeks of treadmill running and compared with data from untrained controls. The terminals were stained with methylene blue. Physical training tended to increase the area and length of the nerve terminals in relation to the corresponding muscle fiber diameter, and to reduce the density of nerve terminal varicosities, but significant differences between the trained group and the control group were obtained only in the extensor digitorum longus muscle. The different degrees of effect on the nerve terminals in the two muscles may be due to different abilities to respond to the training, but may also be due to differences in work load caused by the training. The effect of training on extensor digitorum longus junctions may reflect some transformation from fast to slow morphological characteristics.     

A comparison of fibrillation in denervated skeletal muscle of the anaesthetized rat and guinea-pig

Summary We have used intracellular recordingin vivo to study fibrillation (spontaneous repetitive membrane activity) in extensor digitorum longus (fast twitch) and soleus (slow twitch) muscles of the anaesthetized rat and guinea-pig denervated for periods of about 10 to 60 days. The proportion of fibres fibrillating in the guinea-pig soleus was greater than 50% in most animals up to the longest period of denervation (65 days). Fibrillation was rarely found in rat soleus after three weeks of denervation. Its incidence in the extensor digitorum longus muscles of both species was intermediate. The mean frequency of fibrillation was higher in guinea-pig extensor digitorum longus (16 Hz) and soleus (8 Hz) than in the rat extensor digitorum longus (3 Hz) and soleus (2 Hz). The resting membrane potentials of the denervated muscles were less than normal and correlated inversely with the frequency of fibrillation but not with the incidence of fibrillation: in rat soleus, many fewer fibres were fibrillating at a given membrane potential than in the other three muscles. The incidence of fibrillation was compared with previously reported tensions of the four denervated muscles and was found to have the same rank order. We suggest that fibrillation may reduce atrophy (and hence tension loss) of denervated muscle, which may have implications for artificial stimulation. Fibrillation frequency was directly related to changes in twitch speed of the four muscles after denervation.     

Endocytosis in chronically denervated mouse skeletal muscle. A biochemical and ultrastructural study with horseradish peroxidase.

Endocytosis of horseradish peroxidase was studiedin vivo in innervated mouse extensor digitorum longus and soleus muscles and in the same muscles 6 days after surgical denervation, by a combination of biochemical and morphological techniques. Biochemical determination of peroxidase activity in extensor digitorum longus and soleus muscles at different times after a single intravenous injection of horseradish peroxidase followed by extensive washing of the muscles, showed that peak levels of horseradish peroxidase in muscle tissue were obtained 2 h after injection. The uptake of horseradish peroxidase in denervated extensor digitorum longus and soleus muscles was approximately three times greater than in innervated control muscles. Light and electron microscopic examination of denervated and innervated extensor digitorum longus muscles, demonstrated that horseradish peroxidase was present in the extracellular space, i.e. between muscle fibers and in transverse tubules already 30 min after an intravenous horseradish peroxidase injection. Light-microscopic examination, 2–4 h after an intravenous injection of horseradish peroxidase, revealed that a population of denervated muscle fibers contained horseradish peroxidase in bodies or vacuoles restricted to segments of the fibers. Such bodies, containing horseradish peroxidase, were not observed in innervated muscle fibers. Ultrastructural examination showed that these horseradish peroxidase-bodies were limited by a single membrane and located adjacent to transverse tubules between myofibrils showing early signs of degeneration. Coated or uncoated vesicles of about 50–100 nm appeared to originate from horseradish peroxidase-labelled transverse tubules in denervated muscle fibers.These findings suggest that endocytosis (micropinocytosis) occurs from transverse tubules in segments of denervated muscle fibers where signs of degeneration can be observed. Following the entry of horseradish peroxidase, the tracer accumulates in heterophagosomes and secondary lysosomes of diverse appearance.