In vitro regulation of the innervation pattern of quail muscle fibres by quail and mouse neurons

Myoblasts from rudiments of slow and fast muscle, anterior latissimus dorsi (ALD) and posterior latissimus dorsi (PLD) respectively, of 9-day-old quail embryos were cultured in vitro for a period of up to 60 days in order to give rise to well-differentiated muscle fibres. These fibres were innervated by neurons from either quail or mouse embryo spinal cord and their innervation pattern was examined by the visualization of acetylcholine receptors (ACh-R) and of acetylcholinesterase (ACh-E) activity at the neuromuscular contacts. In the culture system used, quail neurons always innervated muscle fibres at several sites and only when a fast-type activity was imposed on these neurons did a reduction in the number of the previously established neuromuscular contacts take place. In contrast, in the muscle fibres innervated by mouse neurons, a spontaneous reduction in the number of the previously established neuromuscular contacts occurred but this spontaneous reduction depended upon the level of differentiation reached by the muscle fibres in vitro. In the cultures of muscle fibres previously innervated by mouse neurons, the addition of quail neurons did not provoke any modification in the initial innervation pattern, and no quail ACh-R cluster was observed. In contrast, in the muscle fibres previously innervated by quail neurons, the mouse neurons contacted these fibres, resulting in a decrease in the number of quail ACh-R clusters. These results emphasize the part played by neurons in the establishment of the innervation pattern when muscle fibres have reached a high level of differentiation. In vitro, the slow and fast characteristics of the muscle fibres do not influence this pattern.     

Histochemical sub-types of three fibre-types of avian skeletal muscle

Summary The soleus muscle of adult chicken was studied histochemically. Succinic dehydrogenase (SDH) and myofibrillar ATPase reaction, with or without preincubation in K₂-EDTA salt, were compared in serial frozen sections. Based upon the distributions of the above reactions, the three major fibre types distinguished were Type I red, Type II red and Type II white. On the basis of non preincubated ATPase reaction alone two sub-types of type I red fibres could be distinguished. However, following preincubation in a Cold solution of K₂-EDTA, Type II red fibres fell into two sub-types and Type II white fibres fell into three sub-types. Amalgamating the two already existing classifications, a more elaborate classification is presented for characterizing these different sub-types. The presence of two different or a spectrum of staining variations in a seemingly homogeneous population of muscle fibres in a given fibre-type emphasizes the possible correlation between this histochemical data and the heterogeneity of contraction times of the different motor units.     

Molecular forms of the cholinesterases inside and outside muscle endplates

Individual endplates were micro-dissected from chicken fast-twitch muscle, and the molecular forms of acetylcholinesterase and of pseudocholinesterase therein, identified by their sedimentation coefficients, were analysed directly. The forms actually present at the endplate, and those that are non-synaptic9 were established. This analysis was also extended to muscle of the chicken with inherited muscular dystrophy, showing altered distributions of these forms.     

Chick and quail limb bud myoblasts, isolated at different times during muscle development, express stage-specific phenotypes when differentiated in culture

Evidence is presented which shows that myoblasts, isolated at different stages during chick and quail limb bud development, will form, in culture, myotubes which can be distinguished with a combination of morphological as well as biochemical criteria. Hind limb bud myoblasts isolated from 5-day-old embryos form very short myotubes which synthesize a myosin, the light chains of which are predominantly LC1F and LC2S. Myoblasts isolated from the limb buds of 7-8-day-old embryos form large myotubes which synthesize a myosin the light chains of which are predominantly LC1F, LC2S and LC2F. Myoblasts isolated from the thigh muscle of embryos older than 10 days form large myotubes which synthesize a myosin the light chains of which are predominantly LC1F and LC2F. These results have been confirmed by hybridization of the cellular mRNA with a molecular probe specific for LC2F. These results lead us to suggest the existence of at least two classes of myoblasts which appear at different times during limb bud development. The first class, or 'early' myoblasts, is present in the limb buds of 5-day-old embryos, whereas the second class, or 'late' myoblasts, is present in the muscles of embryos older than 8 days. This result, however, is also compatible with the hypothesis that all muscle cells are the same at all times during development, and that the different phenotypes simply reflect differences in the environmental conditions.     

On the time course of surgically induced compensatory muscle hypertrophication of the rat plantaris muscle.

1. The time course of the hypertrophy and hyperplasia of the rat plantaris muscle was determined from measurements of total muscle mass and cross-section analysis of fixed muscle. 2. Muscle enlargement was induced by the surgical removal of the plantaris synergist muscles, the gastrocnemius and the soleus. 3. From the date of surgery through the third post-operative week, muscle enlargement is due to fiber hypertrophication (approximately 100% increase in diameter). After post-operative week three, muscle enlargement is due to a combination of hyperplasia and hypertrophy. At week four the cross-sectional areas return to control values. 4. The neuromuscular junction area was determined by measuring Karnovsky stained post-synaptic membrane. Only a modest 10-30% increase was noted at weeks 2 and 3 with a return to control levels at week 4. The differences were not statistically different.     

The pattern of neurovascular development in the forelimb of the quail embryo

Peripheral nerve and vascular patterns are congruent in the adult vertebrate, but this has been disputed in vertebrate embryos. The most detailed of these studies have used the avian forelimb as a model system, yet neurovascular anatomical relationships and critical vascular remodeling events remain inadequately characterized in this model. To address this, we have used a combination of intravascular marker injection, multilabel fluorescent stereomicroscopy, and confocal microscopy to analyze the spatiotemporal relationships between peripheral nerves and blood vessels in the forelimb of 818 quail embryos from E2 (HH13) to E15 (HH41). We find that the neurovascular anatomical relationships established during development are highly stereotypic and congruent. Blood vessels typically arise before their corresponding nerves, but there are several critical exceptions to this rule. The vascular pattern is extensively remodeled from the earliest stage examined (E2; HH13), whereas the peripheral nerves, the first of which enter the forelimb at E3.5-E4 (HH21-HH24), have a progressively unfolding pattern that, once formed, remains essentially unchanged. The adult neurovascular pattern is not established until E8 (HH34). Peripheral nerves are always found to track close and parallel to the vasculature. As they track distally, peripheral nerves always lie on the side of the vasculature away from the center of the forelimb. Neurovascular patterns have a hierarchy of congruence that is highest in the dorsoventral plane, followed by the anteroposterior, and lastly the proximodistal planes.     

Morphological adaptation of capillary network in compensatory hypertrophied rat plantaris muscle

The aim of this study was to examine the morphological adaptation of the capillary network in hypertrophied plantaris muscles by examining both capillary numbers and luminal circumferences. Hypertrophy of the plantaris muscle was induced by myectomy of the gastrocnemius muscle. This hypertrophy was characterised by increases in muscle mass and fibre cross-sectional area. All capillary parameters were determined using morphometric methods in perfusion-fixed plantaris muscle. Increased capillary-to-fibre ratio was observed in the overloaded plantaris muscle while no change was observed in the capillary luminal circumference. No differences were observed in the capillary density and the capillary-to-fibre perimeter ratio of the normal and the hypertrophied plantaris muscle. These results indicated that chronic overload-induced neocapillarization, but not enlargement of capillary luminal circumference, contributed to the prevention of decreases in the capillary-to-fibre perimeter ratio in the plantaris muscle in the hypertrophied process. Accepted: 13 August 1996     

Calcineurin and heat shock protein 72 in functionally overloaded rat plantaris muscle

The involvement of calcineurin (CaN) and heat shock protein (Hsp) 72 in the regulation of fiber size and/or phenotype in response to functional overload (FO) was investigated. In one FO group, the plantaris muscle was overloaded by cutting the distal tendons (5-10 mm length) of the soleus and gastrocnemius of 3-week-old male Wistar rats. Cyclosporin A (CsA), a CaN inhibitor, was injected daily (5 mg/kg body weight, i.p.) in a second group of FO rats (FO+CsA group) for a 2-week period. Compared to age-matched controls (Con), the absolute and relative plantaris weights were increased in both FO groups: the hypertrophic response was attenuated in FO+CsA rats. The mean cross-sectional area of each fiber type was increased (approximately 2.0-fold) in the plantaris of FO rats: CsA treatment attenuated this effect, although the fibers were still larger than in Con rats. The percent composition of myosin heavy chain (MHC) IIb decreased from 54% in Con to 19% in FO rats, whereas types I, IIa, and IIx MHC increased in the FO rats. CsA treatment blunted the shifts in MHC isoforms: the FO+CsA group showed a smaller decrease in type IIb and a smaller increase in type IIx MHC than the FO group. The levels of CaN-A and -B proteins were higher (approximately 2.5-fold) in FO than Con rats, whereas these values were similar in Con and FO+CsA rats. Hsp72 protein levels were higher in FO (3.6-fold) and FO+CsA (5.2-fold) than Con rats, with the values being significantly higher in the FO+CsA than FO rats. CsA treatment in Con rats had no effects on muscle mass, fiber size, MHC composition, and Hsp72 or CaN levels. Combined, these results suggest that CaN levels are related to changes in both fiber size and phenotype, and that Hsp72 levels are more related to the levels of stress added to the muscle rather than to increases in the slow fiber phenotype in functionally overloaded rat plantaris muscles.     

Dedifferentiation and remodeling of motor endplates following experimental localized lesions of muscle fibers

Local anaesthetics, cardiotoxin and mechanical injuries may cause necrosis of muscle fibres while leaving the motor nerve fibres and their terminals intact. With local injuries to mouse muscles carried out by freezing or cutting we made a point of preserving both the nerve terminals and the muscle fibre portions on which these terminals were located. It was thus possible to follow the changes induced at endplates by these lesions. Within two or three days of the freezing or cutting, the muscle fibres underwent very different degrees of regression of the contractile material and T-system. The neuromuscular junctions also underwent changes, principally affecting their postsynaptic portion, in particular the folds of the subneural apparatus. After dedifferentiation of subsynaptic areas, we observed sprouting of the nerve terminal on muscle fibres which survived the amputation of one end and formed actively new myofibrils. This sprouting restored synaptic connections at the original sites, but with new structural features and correlative changes in the distribution of cholinergic receptors and cholinesterases. It is probable that after a phase of involution followed by a phase of recovery, the injured muscle fibres triggered off the nerve terminal sprouting which led to the remodelling of the endplates.     

Acid phosphatase activity in soleus and plantaris muscle fibres of normal and dystrophic hamsters

Summary The activity of acid phosphatase in skeletal muscle fibres of the plantaris and soleus of normal and dystrophic male hamsters was quantified using a histochemical post-coupling semipermeable membrane technique. Althoug the absolute levels of activity were found to vary widely from one animal to another, the ratio of the mean activities in the two muscles in each animal was virtually constant. In normal muscles, the ratio was about 0.73 and in dystrophic muscles, about 0.77. The activity in plantaris muscle fibres was always significantly lower than that in the corresponding soleus fibres, and in normal fibres compared to dystrophic ones. Another difference was that in normal fibres the mean activity declined to a constant level in mature animals older than about 3 months. In contrast, the activity in dystrophic muscles appeared to fall exponentially throughout life. The functional significance of these findings is discussed.In honour of Prof. P. van Duijn     

Expression profile of Notch-1 in mechanically overloaded plantaris muscle of mice

AimWe investigated the expression pattern of Notch-1 in normal and hypertrophied plantaris muscle of mice.Main methodsWe performed immunofluorescence of both Notch-1 and the Notch-1-linking molecules.Key findingsImmunofluorescence labeling revealed Notch-1 protein in Pax7-positive satellite cells during days 2–6. We observed clear co-localization between Notch-1 and myogenin (4.9 ± 1.3%) in the hypertrophied muscle at 4 days. Several mononuclei (possibly satellite cells) possessed both Notch-1 and Foxo1 in the plantaris muscle subjected to mechanical overloading (4.1 ± 1.2%).SignificanceNotch-1 may play an important role in the maintenance of quiescent satellite cells.     

Adaptation in synergistic muscles to soleus and plantaris muscle removal in the rat hindlimb.

Although the soleus muscle comprises only 6% of the ankle plantar flexor mass in the rat, a major role in stance and walking has been ascribed to it. The purpose of this study was to determine if removal of the soleus muscle would result in adaptations in the remaining gastrocnemius and plantaris muscles due to the new demands for force production imposed on them during stance or walking. A second purpose was to determine whether the mass or the fiber type of the muscle(s) removed was a more important determinant of compensatory adaptations. Male Sprague-Dawley rats underwent bilateral removal of soleus muscle, plantaris muscle, or both muscles. For comparison, compensatory hypertrophy was induced in soleus and plantaris muscles by gastrocnemius muscle ablation. After forty days, synergist muscles remaining intact were removed. Mass, and oxidative, glycolytic, and contractile enzyme activities were determined. Despite its role in stance and slow walking, removal of the soleus muscle did not elicit a measurable alteration in muscle mass, or in citrate synthase, lactate dehydrogenase, or myofibrillar ATPase activity in gastrocnemius or plantaris muscles. Similarly, removal of the plantaris muscle, or soleus and plantaris muscles, had no effect on the gastrocnemius muscle, suggesting that this muscle was able to easily meet the new demands placed on it. These results suggest that amount of muscle mass removed, rather than fiber type, is the most important stimulus for compensatory hypertrophy. They also suggest that slow-twitch motor units in the gastrocnemius muscle play an important role during stance and locomotion in the intact animal.     

Monoclonal antibodies specific to quail embryo tissues: their epitopes in the developing quail embryo and their application to identification of quail cells in quail-chick chimeras.

Quail-chick chimeras have been used extensively in the field of developmental biology. To detect quail cells more easily and to detect cellular processes of quail cells in quail-chick chimeras, we generated four monoclonal antibodies (MAb) specific to some quail tissues. MAb QCR1 recognizes blood vessels, blood cells, and cartilage cells, MAb QB1 recognizes quail blood vessels and blood cells, and MAb QB2 recognizes quail blood vessels, blood cells, and mesenchymal tissues. These antibodies bound to those tissues in 3-9-day quail embryos and did not bind to any tissues of 3-9-day chick embryos. MAb QSC1 is specific to the ventral half of spinal cord and thymus in 9-day quail embryo. No tissue in 9-day chick embryo reacted with this MAb. This antibody binds transiently to a small number of brain vesicle cells in developing chick embryo as well as in quail embryo. A preliminary application of two of these MAb, QCR1 and QSC1, on quail-chick chimeras of neural tube and somites is reported here.     

Quantitative study of motor endplates in muscle fibres dissociated by a simple procedure

Large numbers of single muscle fibres can be obtained reproducibly from glutaraldehyde-fixed skeletal muscle by the method described here. With suitable modifications, one can estimate acetylcholine receptor number (alpha-bungarotoxin binding sites) and endplate area in parallel portions produced from the same muscle sample, so that small differences (e.g. with growth or between muscle types) become detectable. Microdissection further increases the precision of evaluation of junctional, perijunctional and extrajunctional binding sites. Other applications are illustrated.     

Regeneration following orthotopic transplantation of entire plantaris muscle in Sprague Dawley rats

Regeneration of rat plantaris muscle has been studied without predeneration. The original muscle fibres were completely degenerated within one week, but were completely regenerated at the end of experiment. The weight of regenerate showed 51% recovery. The breadth, length and total area of muscle and number of muscle fibres was less by 42, 37, 26 and 29% respectively, compared to control muscle at the end of experiment. The diameter of regenerated fibres was almost normalized at the end of experiment. The number and diameter of nuclei in the regenerate was more than that of control which coincides with the increase in DNA and RNA contents. The protein contents after an initial loss started increasing and continued to do so till the end of the experiment. The better regeneration of the plantaris muscle seems to be due to the presence of supporting gastrocnemius muscle.     

Relationship between myoglobin and succinate dehydrogenase in mouse soleus and plantaris muscle fibres

Summary This report describes a quantitative histochemical study of myoglobin in skeletal muscle fibres. The muscle fibres were classified as fast or slow on the basis of their quantitative myofibrillar ATPase histochemistry. A large range of myoglobin absorbance values was found among fast skeletal muscle fibres. This range was relatively small among slow fibres. The concentrations of myoglobin and the activities of succinate dehydrogenase in individual muscle fibres in serial sections are weakly correlated in both the mouse soleus and plantaris muscle. The myoglobin concentration is higher in fast and slow oxidative soleus muscle fibres and the succinate dehydrogenase activity in these fibres is lower than in oxidative plantaris muscle fibres in the same range of cross-sectional area.     

Effect of hindlimb immobilization and recovery on compensatory hypertrophied rat plantaris muscle

Compensatory hypertrophy of the rat plantaris muscle (PLT) was induced by ipsilateral gastrocnemius muscle ablation. Following 8 weeks (wks) of hypertrophy, hindlimbs were cast immobilized (HI) for 4 weeks after which weight bearing was unrestricted for 8 wks (recovery). Compensatory hypertrophy increased PLT wet weight/body weight ratio (83%), muscle fiber cross-sectional areas (1.5 to 2 fold), and the percent of slow oxidative (% SO) fibers (2 fold) in the experimental compared to the contralateral sham control muscle. PLT protein content and maximal activities of phosphofructokinase (PFK), mitochondrial glycerol phosphate dehydrogenase, and succinate dehydrogenase were unaltered with muscle hypertrophy. HI produced significant decreases in PFK activity (50%) and muscle fiber cross-sectional areas (50%) but did not significantly change the histochemical myofibrillar ATPase profile. Following remobilization, muscle weight/body weight ratio and maximal enzyme activities recovered to that of aged matched controls. Muscle fiber areas returned to pre-immobilization sizes but were approximately 25% smaller than aged matched control hypertrophy muscles. The % SO fibers in the hypertrophied muscle remained higher than controls but did not return to pre-immobilization values. These results indicate that biochemical and histochemical characteristics of hypertrophied rat PLT recover from HI during 8 wks of normal weight bearing similar to that of normal control muscle. However, the recovery time period was insufficient to allow complete compensation of fiber size to that of the age-matched control animals.