Morphometric studies on tenuissimus muscle spindles in the cat

Muscle spindles were studied histochemically in serial transverse sections of 42 cat tenuissimus muscle specimens. Staining for myofibrillar adenosine triphosphatase was employed to identify nuclear bag 1, nuclear bag 2, and nuclear chain intrafusal muscle fibers. The nuclear chain fibers were further subdivided into three categories according to their polar length and the intensity of their staining for nicotinamide adenine dinucleotide tetrazolium reductase. A total of 430 spindle poles were surveyed. The mean spindle content of bag 1, bag 2, and chain fibers was established. The mean polar length of intrafusal fibers as well as that of the intracapsular and extracapsular spindle regions was determined. A cholinesterase (ChE) staining technique was used to demonstrate the termination sites of motor axons along intrafusal fibers. Two types of circumscribed ChE deposits. The "rim" and the "plate," occurred on the fibers. The nuclear chain fibers usually carried both the ChE rims and plates, while most nuclear fibers displayed only the plates. The ChE plates were assessed in term of their appearance, staining intensity, length, and location along the fibers. The mean number of ChE plates found along the fibers was established for each of the various intrafusal fiber types. These histochemical observations are discussed with regard to the current concepts of cat spindle morphology and motor innervation. The results suggest a degree of predictability in the spindle fiber content and in the distribution of motor nerve terminals along intrafusal muscle fibers, at least in the tenuissimus muscle.     

End plate classification and spontaneous sprouting in the sternocostalis muscle of the rat: A new whole mount preparation

Summary The sternocostalis muscle of the rat provides an ideal preparation for examination of neuromuscular junctions in a whole mount. It contains all three morphological end plate types (A, B and C), and its segmental innervation allows ease of experiment, such as partial denervation.The ratio of end plate types remains constant in all individuals of the same age, and there is no variation in this ratio over different regions of the same muscle. Spontaneous sprouting was observed from end plates of all the animals examined: again the ratio of sprouting end plate types remained constant over all muscles examined, and over all regions of the same muscle.     

Innervation and acetylcholine sensitivity of skeletal muscle cells differentiated in vitro from chick embryo

Trypsin-dissociated myoblasts from leg muscle of 12-day chick embryos have been cultured in monolayers. After four days the muscle cultures have been confronted with fragments of the spinal cord of six-day chick embryos. Electrophysiological and morphological analysis demonstrate that characteristic neuromuscular transmission can develop in these cultures. Electrical stimulation of the cord fragment evokes contractions of innervated muscle fibers, from which end plate potentials and miniature end plate potentials with average frequency around one per second or more can be recorded. D-tubocurarine (1 μg/ml) suppresses reversibly these synaptic potentials. Non-innervated muscle fibers are sensitive to acetylcholine over all their surface, while innervated muscle fibers are sensitive at the regions where structures suggestive of motor end plate (“bulb-type”) are found. We can conclude that neuromuscular connections developed in vitro in our experiments are functional in respect of transmission of impulses but also in respect of neurotrophic influences for restriction of chemosensitivity.     

About the T-system in the myofibril-free sarcoplasm of the frog muscle fibre

Previous investigations of the T-system in skeletal muscle fibres described the inter-myofibrillar relationships between T-tubules and the sarcoplasmic reticulum. They disregarded the arrangement of the T-system in the myofibril-free sarcoplasm in the area of muscle fibre nuclei. In the present investigation, the T-system was filled by means of lanthanum incubation and the myofibril-free sarcoplasm was ultrastructural examined by means of thin (< or = 100 nm) as well as thick sections (> 300 nm-1 microm) with the electron microscope. The investigation of thick sections revealed that T-tubules meander through this myofibril-free sarcoplasm and tangle up at the poles of muscle fibre nuclei and in the area of fundamental nuclei of the motor end plate. They are, far from myofibrils, in proximity to these nuclei, the Golgi apparatus and mitochondria. On basis of this proximity and their openings at the muscle fibre surface, a contribution at the drainage of metabolic products and at the local calcium control is discussed.     

Morphologic and histochemical characteristics of frog skeletal muscle following application of colchicine to a motor nerve]

Experiments with application of colchicine to the muscle motor nerve carried out; this was done for the purpose of disturbance of rapid axoplasmic transport. A reduction of the areas of transverse sections of the muscle fibers, an increase in the number of fibers with a low succinic dehydrogenase (SDH) activity a greater homogeneity of the muscle fibers by the degree of optic density in staining for detection of the SDH activity was noted. Analogous changes were revealed under conditions of section of the motor nerve. However, denervation was accompanied by the block of conductivity and by degenerative changes in the nerve endings. As to the preparations treated with colchicine, transmission of excitation in the nerve and through the synapse was retained and was recorded by the end plate miniature potentials, end plate potentials and the action potentials of the muscle fibers. A conclusion was drawn that rapid axoplasmic transport brought substances maintaining differentiated state of the muscle fibers.     

Structure of muscle fibres and motor end plates in extraocular muscles of the grass snake, Natrix natrix L.

Six extraocular muscles of the grass snake, Natrix natrix L. together with their motor end plates were examined in the light and electron microscope, and the measurements of the diameter of muscle fibres and the area of their motor end plates were performed. Morphologically, two types of muscle fibres: tonic and red phase ones were distinguished. The former fibres, 2,3 to 14,5 mum in diameter possess single or multiple (up to five on a single fibre) "en grappe" motor end plates, without postsynaptic junctional folds. The latter fibres, 10...40 mum in diameter have single, "en plaque" motor end plates, with numerous postsynaptic junctional infoldings. The morphological features of muscle fibres and motor end plates as well as the correlation between the diameter of muscle fibres and the area of motor end plates are discussed.     

Specificity and development of innervation pattern of Xenopus pectoralis muscle

Multielectrode recordings were used to identify and measure the axonal inputs to each end plate on contiguous surface fibers covering about 25% of the Xenopus pectoralis muscle in mature and developing animals. The mature innervation pattern was remarkably precise. Individual axons tended to innervate fibers of similar input resistance (R_in) in compact motor units restricted to only a portion of the region studied. Motor units comprising fibers of similar R_in overlapped mainly near their borders. Most fibers had two end plates. In more than 80% of these fibers, both end plates received input from the same axon. In 57%, this was the only input to both end plates. This implies a powerful mechanism for excluding or eliminating inputs from other axons. About 16% of the mature junctions showed focal polyneuronal innervation, with the weaker end plate potential component often less than 1 mV in noncurarized preparations. However, we have no evidence that the weaker inputs were being eliminated. During development, motor units became more compact, which was associated with synapse elimination; but from the earliest times studied, soon after metamorphosis when many fibers were adding second end plates, a majority of those that had two end plates were innervated at both sites by the same axon. © 1996 John Wiley & Sons, Inc.     

Reinnervation of murine muscle following fetal sciatic nerve transection

A technique is reproted that permits transection of the sciatic nerve of mouse fetuses without interfering with fetal viability. Sciaticotomy was performed on Swiss Webster mice at day 17 of gestation; the contralateral side served as a control. Six weeks later the extensor digitorum longus (EDL) muscles on both sides were injected with horseradish peroxidase (HRP). Examination of the lumbar spinal cord revealed that while a substantial number of motor neurons in the region of the spinal cord giving rise to the sciatic nerve died, the EDL muscle did become reinnervated. The size of the EDL motor neuron pool on the denervated-reinnervated side was ～43% of that seen on the control side. While the control EDL motor neuron pool was located in lumbar segments L3–L5, the location of the pool to the denervated-reinnervated EDL was shifted cranially to L2–L4. Denervated-reinnervated EDL muscles were analyzed immunohistochemically to study the effect of fetal denervation on the neuronal cell adhesion molecule (N-CAM) expression. At 2 weeks postnatal, N-CAM immunoreactivity in control muscle was segregated to the motor end plate region, while fetally denervated muscle continued to express N-CAM along the length of the sarcolemma. Thus fetally denervated muscle does not develop the same pattern of N-CAM expression as normal, innervated muscle. By 6 weeks of age, the denervated-reinnervated muscle showed the same level and distribution of N-CAM immunoreactivity as did age-matched control muscle, indicating that most, if not all, of its myofibers had been reinnervated.     

Morphology of muscle fibres in amphibian submandibular muscle

The microscopic organization and ultrastructure of the submandibular muscle of 10 species of Amphibia were compared. Among other fibre features the diameter of fibres, their content of mitochondria and fat, organization of sarcomeres: morphology of Z-line, M-band and sarcoplasmic reticulum were taken into consideration and 4 main types of muscle fibres were distinguished. They correspond to tonic (slow) and phasic (red, white and intermediate) ones. Slight variety of fibre morphology and of fibre elements among the examined species was found. Special attention to the variety of fibre morphology among the established types has been paid and the existence of continuous "spectrum" of fibres was suggested. The correlation of frequency of fibres of particular types with the body size, gular oscillation frequency, and some other characteristics of the submandibular muscle in the examined species was discussed. Also the zonal arrangement of muscle according to the fibre types, as well as possible dynamic nature of muscle fibres were emphasised.     

Electron microscope radioautography as a quantitative tool in enzyme cytochemistry. I. The distribution of acetylcholinesterase at motor end plates of a vertebrate twitch muscle

Tritiated diisopropylfluorophosphate (DFP) was used to phosphorylate acetylcholinesterase (AChase) in the motor end plate of mouse sternomastoid muscle, and its distribution within the end plate was evaluated quantitatively by electron microscope radioautography. With the use of emulsion layers whose sensitivity to tritium had been calibrated, the density of AChase in different components of the end plate was calculated. The AChase was primarily localized (85%) in the junctional fold region. The concentration of AChase there was more than 20,000 active sites per cubic micron of tissue. The resolution of the technique was not sufficient to determine whether there was some AChase in the nerve end bulb; however, if there is any there, the concentration must be less than 10% of that at the junctional fold region.     

Extraocular muscles in the lamprey, Lampetra fluviatils L. II. Motor end plates

Motor innervation and particularly the structure of motor end plates (MEPs) was studied in the extraocular muscles of the lamprey, Lampetra fluviatilis L., by light and electron microscopy. Each muscle is supplied with numerous thin motor nerve fibres. Motor end plates are located at their ends or along their course. Two motor end plate types were distinguished: en grappe-like plates with a low acetylcholinesterase (AChE) activity were observed on thin muscle fibres, whilst en plaque-like plates with a high AChE activity were found on thick mitochondria-rich and thick multifibrillar muscle fibres. The postsynaptic membrane of the former MEP type does not show the presence of infoldings, MEPs located on thick mitochondria-rich fibres show occasional infoldings, whereas the postsynaptic membrane of MEPs present on thick multifibrillar fibres reveals numerous infoldings. Motor end plates present in the extraocular muscles in the lamprey possess features typical for higher vertebrates and elasmobranch fishes, as well as for Tunicata.     

The ultrastructure of oesophageal striated muscle in the guinea-pig and marmoset

The ultrastructural characteristics of oesophageal striated muscle from guinea-pig and marmoset have been examined using transmission electron microscopy and compared with ultrastructural features of skeletal muscle. The findings show that, although similar to skeletal muscle, oesophageal striated muscle exhibits important differences in the arrangement of its muscle fibres and their motor innervation. It was also found impossible to classify oesophageal striated muscle according to the usual ultrastructural criteria since its motor endplates bore a greater resemblance to those of intrafusal skeletal muscle fibres. The consideration of these differences in conjunction with characteristics revealed elsewhere by other techniques confirms the unique nature of oesophageal striated muscle.     

Early ablation of target muscles modulates the arborisation pattern of an identified embryonic Drosophila motor axon.

The Drosophila RP3 motor axon establishes a stereotypic arborisation along the adjoining edges of muscles 6 and 7 by the end of embryogenesis. The present study has examined the role of the target muscles in determining this axonal arborisation pattern. Target muscles were surgically ablated prior to the arrival of the RP3 axon. Following further development of the embryo in culture medium, the morphology of target-deprived RP3 motor axons was assayed by intracellular injection with the dye Lucifer Yellow. Axonal arborisations were formed on a variety of non-target muscles when muscles 6 and 7 were removed and these contacts were maintained into stage 16. The pattern of axonal arborisations over non-target muscles varied between preparations in terms of the number of muscles contacted, and the distribution of arborisations on individual muscles. Following removal of muscle 6, the RP3 motor axon frequently contacted muscle 7, and axonal arborisations were present along the distal edge of the muscle. In the absence of muscle 7, the RP3 axon often did not contact muscle 6 and when muscle 6 was contacted, the arborisation of RP3 was poorly developed. Axonal processes were retained on non-target muscles when only one target muscle was present. Therefore, the establishment of a stereotypic arborisation by the RP3 motor axon is apparently dependent on growth cone contact with both target muscles.     

The origin of the muscle fasciculation caused by funnel-web spider venom

The origin of the fasciculation of skeletal muscle produced by funnel-web spider venom (FSV) has been examined in mouse phrenic nerve hemi-diaphragm preparations, FSV from male spiders at concentrations greater than 10(-6) g/ ml invariably produced muscle fasciculation which could be prevented by d-tubocurarine (14micron), tetrodotoxin (0.3 micron) or by increasing the external magnesium concentration or calcium concentration. Diphenyl hydantoin (3-6 X 10(-5) M) was able to reduce these fasciculations in some experiments. In curarized preparations, multiple end plate potentials (EPPs) in response to single stimuli and bursts of spontaneous EPPs were seen in the presence of FSV (10(-5) g/ml). Extracellular recordings from phrenic nerves in the presence of FSV (10(-5) g/ml) revealed additional components in compound action potentials elicited by single stimuli, and "spontaneous" electrical activity was observed in unstimulated nerves. This spontaneous activity was abolished by raising the divalent cation concentration in the bathing solution. These results suggest that a primary site of action of FSV is the surface membrane of nerve fibres and that muscle fasciculation arises as a consequence of spontaneous action potentials produced by the venom in motor nerves.     

End plate spikes in the human electromyogram. Revision of the fusimotor theory.

'End plate spike' (EPS) is a spontaneous action potential of a normal striated muscle. EPSs are found in local 'active spots' of the muscle. The prevailing hypothesis about the origin of EPSs states that when a needle electrode affects a motor nerve branch near the neuromuscular junction at the end plate zone, an increased leakage of acetylcholine to the synaptic cleft ensues. This elicits postsynaptic action potentials of the muscle fibre which can be recorded as EPSs with the same needle electrode. Thus EPSs are thought to be caused by needle injury or irritation of the motor axon. We suggest that EPSs are action potentials of intrafusal muscle fibres and that 'active spots' are in fact muscle spindles. Waveform analysis reveals three types of EPSs: small EPSs, not propagated outside the active spot either: i) with negative onset; or ii) with short positive initial deflection; and iii) large EPSs resembling propagated motor unit potentials (MUPs) but with a typical EPS firing pattern, distinctly different from that of the MUPs. Study of EPS activation in different manoeuvres associates small EPSs with intrafusal gamma motor units and large MUP-like EPSs with beta motor units.     

Recovery of Acetylcholinesterase in the Diaphragm, Brain, and Plasma of the Rat After Irreversible Inhibition by Soman: A Study of Cytochemical Localization and Molecular Forms of the Enzyme in the Motor End Plate

Abstract Recovery of AChE activity in the motor end plate region and end plate free region of the rat diaphragm was studied after irreversible inhibition by soman. Recovery was slow during the first 2 days and only 4 S and 10 S molecular forms of AChE were present in the end plate region. However, cytochemical evidence indicates that synaptic AChE has already started to accumulate and that the synthesis of AChE in muscle and Schwann cell might even be enhanced. Tubular structures, observed underneath the motor end plate, may serve to transport the enzyme from its sites of synthesis in the sarcoplasmic reticulum. Asymmetric molecular forms of AChE in the end plate region appeared later during recovery and, one week after poisoning, their activity was only about 50% of normal value. The limited ability of newly synthesized AChE to attach to the subcellular structures and, therefore, to be retained in the muscle, may explain the phase of slow recovery. In accordance with this view, AChE activity in brain recovered in a similar way as in muscle, whereas soluble plasma cholinesterases recovered faster, apparently without a slow initial phase.     

Penis-retractor muscle of Aplysia: excitatory motor neurons

Cobalt axonal iontophoresis and intracellular recordings were used to identify a cluster of several motor neurons innervating the penis-retractor muscle of Aplysia. Intracellularly recorded motor neuron action potentials elicited direct, one-for-one, constant latency excitatory junctional potentials (ejps) in individual muscle fibers. The axons of motor neurons could be recorded extracellularly in the penis-retractor nerve and stimulation of the nerve backfired the motor neurons. Perfusion of the ganglion, the muscle, or both with solutions of either increased Mg++/decreased Ca++ or increased Ca++ sea water indicated that the presumed motor neuron impaled was not a sensory cell and that interneurons were not intercalated in the pathway. Innervation of muscle fibers was found to be functionally polyneuronal and diffuse. The ejps were found to undergo marked facilitation with repetitive motor-neuron stimulation. The motor neurons were isolated in a distinct cluster in the right pedal ganglion. Their electrical activity was characterized by spontaneous irregular action potentials and a moderate input of postsynaptic potentials.     

Anatomic and physiological characteristics of the ferret lateral rectus muscle and abducens nucleus.

The ferret has become a popular model for physiological and neurodevelopmental research in the visual system. We believed it important, therefore, to study extraocular whole muscle as well as single motor unit physiology in the ferret. Using extracellular stimulation, 62 individual motor units in the ferret abducens nucleus were evaluated for their contractile characteristics. Of these motor units, 56 innervated the lateral rectus (LR) muscle alone, while 6 were split between the LR and retractor bulbi (RB) muscle slips. In addition to individual motor units, the whole LR muscle was evaluated for twitch, tetanic peak force, and fatigue. The abducens nucleus motor units showed a twitch contraction time of 15.4 ms, a mean twitch tension of 30.2 mg, and an average fusion frequency of 154 Hz. Single-unit fatigue index averaged 0.634. Whole muscle twitch contraction time was 16.7 ms with a mean twitch tension of 3.32 g. The average fatigue index of whole muscle was 0.408. The abducens nucleus was examined with horseradish peroxidase conjugated with the subunit B of cholera toxin histochemistry and found to contain an average of 183 motoneurons. Samples of LR were found to contain an average of 4,687 fibers, indicating an LR innervation ratio of 25.6:1. Compared with cat and squirrel monkeys, the ferret LR motor units contract more slowly yet more powerfully. The functional visual requirements of the ferret may explain these fundamental differences.     

Methodological issues related to thickness-based muscle size evaluation

The purpose of this study was to highlight the issues related to thickness-based muscle size evaluation that is commonly done in field studies. The cross-sectional area, thickness (the vertical distance from the upper end of the elbow flexors to that of the humerus) and width (the horizontal distance from the left to the right end of the elbow flexors) of the elbow flexors at levels from the reference site (60% of the upper arm length from the acromial process of the scapula to the lateral epicondyle of the humerus) to 5 cm distal to it were determined in 11 young men using magnetic resonance imaging, both at rest and during isometric elbow flexion at 30% of maximal voluntary contraction. During 30% of maximal voluntary contraction, the thickness increased but the width decreased at each measurement site compared with those at rest. This was possibly due to difference in muscle slackness between both conditions. The correlation coefficients between the thickness and cross-sectional area for the elbow flexors were significantly lower at rest (r=0.551-0.856) than during 30% of maximal voluntary contraction (r=0.711-0.922). The present findings indicate that the thickness-based muscle size measurement at rest includes errors owing to the slackness of the resting muscles.     

Contraction properties and motor nucleus morphology of the two heads of the cat flexor carpi ulnaris muscle

Previous studies have examined the isometric contraction properties of the two heads of the cat flexor carpi ulnaris acting as a single unit. In this study, the contraction properties and fiber architecture of each head of the flexor carpi ulnaris were determined separately and related to previous reports on the histochemical characteristics of this muscle. The morphology of retrograde-labeled motor nuclei for the two heads of the muscle was also examined. The humeral head had a significantly longer contraction time (48 msec) than the ulnar head (36 msec) as well as a significantly lower tetanic fusion frequency (28 Hz vs. 35 Hz). The maximum tetanic tension per gram of muscle tissue was 71% greater in the ulnar head. Motoneurons of the flexor carpi ulnaris formed a column 12 mm long and 0.5 mm wide in the center of the ventral grey in spinal segments C8 and T1. The ulnar head had alpha-motoneurons with greater soma diameters than those in the humeral head. The smaller soma diameter, slower contraction time, and weaker contraction in the humeral head correlate with the preponderance of oxidative-metabolic muscle fiber types found in the humeral head by other workers. These correlations suggest that the humeral head plays a major role in maintaining a sustained antigravity tension that prevents the wrist from buckling during standing.