Pattern formation of the sensible nerves in the hand of mouse embryos with special reference to the posterior-anterior developmental gradient

The development of the sensible innervation of the hand was investigated. For this reason normal forelimbs and limb bud cultures from 12- to 14-day-old mouse embryos were stained in toto using the cholinesterase technique. The dorsal side of the hand is mainly innervated by sensible branches of the radial and the musculocutaneous nerve, which penetrate the fascia in the region of the elbow. From here they grow in a distal direction forming the dorsal digital nerves. On the ventral side the median nerve grows in a subfascial compartment towards the palmar side of the hand. While passing the wrist the median nerve exhibits a frayed appearance. A net of branching and anastomosing small nerve fibre bundles is visible. This implies that on the palmar side of the metacarpal region of the hand-plate no specific highways for the growing nerve fibres exist. From late day 12 to early day 14 of embryonic development this diffuse nerve net is organized. In a posterior-anterior (ulnoradial) developmental gradient the common palmar digital nerves were formed, and these nerves divide at their tip into the proper digital nerves. However, the proper digital nerves again follow special pathways during their outgrowth.     

Expression of cholinesterase in the Schwann cells of peripheral nerves during development

In peripheral nerves of mouse embryos Schwann cells exhibit a high activity of unspecific cholinesterase. At first (day 12 of embryonic development) this enzyme occurs in the nuclear envelope and in the granular endoplasmic reticulum. Thus, it is possible to differentiate between Schwann cells and fibroblasts which lack cholinesterase. Later on (day 16) the cholinesterase has shifted to the cell membrane of the Schwann cells. However, only that part of the plasmalemma which encircles single axons and the mesaxons exhibits an irregular deposition of the reaction end product. In newborns the first loops of the just formed myelin sheath are still stained. With maturation of the myelin sheath the enzyme activity disappears. The functional role of cholinesterase is unclear. Possible roles are discussed. The expression of cholinesterase in Schwann cells depends on the integrity of the axons. After a few hours, the cultivation of amputated limbs results in a reduction of the enzyme activity. After 1 day in culture cholinesterase disappears totally.     

Changes in the muscle ultrastructure and electrolyte composition of rats in embryogenesis. II. X-ray spectrum microanalysis of the intracellular concentrations of potassium, sodium and phosphorus in skeletal and heart muscle

Concentrations of K, P and Na were determined in skeletal and cardiac muscle cells of rat embryos. High K and P levels--133 and 166 mmole/kg wet weight, resp.,--were found in skeletal muscles of 13 day old embryos, the concentration of Na in these cells being 81 mmole/kg w. w. On the 18th day of development, K and P in skeletal muscle cells decreased down to 79 and 118 mmole/kg w. w., resp., while the concentration of Na increased to 165 mmole/kg w. w. In 19 day old embryos, the concentrations of K and P increased, although they did not reach the level typical of skeletal muscles of adult rats. The concentrations of K and P in cardiac muscle cells of 13 day embryos were found equal to 100 and 108 mmole/kg w. w., resp., on the 19th day of development these concentrations reached the level typical of the cardiac muscle cells of adult rats.     

Expression of nestin,desmin and vimentin in intact and regenerating muscle spindles of rat hind limb skeletal muscles

We describe the expression and distribution patterns of nestin, desmin and vimentin in intact and regenerating muscle spindles of the rat hind limb skeletal muscles. Regeneration was induced by intramuscular isotransplantation of extensor digitorum longus (EDL) or soleus muscles from 15-day-old rats into the EDL muscle of adult female inbred Lewis rats. The host muscles with grafts were excised after 7-, 16-, 21- and 29-day survival and immunohistochemically stained. Nestin expression in intact spindles in host muscles was restricted to Schwann cells of sensory and motor nerves. In transplanted muscles, however, nestin expression was also found in regenerating “spindle fibers”, 7 and 16 days after grafting. From the 21st day onwards, the regenerated spindle fibers were devoid of nestin immunoreactivity. Desmin was detected in spindle fibers at all developmental stages in regenerating as well as in intact spindles. Vimentin was expressed in cells of the outer and inner capsules of all muscle spindles and in newly formed myoblasts and myotubes of regenerating spindles 7 days after grafting. Our results show that the expression pattern of these intermediate filaments in regenerating spindle fibers corresponds to that found in regenerating extrafusal fibers, which supports our earlier suggestion that they resemble small-diameter extrafusal fibers.     

Complete Staining of Nerve Fiber and Myoneural Junctions with Acetylthiocholine and Silver

We describe a combined stain for simultaneous demonstration of the preterminal axons and cholinesterase activity at myoneural junctions of mammalian muscles. This technique employs acetylthiocholine iodide as the substrate for cholinesterase activity and silver nitrate impregnation of preterminal axons. The procedure is rapid, simple and Uses fresh muscles. Intramuscular nerves, preterminal axons and myoneural junctions are stained simultaneously brown or black with minimal background staining of connective tissue and muscle fibers.     

Complete Staining of Nerve Fiber and Myoneural Junctions with Acetylthiocholine and Silver

We describe a combined stain for simultaneous demonstration of the preterminal axons and cholinesterase activity at myoneural junctions of mammalian muscles. This technique employs acetylthiocholine iodide as the substrate for cholinesterase activity and silver nitrate impregnation of preterminal axons. The procedure is rapid, simple and Uses fresh muscles. Intramuscular nerves, preterminal axons and myoneural junctions are stained simultaneously brown or black with minimal background staining of connective tissue and muscle fibers.     

Upright posture of man and morphologic evolution of the musculi extensores digitorum pedis with reference to evolutionary myology. III

The following anatomical objects were studied with regard to myology during evolution: M. extensor hallucis longus (MEHL), M. extensor digitorum longus (MEDL) with M. peroneus tertius (MP III), M. peroneus brevis (MPB) with M. peroneus digiti V (MPD V), M. extensor hallucis brevis (MEHB), M. extensor digitorum brevis (MEDB), and the Retinaculum musculorum extensorum imum (RMEI). The study was carried out by the preparation of 3 different groups of material. The 1st group consists of lower extremities of humans. The number of the extremities differs for the particular objects between 151 and 358 (see page 381). The 2nd group of material consists of 122 Membra pelvina from Marsupialia, Insectivora, and Primates. Table 1 shows as well the mammalian species as the number of the studied extremities. The extremities of the 1st and 2nd group were preserved in an manner suitable for a macroscopic preparation. The 3rd group of material consists of 71 lower extremities from embryos and fetus. The lower legs and feet were stained either according to the method described by Morel and Bassal with eosin added or according to Weigert. From this material, complete series of cross sections were prepared. Table 2 shows the age of the embryos (VCL [mm]) as well as the number of the studied extremities. It is important that up to the age of 46 mm VCL the difference in the age of the embryos usually amounts from 0.5 to 1.0 mm. This small difference in the age of the embryos and fetus allows a very good follow up of the changes in construction during the organogenesis. The comparison of the 3 different groups shows the following changes for the above mentioned muscles: The M. extensor hallucis longus (MEHL) is a muscle which is not split. The same result applies for its tendon which inserts at the distal phalanx of the hallux. This primitive form of the muscle amounts actually to 51.12% in human beings. In 48.88% of the cases, additional tendons and muscles are formed by the MEHL. Most of these supplements are positioned on the medial side of the main tendon, only a few lie to the lateral side. For the supplement tendons, the medial one as well as the lateral one occasionally possess a muscle belly. The muscle of the medial tendon is split off from the proximal margin of the MEHL. The muscle of the lateral tendon is split off from the distal margin of the MEHL.(ABSTRACT TRUNCATED AT 400 WORDS)     

Prenatal alcohol exposure and early postnatal changes in the developing nerve-muscle system

BACKGROUND: Extensive research on prenatal alcohol exposure has proven the potent teratogenicity of this substance of abuse. Children born to alcoholic mothers are often diagnosed with fetal alcohol syndrome (FAS). Those afflicted with FAS often have muscle weakness, muscle wasting, and atrophy. This study assessed the effects of prenatal alcohol exposure on the developing rat neuromuscular system. METHODS: Pregnant Sprague-Dawley rats were injected intraperitoneally with 1.0 ml of 20% ethyl alcohol/100 gm body weight. Unexposed rats served as controls. The offspring were killed 2, 3, 4, and 5 weeks after birth, and their body weights were recorded. The tibialis anterior (TA) and extensor digitorum longus (EDL) muscles were recovered and weighed. The TA muscles were histochemically stained by silver cholinesterase in order to study the pattern of innervation. The EDL muscles were processed and stained by hematoxylin-eosin. The number and size of the EDL muscle fibers was quantified. The sciatic nerve was also removed and stained by Swank and Davenport's method to demonstrate the myelin pattern. RESULTS: Assessment at the neuromuscular junction showed a higher proportion of endplates polyneuronally innervated in the alcohol-exposed rats. The muscle weights, as well as the number and size of the muscle fibers, were significantly reduced in these animals. A light-microscopy examination of the nerve sections revealed alterations in the connectivity of myelin. CONCLUSIONS: The finding that a higher proportion of endplates were polyneuronally innervated in the alcohol-exposed rats indicates that the maturation process of the neuromuscular system was delayed, thus confirming the deleterious effects of alcohol on growth and maturation of the nerve-muscle system.     

Studies on the tendinous compartments of the extensor muscles on the back of the human hand and their tendon sheaths. I

In 47 dissected right and left hands of adults of both sexes, kept in a moist condition, significant practical-clinical investigations of the transitional zone between forearm and hand were undertaken. In particular it was sought to determine the characteristic sizes of the extensor retinaculum, the osteofibrous tunnels, the insertion tendons of the hand and finger extensor muscles, and their tendon sheaths. Together with the palmar carpal ligament, the 2 to 3 cm wide extensor retinaculum annularly surrounds the whole circumference of the carpus. It extends obliquely from radial-proximal to ulnar-distal and conducts the extensor tendons over the carpal articulations. According to recent studies, it is divided into a superficial and a deep fibrous layer. From the undermost surface, vertical and oblique septa run to the plane of the forearm and carpal bones. They separate the fibrous portion of the 6 tendinous compartments of the dorsum manus. In 8.5% of cases, an accessory and completely independent tunnel of the extensor pollicis brevis muscle exists in the material investigated, and in 2.2% of cases, there is an additional tunnel for the extensor carpi radialis muscle. Hence, one occasionally finds 8 separate osteofibrous gliding compartments for the extensor muscles in the dorsal hand region. The longest tunnel belongs, as a rule, to the extensor digiti minimi muscle, whilst the widest pertains to the extensor digitorum muscle. Within the tunnel and also proximal and distal to it, the extensor tendons are surrounded by synovial sheaths. Because of its wide encroachment on the dorsum of the hand, the insertion tendon of the extensor digiti minimi muscle possesses the longest tendon sheath, measuring 68.8 mm. The next longest sheath, that of the extensor pollicis longus muscle, which measures 56.2 mm, begins further proximal to the gap of the radiocarpal articulation. In 12.8% of cases, there are divided sheaths of the abductor pollicis longus and of the extensor pollicis brevis muscle. The tendon sheath of both extensor carpi radiales muscles is frequently divided into 2 compartments which, in 2/3 of cases, communicate. The compartment of the extensor carpi radialis brevis muscle, in 91.5% of cases, shares a window-like opening with the roof of the synovial vagina of the extensor pollicis longus muscle. The tendon sheath of the long extensor muscles of the fingers originates 5 mm proximal to the forearm border of the extensor retinaculum and has a communal recess. The IVth tendon sheath opens distally and splays out in a glove-like manner to some distal recesses.(ABSTRACT TRUNCATED AT 400 WORDS)     

Axon growth from limb motorneurons in the locust embryo: the effect of target limb removal on the pattern of axon branching in the periphery

Metathoracic limb buds have been unilaterally ablated from locust embryos at 25 to 30% of embryonic development and the effect of this operation on the axon morphology of the motorneuron fast extensor tibiae (FETi) observed at later embryonic stages. In control embryos this neuron sends a single axon out the main leg nerve, nerve 5, to the extensor tibiae muscle in the femur. In limb ablated embryos the axon of FETi is found in a wide variety of aberrant peripheral nerve pathways and projects to a wide range of foreign muscles. There is a degree of apparent selectivity, but no rigid hierarchy, in the choice of pathway and muscle made by FETi. A high degree of variability is found between one embryo and another in the extent and pattern of axon branching. The axon of FETi is generally found in pathways that correspond to nerves in control embryos but on occasion grows along novel routes. An anteriorly directed dendritic branch, seldom seen in control FETi neurons, is frequently seen in experimental FETis. These findings are discussed in terms of the rules for specific axon growth in normal development.     

Migration and proliferation of primordial germ cells in the rat

Information about early primordial germ cell (PGC) formation and migration in rats is lacking. In utero developed and in vitro cultivated whole rat embryos were studied on days 10-13 postcoitum (p.c.). The development of the PGCs was investigated in serial sections stained for alkaline phosphatase activity. On postcoital day 10, PGCs were found in the invaginating visceral yolk sac endoderm and at the base of the allantois. At day 11 p.c. PGCs were mostly found in the ventral and lateral gut wall or in the mesenchyme between the gut and the future genital ridges. At day 12 p.c. most of the PGCs (94%) could be localised in the mesenchyme or in the future genital ridges. On postcoital day 13 almost all PGCs had reached the now-well-developed genital ridges. Quantitative measurements showed an increase in the number of PGCs from 84 at day 10 p.c. up to 2,768 at day 13 p.c. Only slight differences were found between in vivo and in vitro embryos with respect to the number of PGCs and their developmental pattern. The in vitro culture of whole rat embryos enables the discrimination between the effects of indirect (maternal) and direct action of PGC-toxic agents.     

Primary cultures of endothelial cells of the rat liver

Summary In the soleus muscle of the normal rat the number of cells containing fast troponin I decreased and those containing slow troponin I increased after birth until less than 10% stained for the fast form in the adult muscle. On denervation of soleus muscle this pattern of change was reversed with the result that the majority of cells stained for fast troponin I. The change was more rapid when denervation was carried out at 12 weeks rather than at 52 weeks of age. Denervation of extensor digitorum longus and tibialis anterior muscles produced little change in the distribution of fast and slow troponin I over a period of 12 weeks. After long periods (>24 weeks) of denervation of these fast muscles, fast troponin I was observed in cells in which originally only slow troponin I could be detected. Similar results to those obtained with troponin I in both fast and slow muscles were obtained using antibodies to the fast and slow forms of troponin C and troponin T.     

On the spatial relationship between fibroblasts and myogenic cells during early development of skeletal muscles

The spatial relation between fibroblasts and myoblasts as well as myotubes in the extensor muscles of the forearm of mouse embryos was investigated with routine electron microscopic techniques. From day 13 of embryonic development onwards, fibroblasts and myogenic cells may be distinguished by their ultrastructure from each other. Above all the fibroblasts are characterized by their long cell processes. Specialized cell contacts between them are visible. Fibroblasts often encircle some myogenic cells indicating the division of the muscles into smaller bundles. Over long distances, the intercellular gap between both cell types is narrow and measures about 15 nm. Sometimes specialized intercellular junctions can be seen. They resemble simple desmosomes and seem to be mechanical cell junctions. After the establishment of the basal lamina around the myogenic cells such specialized cell contacts are no longer visible. It seems possible that the fibroblasts act as mechanical counterpart for the organization of the myogenic cells.     

Heterogeneity and distribution of fast myosin heavy chains in some adult vertebrate skeletal muscles.

Three monoclonal antibodies, LM5, F2 and F39 raised to chicken fast skeletal muscle myosin, specific for myosin heavy chain (MHC) subunit, were used to study the composition and distribution of this protein in some vertebrate skeletal muscles. These antibodies in immunohistochemical investigations did not react with the majority of the type I fibres in most muscles. Antibodies LM5 and F39 stained all the type II fibres in all the adult chicken skeletal muscles studied. Antibody F2 also stained all the type II fibres in most chicken skeletal muscles tested except in gastrocnemius in which a proportion of both the type IIA and IIB fibres either did not stain or stained only weakly. Antibody F2 unlike LM5 and F39 stained most of the type IIIB fibres in anterior latissimus dorsi (ALD) and IB fibres in red strip of chicken Pectoralis muscle. Antibodies LM5 and F2 in the rat diaphragm reacted with all the type IIA and IIB fibres, while antibody F39 stained only the type IIB fibres darkly with most IIA fibres being either not stained or only weakly stained. In the rat extensor digitorum longus (EDL) and tibialis anterior (TA) muscles, antibody LM5 stained all the IIA and IIB fibres. Antibody F2 in these muscles stained all the type IIA fibres but only a proportion of the IIB fibres. The remaining IIB fibres were either unstained or only weakly positive. Antibody F39 in rat EDL and TA muscles did not only distinguish subgroups of IIB fibres (dark, intermediate and negative or very weak) but also of the IIA fibres. These three antibodies used together therefore detected a great deal of heterogeneity in the myosin heavy chain composition and muscle fibre types of several skeletal muscles.     

Brachial muscles of dystrophic chick embryos atypically sustain interaction with thoracic nerves

Previous analyses of experimental chick embryos of normal lineage demonstrate the inability of brachial muscles to sustain a successful union with foreign nerves derived from a thoracic neural tube segment transplanted to the brachial region at day 2 in ovo (day 2E). The present experiments were performed to determine if mutant chick embryos afflicted with hereditary muscular dystrophy would respond similarly to this experimental manipulation. Using the same criteria applied to our analysis of experimental normal embryos, our results demonstrated that dystrophic brachial muscles were capable of maintaining a compatible union with foreign thoracic nerves throughout the experimental period analysed. Significant muscle growth occurred, intramuscular nerve branches were maintained, motor endplates formed and wing motility was equivalent to that of unoperated dystrophic embryos. Thus, foreign nerves rejected by normal brachial muscles were accepted by brachial muscles of the mutant dystrophic embryo.     

Differentiation of muscle fiber types in aneurogenic brachial muscles of the chick embryo

Cross-reinnervation studies performed ex ovo with newly hatched chicks demonstrate that peripheral motor neurons control the phenotypic characteristics of avian muscles. The present experiments were designed to determine whether or not nerves play a similar role during the initial expression of muscle fiber types. Previous experiments indicated that differentiation of specific fiber types occurs during the first week of embryogenesis, temporally coincident with the penetration of nerves within muscle masses. These observations suggested that peripheral nerves may be associated with the initial differentiation of fiber types. To test this hypothesis directly, anterior limb buds of the chick embryo were rendered aneurogenic by deletion of the brachial segment of the neural tube. To ensure a completely aneurogenic environment for developing brachial muscles, surgery was performed at day 2 in ovo before the exit of ventral root fibers. Experimental and control embryos from Stage (St) 25 (4.5 d) through St 45 (19d) were analyzed histochemically by a silver-cholinesterase reaction to detect nerves and by the myosin ATPase reaction, following alkali and acid preincubation, to determine the fiber type composition of the muscles. In addition, the total volume of aneurogenic and control muscles was compared. Results demonstrate that the characteristic myosin ATPase profiles of individual aneurogenic and innervated (control) muscles were identical throughout the entire period analyzed. Therefore, we conclude that these enzymic profiles are endogenously expressed and are not under neuronal control during early embryogenesis. Furthermore, the entire sequence of events from the migration of myogenic cells to the anterior limb bud through the division of the primary muscle masses to form individual brachial muscles proceeded on schedule in the absence of nerves. Since the growth of aneurogenic muscles was impaired, we conclude that during embryogenesis peripheral motor nerves are necessary initially for the proper growth of muscles and ultimately, for their survival. They are not involved, however, with either the initial formation or initial differentiation of individual brachial muscles.     

Morphological effects of ciliary neurotrophic factor treatment during neuromuscular synapse elimination

In adult skeletal muscles, exogenous ciliary neurotrophic factor (CNTF) induces axons and their nerve terminals to sprout. CNTF also regulates the amount of multiple innervation in developing skeletal muscles during synapse elimination, maintaining multiple innervation of muscle fibers. While CNTF may maintain multiple innervation by regulating developmental synapse elimination, it is also possible that CNTF induces the formation of new multiple innervation through a sprouting response. In this study I examined morphologically the effects of CNTF during synapse elimination in the extensor digitorum longus (EDL) muscle. Rat pups received injections of CNTF in one leg and vehicle in the other either early [postnatal day 7 (P7)-P13] or late (P14–P20) in development. The early treatment period corresponds to that time when the pattern of innervation in the EDL is converted from predominantly multiple to single innervation. The late treatment period is at the end of synapse elimination for the EDL but corresponds to the major period of synapse elimination in the levator ani (LA), allowing a comparison of effects on these two muscles from the same animals. On the day after the final injection, EDL muscles were dissected and stained with tetranitroblue tetrazolium and the resulting pattern of innervation was assessed. The present findings indicate that only the early CNTF treatment regulates the level of multiple innervation in the EDL. Moreover, the effect of early CNTF treatment was local, affecting multiple innervation only in the EDL from the CNTF-treated leg. CNTF injected during the late treatment period had no apparent effects on the EDL but had a potent effect on the pattern of innervation in the LA, significantly increasing the level of multiple innervation in this muscle. Thus, CNTF affected multiple innervation in these two muscles only if provided during the period when single innervation normally develops. There was no evidence to indicate that CNTF induced axons or their terminals to sprout during either treatment period. In conclusion, CNTF increases the level of multiple innervation, probably by regulating synapse elimination, and skeletal muscles themselves may be an important target site for CNTF action. Presumably, the sprouting response to CNTF found in adult muscle develops sometime after P21. © 1996 John Wiley & Sons, Inc.     

Muscle force is determined also by muscle relative position: isolated effects

Effects on force of changes of the position of extensor digitorum longus muscle (EDL) relative to surrounding tissues were investigated in rat. Connective tissue at the muscle bellies of tibialis anterior (TA), extensor hallucis longus (EHL) and EDL was left intact, to allow myofascial force transmission. The position of EDL muscle was altered, without changing EDL muscle-tendon complex length, and force exerted at proximal and distal tendons of EDL as well as summed force exerted at the distal tendons of TA and EHL muscles (TA+EHL) were measured. Proximal and distal EDL forces as well as distal TA+EHL force changed significantly on repositioning EDL muscle. These muscle position-force characteristics were assessed at two EDL lengths and two TA+EHL lengths. It was shown that changes of muscle force with length changes of a muscle is the result of the length changes per se, as well as of changes of relative position of parts of the muscle. It is concluded that in addition to length, muscle position relative to its surroundings co-determines isometric muscle force.     

The mammalian myotome: a muscle with no innervation

SUMMARY The segmented muscular myotome is the first muscle to form in all vertebrates. In fish and amphibian embryos, the myotome becomes innervated very early and is essential for larval swimming. Its role in birds and mammals, however, is not clear. Using immunohistochemistry on sections and whole mounts of rat embryos, we demonstrate that the mammalian myotome differentiates and develops over a period of 3 days without being invaded by the outgrowing spinal nerves. In contrast, the limb muscle masses become filled with fine nerve branches from the first time that myocyte differentiation can be detected. Additionally, we show that the mammalian myotome does not express clustered acetylcholine receptors until after embryonic day 13.5, which corresponds to the beginning of its transformation into the adult epaxial muscles, showing that there are no functional myotomal neuromuscular junctions before this age. We suggest that the mammalian myotome has entirely lost the function of neurally controlled muscular contraction: its remaining functions are likely to be as a signaling tissue, as a structural scaffold, and as an incubator for myogenic precursors of the deep back, abdominal, and intercostal muscles.     

POMC-derived peptides in the neuromuscular system of streptozotocin-diabetic mice.

Immunoreactivity for beta-endorphin and alpha-MSH/ACTH was demonstrated in intramuscular nerves in soleus, extensor digitorum longus, and diaphragm muscles of normal and streptozotocin-diabetic mice. There was a higher incidence of immunoreactive nerves in the muscles of the diabetic mice. Specific binding for [125I]ACTH was detected in a proportion of the muscle fibers, using autoradiography. There were significantly more fibers with specific [125I]beta-endorphin sites and specific [125]ACTH sites in some muscles in the diabetic mice. The increased expression of POMC-derived peptides and their receptors in the neuromuscular system of streptozotocin-diabetic mice may indicate early neuropathic change.