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Effects of innervation on acetylcholine sensitivity of developing muscle in vitro.

1. Chick embryo skeletal muscle fibres were grown in culture. The acetylcholine (AACh) sensitivity of non-innervated fibres was compared with that of fibres innervated in vitro by chick embryo ciliary ganglion neurones. 2. The general pattern of ACh sensitivity was unchanged by innervation: ACh hot spots were superimposed on a background of uniform ACh sensitivity. 3. Quantitative comparisons revealed two differences between non-innervated and innervated fibres. First, hot spots were encountered about one third more often on innervated fibres. Secondly, about one-third of the hot spots on innervated fibres had significantly higher ACh sensitivities than the remainder, which were similar to those on control fibres. 4. Apossible explanation of these results is that nerves which form synapses induce the appearanceof end-plates which have higher ACh sensitivities than the pre-existing ACh hot spots.

Acetylcholine

Synthesis of the calcium transport ATPase of sarcoplasmic reticulum and other muscle proteins during development of muscles cells in vivo and in vitro.

The effect of medium Ca2+ concentration upon the concentration and the rate of synthesis of muscle proteins was investigated in chicken pectoralis muscle cultures. There is an easily identifiable class of muscle protein which includes the Ca2+-ATPase of sarcoplasmic reticulum, myosin, troponin C, ATP : creatine phosphotransferase, muscle specific actin, tropomysin 1 and 2, and muscle hemagglutinin, which show a large increase in concentration during normal development. The increased synthesis of these proteins was inhibited, without inhibition of cell proliferation, in culture media of relatively low Ca2+ concentration, 0.05--0.3 mM, where fusion was prevented. Similar medium Ca2+ concentration was required for the expression of all these proteins, suggesting their coordinate regulation. The proteins are denoted as 'calcium-modulated proteins'. The increased Ca2+ transport activity of sarcoplasmic reticulum in cultured chicken pectoralis muscle cells during development at 1.8 mM medium calcium concentration represents de novo synthesis of the Ca2+ transport ATPase, as shown by immunoprecipitation, active site labeling and direct identification of the Ca2+ transport ATPase on two-dimensional gel electropherograms of whole muscle homogenates. The concentration and the turnover rate of the majority of the muscle proteins is not affected significantly by medium Ca2+ concentration between 0.06 and 1.8 mM. It is proposed that increase in cytoplasmic free Ca2+ concentration during fusion plays a central role in the regulation of the synthesis of calcium-modulated proteins.

Acetylcholinesterase

Transitional stages in the histochemical development of muscle fibres during post-natal growth.

Serial frozen sections of longissimus dorsi muscles from seven pigs at different live weights (13 to 127 kg) were reacted for ATPase by the calcium method at an alkaline pH and for NADH oxidative activity. One hundred muscle fibres from each animal were identified individually in serial sections and their staining intensity was measured with a microscope photometer at 600 nm. For each section, staining intensity of fibres (% tranmission) was measured and converted to the nearest one-tenth unit of the range from the darkest to the lightest staining fibres. Frequency of occurrence of fibre types was plotted on a 10 X 10 grid using the range co-ordinates for NADH oxidative activity (vertical) and ATPase activity (horizontal). The commonly recognized histochemical fibre types in this muscle appeared as crowded areas in the grid but, in many cases, these areas were part of a continuous 'L' shaped range, a continuous but skewed distribution with regard to NADH oxidative activity was detected. In fibres with NADH oxidative activity of 0.6 to 1.0 units of the range, a continuous but irregular distribution with regard to ATPase activity was detected. Within this range, there was some evidence of a growth-related shift towards weaker ATPase activity.

Adenosine Triphosphatases

Postnatal cytochemical development of muscle fibers in segmental tail muscles of the rat.

Postnatal development of extrafusal and intrafusal muscle fibers was examined histochemically in segmental tail muscles of the rat. At birth all fibers show a strong reaction for myosin ATPase, uniformity in diameter, and homogeneity in staining intensity. During the first postnatal week, the muscle fibers undergo gradual hypertrophy and hyperplasia but they all maintain the same intense homogeneous staining pattern for the enzyme. By day 9, further differentiation of the muscle fibers results in the formation of a second intrafusal fiber type while the extrafusal fibers are still relatively homogeneous. Finally, two kinds of extrafusal fiber and a third type of intrafusal fiber can be distinguished by day 21. This histochemical fiber pattern is essentially maintained in the adult. These findings show that fiber type development in rat tail muscles lags behind the usual time course of myogenesis known to occur in more rostral regions of the animal. It also indicates that histochemical differentiation of intrafusal fibers in these muscles does not parallel that which occurs in extrafusal fibers. It is likely that arrival and initial contact of sensory nerve terminals on developing intrafusal fibers at day 7 directly influences their relatively early histochemical heterogeneity.

Adenosine Triphosphatases

Ultrastructure of human intramuscular blood vessels in development.

Muscles from human foetuses of nine weeks to nine months development were examined by electron microscopy. Capillaries, arteries and veins are frequent in the human foetal muscle at all stages of development. At nine weeks the vessels have the appearance of capillaries and basement membrane lies around the endothelial cells forming them. The capillaries are of continuous type which do not have apertures in their walls. Tight junctions are seen at some zones of adjacent endothelial cells of the capillaries. Sometimes, pericytes are also seen. At sixteen weeks vessels having the features of veins and of arteries can be identified between the muscle cells. Regarding the arteries, some of the endothelial cells are united to the smooth muscle cells and the intimal elastic lamina is interrupted where these cells approximate. The significance of this junction may be to anchor the intima to the media.

Arteries

The fine structure of developing locomotor muscles of the pelagic tunicate, cyclosalpa affinis (Thaliacea: Salpidae).

Salps are free-swimming tunicates whose peculiar life history renders them ideal for developmental studies. The solitary salp reproduces asexually by budding a stolon containing the complete developmental sequence of the aggregate generation. The ultrastructure of developing locomotor muscle of the aggregate generation of Cyclosalpa affinis was studied. The early muscle contains essentially non-striated myofibrils. However, in transverse sections, , areas indicating early I-band A-bands can be recognized. As development continues, the number of fibrils increases, the Z-line appear, and the fibrils contain more recognizable striations. The fully developed muscle has the caracteristic structure of striated muscle. Longitudinal sections show sarcomeres with irregular and discontinuous (perforated) Z-lines; H-zones are not apparent. No M-lines are seen. Throughout development, the ratio of thin to thick myofilaments is always 2:1, the ratio found in all vertebrate striated muscle. Other finding in C affinis suggest that: (1) multinucleated muscle cells are formed by the fusion of mononucleated cells, (2) membranes of adjacement mononucleated cells destined to fuse form myelin figures, and (3) these myelin figures become closely associated with mitochondria.

Animals

Muscle abnormalities in coeliac disease: studies on gross motor development and muscle fibre composition, size and metabolic substrates.

In 11 children with coeliac disease gross motor development was assessed before and during diet treatment using the gross motor subscale of the Denver developmental screening test. ATP, creatine phosphate (CP), glycogen and lactate concentrations, muscle fibre size and fibre composition were measured in specimens obtained by needle biopsy from the vastus lateralis muscle. Before treatment, gross motor development was delayed. ATP, and to a lesser extent, CP and glycogen concentrations were lowered compared to a control group. After treatment, gross motor development was normal and no differences in ATP, CP or glycogen concentrations were found compared to the control group. Fibre size seemed unaffected by the disease. The percentage of type 1 fibres was significantly lower before treatment, compared to values obtained during treatment and from the control group. Whether these metabolic changes were due to the coeliac disease per se or the inactivity which it causes was not possible to establish. In humans, only altered neurogenic influence on the muscles has been previously shown to give changes in fibre composition.

Adenosine Triphosphate

Ca+2-accumulating components in developing skeletal muscle.

This ultrastructural study on the localization of Ca+2 in developing skeletal muscle indicates that the formation of calcium-accumulating components begins during embryonic development. Both oxalate and pyroantimonate techniques are used to localize Ca+2 in distinct cellular components of chick pectoral and sartorius muscles. Two major sites for Ca+2 accumulation are present in ultrathin sections of embryonic and post-embryonic muscles: the terminal cisternae of the sarcoplasmic reticulum and specific lines in the I-bands. Calcium oxalate-accumulating vesicles are present in the smallest recognizable myotubes at the twelfth day of incubation, but calcium-accumulating components are not seen at myofibrillar I-band sites until the fourteenth to seventeenth days of incubation. The fact that myofibrils first form and later in development accumulate a Ca+2-binding component suggests that this Ca+2-binding component is not necessary for the formation of myofibrils, but is added to myofibrils before hatching to serve a probable regulatory role in contraction.

Age Factors

Mechanics and electrolyte composition of arterial smooth muscle in developing dogs.

The effects of age on arterial smooth muscles (SM) mechanics and electrolyte composition were studied during growth and development in six litters of puppies and their mothers from isolated segments of carotid, renal, mesenteric, and iliac arteries. An increase in the maximum wall stress following both norepinephrine (NE) and potassium (K) was found with age at all sites. The ratio of the stress response for K/NE was found to decrease with age. The maximum stress response for both NE and K shifted to lower values of wall strain with age in the same manner that passive stress-strain curves were shifted. The maximum diameter re3 (3 SM activator to SM activator were not significantly altered with age, but they were better maintained at higher transmural pressure in older animals. Chemical analysis indicated "cell" water, Mg, and K contents decreased with age, as did extracellular water, Na, and Cl. Na anhe suggest that changes in SM excitation-contraction coupling occur with age. Passive wall elements appear to interact with the contractile elements in determining wall responses to SM activation.

Aging

Indirect fluorescence of primary and secondary myofibers in developing porcine muscle.

Cytochemical differentiation of two populations of developing skeletal myofibers has been demonstrated in fetal muscle with metachromatic fluorescence of ribonucleic acid and deoxyribonucleic acid by staining fresh frozed cryostat sections of developing porcine skeletal muscle with acridine orange (CL. 46005). Evidence is presented that supports the hypothesis that first-formed myofibers (primary myofibers) serve as a structural framework upon which myoblasts proliferate, fuse in linear sequence and give rise to a second population (secondary myofibers) of myofibers.

Animals

The formation of synapses in amphibian striated muscle during development.

1. A study has been made of the formation of synapses in developing reinnervated and cross-reinnervated amphibian twitch muscles which receive either a focal (iliofibularis) or a distributed (sartorius) innervation from 'en plaque' nerve terminals using histological, ultrastructural and electrophysiological techniques. 2. During the development of the tadpole through metamorphosis to the adult frog, the sartorius myofibres increased in length at about twice the rate of the iliofibularis myofibres, due to a fast rate of growth at their insertions on to the pelvic tendon. 3. The short iliofibularis and sartorius myofibres of young tadpoles (800 mum long) possessed only a single synapse and the iliofibularis myofibres did not receive any further innervation during development. However the sartorius myofibres received further transient innervation on the new muscle laid down during development at the fast growing pelvic insertion, until the distance between the original synapse formed on the myofibres and the synapse at the pelvic end of the muscle was about 12 mm. 4. During development synapses possessed either skewed, multimodal, or unimodal m.e.p.p. amplitude-frequency distributions; the intervals between m.e.p.p.s. were not distributed randomly according to a Poisson process, as m.e.p.p.s. of similar amplitudes tended to be separated by very short intervals; the unit-size e.p.p. had a similar amplitude-frequency distribution as the m.e.p.p.s. if these had a unimodal distribution. 5. Reinnervation or cross-reinnervation of the sartorius and the iliofibularis muscles in adults or at a late stage of development simply reconstituted the normal focal and distributed innervation patterns of the muscles, as found in the control muscles of the contralateral and unoperated legs. 6. These observations on synapse formation in amphibia are consistent with the hypothesis that during development the axon making the initial synaptic contact on the muscle cells induces a property over a length of muscle membrane adjacent to this site which makes it refractory to synapse formation; thus during reinnervation or cross-reinnervation of adult muscles this refractory property constrains synapse formation to these sites.

Amphibians