Effects Of Innervation Research Articles

Effect of Denervation on the Isoform Transitions of Tropomyosin, Troponin T, and Myosin Isozyme in Chicken Breast Muscle1

The effect of innervation on the transition of tropomyosin, troponin T, and myosin isozyme during chicken breast muscle development was examined by denervating the muscle at various ages after hatching. The types of proteins were characterized by 2-D electrophoresis for tropomyosin, immunoblotting for troponin T and pyro-phosphate acrylamide gel electrophoresis for myosin isozymes. As judged by the types of these three proteins, when neonatal muscle was denervated, the protein isoform transition from the neonatal to adult state was interrupted, whereas the denervation of mature muscle caused the reappearance of the neonatal forms of proteins. The present results indicate that differentiation from the neonatal state to the adult state and the maintenance of the adult state are controlled by some factors related to nerves.

Embryonic growth and innervation of rat skeletal muscles. I. Neural regulation of muscle fibre numbers.

Individual rat embryos were chronically paralysed during development in utero by insertion of small slow-release capsules containing tetrodotoxin (TTX). In others, motoneurons were destroyed by individual injections of P-bungarotoxin (P-BTX). These embryos were compared with normal controls so that the effects of innervation and of use on embryonic development of skeletal muscles could be defined. The use of Sirius Red to stain muscle basal lamina made it possible to count muscle units (one or more myotubes contained within a single basal lamina sheath) with light microscopy. (i) In left hemidiaphragm muscles from control embryos about 20 % of the adult number of muscle units was present on day 17 of gestation, the time at which formation of secondary myotubes was first observed, and about 70 % at birth. New units continued to form during the first postnatal week. (ii) Skeletal muscles still formed following destruction of muscle innervation at day 14 of gestation, although at day 21 of gestation (birth) they were smaller and thinner than controls. The number of units in these muscles remained constant from day 18 through day 21 of gestation, and was similar to that in day 17 controls. No secondary myotubes were seen histologically. (iii) Following destruction of motoneurons at day 16, or 18 of gestation, new muscle units continued to form for about 1 1/2 d, indicating that the temporary presence of innervation affected later development of muscle tissue. The denervated muscles contracted spontaneously during this time. (iv) TTX-induced paralysis from day 16 onwards was at least as effective as denervation in inhibiting the generation of new muscle units. There was a dose—response relation in the ability of TTX to reduce the generation of new muscle units beyond the amount necessary to paralyse nerves, and it is suggested that the higher concentrations also blocked myogenic contractions. High doses of TTX stopped generation of new units within half a day. (v) Even the slightest degree of recovery from paralysis was accompanied by restoration of the proper number of muscle units. This shows that there is no ‘ critical period5 for muscle fibre development within the last five prenatal days in the rat. (vi) It is concluded that development of primary myotubes is autonomous, but that development of secondary myotubes, which form the majority of muscle cells in the adult, is dependent on innervation. Even when nerves are present, new muscle fibres cannot be generated in the absence of muscle contraction.

Effects of innervation by ciliary ganglia on developing muscle in vitro

Electrophysiological and pharmacological properties of neuromuscular junctions formed in tissue culture between chick ciliary ganglia and chick skeletal muscle cells have been studied. Functional neuromuscular junctions are formed already within 24 h. No functional acetylcholine (ACh) esterase is present at these endplates. The neurites conduct action potentials to the neuromuscular junctions, where EPPs are generated. Tetrodotoxin (TTX) blocks this nerve conduction but in the presence of TTX MEPP-like potentials remain whose amplitudes are lowered when theMg 2+Ca/ 2+ ratio in the medium is raised. It is speculated that these large TTX-resistant potentials are multiquantal. The relatively high resting membrane potential in non-innervated muscle fibers was not changed by innervation. ACh-sensitivity was determined by iontophoretical application of ACh to the myotubes. Non-innervated myotubes exhibited an evenly distributed ACh-sensitivity. Local differences in ACh-sensitivity were always gradual and never exceeded a factor of 3. Innervation did not alter the overall ACh-sensitivity, but on functionally innervated muscle cells loci hypersensitive to ACh were found. Hypersensitivity was located within sharply defined areas. Apparentlu the parasympathetic neurons of the ciliary ganglion are able to form functional neuromuscular junctions with skeletal muscle cells in tissue culture and to induce the formation of regions of high ACh-sensitivity.

The physiology, pharmacology, and trophic effectiveness of synapses formed by autonomic preganglionic nerves on frog skeletal muscle.

1. Frog sartorius muscles, newly denervated and transplanted to the lymph sac of the back, are reinnervated by implanted cholinergic nerves (spinal somatic motor nerves or the preganglionic sympathetic splanchnic nerve), but not by nerves). 2. Foreign somatic motor nerves (s.m.n.s) form synapses that resemble normal sartorius neuromuscular junctions electrophysiologically. 3. Axons of the sympathetic preganglionic splanchnic nerve (s.p.n.) grow throughout the muscle, but only a small percentage of fibres form synapses. Most e.p.p.s are of low quantal content, generally subthreshold. Long onset latencies and multiple post-synaptic responses indicate that innervation is multiple, multi-terminal, and by unmyelinated axons. 4. Spontaneous miniature e.p.p.s at splanchnic junctions occur at an average rate under 0.1/sec. Their average amplitude and time course are about the same as for control muscles, but the variability of amplitudes is greater than for control muscles. 5. The amount of facilitation shown by s.p.n.-evoked e.p.p.s is the same as by s.m.n. e.p.p.s, but the time course is almost twice as long. 6. S.p.n.-reinnervated fibres show dramatic post-tetanic potentiation preceded by depression, following as few as 20--50 stimuli. 7. As judged by standard physiological and histochemical criteria, AChEsterase is absent at s.p.n. junctions. 8. The pharmacological responses of the s.p.n. junctions are similar to those of normal or foreign s.m.n. innervated neuromuscular junctions in their sensitivity to the cholinergic blocking agents D-tubocurarine and hexamethonium. 9 The s.p.n. is capable of restricting ACh sensitivity to the sites of nerve contacts, although this restriction occurs more slowly and less completely than with s.m.n. reinnervation. The loss of extrajunctional ACh sensitivity can be correlated with effectiveness of innervation; but significant restriction occurs even in s.p.n. reinnervated fibres that probably never contract to nerve stimulation.

A rapid silver impregnation for central and peripheral nerve fibers in paraffin and frozen sections

The direction of known staged process of regeneration of free muscle grafts was inverted in our experimental rat model from a centripetal to a centrifugal by central implantation of blood vessels into isolated free muscle grafts.The effects of innervation, reinnervation and exercise on muscle fiber regeneration were analyzed at various intervals from 4 to 90 days by morphological and morphometric methods. Reinnervation occurred as well in grafts with the motor nerve left intact as it did in grafts with a severed and reimplanted nerve. Reinnervation proved to be prerequisite for a lasting muscle regeneration. Denervated muscle grafts even after neovascularization underwent irreversible fibrosis. A positive effect of exercise on the early states (30 days) of muscle regeneration was revealed by morphometrical analysis. In the long term (90 days) fiber diameter assimilated in all groups. The animal model mimics a clinical situation of flap prefabrication demonstrating the relationship of functional tissue regeneration and neovascularization. It can be transferred into the acute clinical situation as well as in tissue engineering.

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.

Effects of innervation on the distribution of acetylcholine receptors on cultured muscle cells.

1. Myotomal muscle cells from embryos of Xenopus laevis were cultured as a monolayer either alone or together with neural tube cells from the same embryos. 2. Spontaneous twitching and contractions evoked by electrical stimulation of neural perikarya were observed only in nerve-contacted muscle cells, and could be abolished by curare or alpha-bungarotoxin. 3. Within 2 days in culture muscle cells not contacted by nerve developed one or more discrete patches of acetylcholine (ACh) receptors as revealed by staining with fluorescent conjugates of alpha-bungarotoxin. Similar patches were also seen when staining was carried out after paraformaldehyde fixation, suggesting that they were not induced by the dyetoxin conjugate. 4. Radioautography after labelling with [125I]alpha-bungarotoxin revealed patches with grain densities approximately twenty-five-old greater than over the remainder of the cell. 5. Fluorescent stain on innervated cells was restricted to the path of nerve-muscle contact and sometimes extended for greater lengths than the largest patches seen on non-contacted muscle cells. 6. Similar long bands of stain associated with nerve--muscle contacts were observed when cultures were grown in high concentrations of curare and carbachol which prevented spontaneous twitching. They were also seen in cultures in which the addition of neural tube cells was delayed for 2-3 days. 7. It is concluded that innervation caused receptors to accumulate at sites of nerve-muscle contact and that this process can operate independently of muscle activity.

Analysis of afferent responses from isolated semicircular canal of the guitarfish using rotational acceleration white-noise inputs. II. Estimation of linear system parameters and gain and phase spectra

Quantitative estimates were computed for exponential coefficients and rate constants contributing to afferent unit impulse responses obtained from bundles innervating specific regions of the semicircular canal. The grouping of these estimates into specific response classes provided quantitative correlations with specific anatomical regions of innervation of the crista. Linear system gain and phase spectra were computed also, by applying Fourier transformations to unit impulse responses, for purposes of comparison with previous studies employing frequency domain analyses. Responses fitted by third-order linear system equations were specific to afferents innervating the crest and transition regions of the crista; whereas those fitted by overdamped, second-order equations were specific to afferents innervating the slopes and transition crista regions. It was concluded that strictly mechanical models of the transduction process are inadequate to account for the diverse and spatially distributed classes of observed responses and, moreover, structural features such as different hair cell types or efferent innervation effects could be excluded as inoperative in this preparation. The alternative hypothesis was suggested that certain of the observed subcomponents could be direct reflections of the initial mechanical stimulus, but that other subcomponents were reflections of more complex filtering mechanisms operating at the cellular or synaptic levels.

Some Aspects of Muscle Regeneration

Publisher Summary This chapter discusses some aspects of muscle regeneration. Regeneration of striated muscle must produce, to be complete, a multinucleated fiber attached at both ends containing myofibrils that can contract under a nervous stimulus. This is a minimum requirement for a functional restoration. But muscle tissue, being one of the most specialized in both form and function, is also one for which regenerative processes must occur in a delicately coordinated sequence. Whatever the method of destruction of the muscle (cutting, chemical or physical necrosis, ischemia), regeneration phenomena begin before destruction and phagocytosis of the destroyed parts are completed. In all cases of muscle regeneration, there is an early stage in which free cells are particularly numerous. All types of connective tissue cells are present. Part of the fibroblast like cells are myoblasts in the sense just defined but they cannot readily be recognized in sections with the usual histological techniques. The synthesis of the muscle proteins and the building up of myofibrils occur essentially in the sarcoblast. The sarcoblast includes all polynucleated stages before the muscle fiber is well differentiated. Thus its aspects change with time and may be variable. The chapter also discusses the formation of myohbrils, internal organization of the muscle fiber, the relation of the regenerated musclc fiber to the con nective tissue, and effect of innervation on muscle regeneration. Taking into account both in vivo and in vitro experiments, it seems that differentiation of regenerating muscle occurs in the absence of nerves. But later growth needs some influence from the nervous system.