Role of innervation on the embryonic development of skeletal muscle

Summary The extent to which the motor innervation regulates the embryonic development of skeletal muscle was investigated by comparing changes in normal, aneural, and paralyzed superior oblique muscle of the duck embryo. The muscle was made aneural by permanently destroying the trochlear motor neurons with electrocautery on day 7 i.e., three days prior to innervation. Embryos were paralyzed by daily application of -bungarotoxin onto the chorioallantoic membrane from day 10 onwards. The differentiation of myoblasts and myotubes in the aneural muscle was severely affected and did not progress to the myofiber stage. A mass of dead cells in the aneural muscle was replaced by connective tissue. Although the differentiation of myoblasts and myotubes was also retarded in the paralyzed muscle, numerous muscle cells progressed to the myofiber stage. Neuromuscular junctions of normal ultrastructure were seen in all paralyzed muscles. Degeneration of some cells in the paralyzed muscle occurred but there was no evidence of a massive wave of cell death similar to that observed in the aneural muscle. These observations suggest that both the trophic factors from the nerve and the nerve-evoked muscle activity are essential for the execution of the developmental program of the muscle. Trophic factors may play a larger role in differentiation, and maintenance of the muscle than muscle activity.Supported by a grant from the Muscular dystrophy Association and a grant from NIHWe are grateful to Beth McBride and Greg Oblak for their technical assistance     

Neural control of embryonic acetylcholine receptor and skeletal muscle

The manner by which motor neurons exert control over the distribution and number of acetylcholine receptors, and muscle development was investigated in the superior oblique muscle of white Peking duck embryos. Clusters of receptors in the normally developing muscle first appeared on day 10 of incubation as determined with I125 alpha-bungarotoxin autoradiography. The initial appearance of receptor clusters coincided with the arrival of motor nerve fibers in the muscle. Clusters of receptors also appeared in normal fashion in muscles made aneural by destruction of motor neurons on day 7. But after day 14 these clusters had disappeared and no new clusters were seen thereafter in the aneural muscle. Receptor clusters persisted throughout development in muscle in which neuromuscular transmission was blocked with either curare or botulinum toxin and in muscles denervated on day 10.5, i.e., shortly after the initial nerve-muscle contact but prior to the onset of muscle activity. A progressive increase in the total number of receptors and in the total amount of protein occurred during the course of normal development. However, the specific activity of the receptor protein declined sharply following innervation on day 10. The total number of receptors and the specific activity of the receptor was affected depending on whether the motor neurons were destroyed before or after innervation and following chronic blockade of neuromuscular transmission. The half-life of the receptor protein was similar in normal, aneural, and paralyzed muscles (26, 25, 26 h, respectively). Measurements of total protein indicated that essentially no muscle growth occurred in the complete absence of innervation. Paralyzed muscles continued to develop but at a slower pace.     

Neurotrophic control of 16S acetylcholinesterase from mammalian skeletal muscle in organ culture

The effects of rat obturator nerve extracts on total and 16S acetylcholinesterase (AChE) activity were studied in endplate regions of denervated anterior gracilis muscles maintained in organ culture for 48 hr. The decrease of total AChE activity in cultured muscles was similar to that observed in denervated muscles in vivo. This decrease in activity was partly prevented by addition of either 100 or 200 μl nerve extract (2.7 mg/ml protein) to the nutrient medium. Nerve extract treatment also decreased the release of AChE activity from the muscle into the bathing medium. Conversely, rat serum (20 μl; 90 mg/ml protein) had no effect on total AChE activity in muscle endplates, nor on release of the enzyme by the muscle. The 16S form of AChE was confined to motor endplate muscle regions and its activity was drastically decreased by denervation in both organ culture and in vivo preparations in a comparable manner. Nerve-extract supplemented cultures contained a significantly (p ? 0.001) larger amount of endplate 16S AChE activity (140–145%) than the corresponding controls (100-). Our results suggest that some nerve soluble substance, other than serum contaminants or 16S AChE itself, affects the maintenance of 16S AChE at the neuromuscular junction.     

Acetylcholinesterase molecular forms in the fast or slow muscles of the chicken and the pigeon

Molecular forms of acetylcholinesterase (AChE) were examined in various skeletal muscles of the chicken and the pigeon. In chicken pectoralis m., AChE was found to be restricted to endplate containing segments, and no asymmetric form could be detected in aneural samples. In the chicken muscles studied, a relation has been established between globular (G1,G2,G4) forms or asymmetric (A8,A12) forms, and muscle fibre types. Asymmetric forms are preponderant in fast-twitch muscles, whereas in slow tonic muscles 80% of the AChE activity is due to globular forms. However, comparison with pigeon muscles shows that AChE chicken muscle patterns may not be generalized.     

Autonomy of acetylcholinesterase differentiation in muscle lineage cells of ascidian embryos

The two muscle lineage blastomeres were removed surgically from Ciona intestinalis embryos at the eight-cell stage and allowed to develop in isolation. Acetylcholinesterase, an enzyme that occurs only in muscle cells of the developing larva, was detected histochemically in progeny cells of these isolated blastomers. Acetylcholinesterase differentiation in muscle lineage cells is not, therefore, dependent on inductive interactions with embryonic tissues derived from other eight-cell stage blastomeres.     

Sciatin: a myotrophic protein increases the number of acetylcholine receptors and receptor clusters in cultured skeletal muscle

Factors present in neural extracts or in media conditioned by neurons have been shown by others to increase both the number of acetylcholine receptors (AChRs) and the number of receptor clusters in cultures of embryonic skeletal muscle. We have recently shown that the glycoprotein, sciatin, exerts trophic effects on developing muscle in vitro. In the present study, we investigated the effect of sciatin on AChRs in aneural cultures of chick skeletal muscle. Sciatin caused a significant increase in the number of AChRs/dish as measured by binding of ¹²⁵I-α-bungarotoxin (α-Btx) and in acetylcholinesterase (AChE) activity/dish in differentiating muscle cells. The increase in AChRs elicited by sciatin was due solely to increased receptor synthesis and incorporation. The rate of AChR synthesis in sciatin-treated cultures was as much as five times the control rate and was significantly reduced by cycloheximide (10 μM). AChR degradation was unaffected by the myotrophic protein. Although the number of AChRs/dish was increased by sciatin during myogenesis, AChR specific activity, expressed as picomoles ¹²⁵I-α-Btx bound/mg cell protein, was only transiently increased by the myotrophic protein. This contrasted with AChE specific activity in sciatin-treated cultures which remained elevated throughout differentiation. Autoradiographs of ¹²⁵I-α-Btx-labeled cultures showed that sciatin caused an increase in the number and size of AChR “hot spots” and maintained the integrity of these AChR clusters in aneural muscle cultures for up to 5 weeks. At this time control cultures had completely degenerated. The mechanism by which sciatin enhanced the synthesis of AChRs appeared to be distinct from that of tetrodotoxin (TTX), an agent which abolishes muscle activity. However, like theophylline, sciatin might evoke increased synthesis of AChRs via regulation of cyclic AMP since the myotrophic protein increased cAMP both in cells and in conditioned medium. The results of this study suggest that sciatin may be related to the diffusible factor(s) from motor neurons described by others which has trophic effects on AChRs. Furthermore, we suggest that this myotrophic protein may be responsible for the clustering of AChRs and maintenance of receptor clusters at neuromuscular junctions in developing avian muscle.     

THE LOCALIZATION OF ACETYLCHOLINESTERASE AT THE AUTONOMIC NEUROMUSCULAR JUNCTION

Acetylcholinesterase has been localized at the autonomic neuromuscular junction in the bladder of the toad (Bufo marinus) by the Karnovsky method. High levels of enzyme activity have been demonstrated in association with the membranes of cholinergic axons and the adjacent membranes of the accompanying Schwann cells. The synaptic vesicles stained in occasional cholinergic axons. After longer incubation times, the membrane of smooth muscle cells close to cholinergic axons also stained. Axons with only moderate acetylcholinesterase activity or with no activity at all were seen in the same bundles as cholinergic axons, but identification of the transmitter in these axons was not possible.     

In vitro maintenance of nematode parasites assayed by acetylcholinesterase and allergen secretion.

A comparison was made of the ability of Nippostrongylus brasiliensis and Necator americanus to synthesize and secrete acetylcholinesterase when they were maintained in different in vitro culture media. The amount of allergen released by N. brasiliensis was also studied. The adult and fourth stage larval stages (but not the infective larvae) of Necator and adult N. brasiliensis secreted from their anterior glands up to 40 times as much acetylcholinesterase as they contained at the outset of culture. In contrast, allergen, which is less easy to quantitate, was secreted into the same media at about one-third the rate of secretion of acetylcholinesterase. Acetylcholinesterase was synthesized and released by both worms in media containing protein and the enzyme did not lose activity when kept for several days at 37 C. The secretion of this enzyme by nematodes kept in culture provides a simple, sensitive, rapid, and quantitative assay for measuring the ability of these nematodes to synthesize and secrete antigens in culture.     

Lineage segregation and developmental autonomy in expression of functional muscle acetylcholinesterase mRNA in the ascidian embryo

Acetylcholinesterase is a histospecific marker of cell differentiation occurring only in the muscle and mesenchyme tissues of the ascidian embryo. The distribution of functional mRNA coding for this enzyme has been investigated and it is shown here that only cells of muscle and mesenchyme lineages possess such a template. Blastomeres of four cell lineage quadrants were separated microsurgically from eight-cell-stage embryos of Ciona intestinalis and raised in isolation until muscle development was well advanced. Measurement of enzyme activity in the resulting partial embryos revealed that acetylcholinesterase was limited to descendants of one blastomere pair, the B4.1 blastomeres containing muscle and mesenchyme lineages. To study the tissue distribution of acetylcholinesterase mRNA, RNA from partial embryos was translated in Xenopus laevis oocytes. When oocytes were injected with an appropriate template, they synthesized a biologically active acetylcholinesterase that could be selectively immunopurified with an antiserum to the ascidian enzyme. Under the conditions used the quantity of acetylcholinesterase mRNA was directly related to the enzyme activity in immunoprecipitates. Acetylcholinesterase mRNA was found only in B4.1 lineage partial embryos where it occurred in approximately the same amount as in whole embryos of the same age. Since there is a limited period from gastrulation until the middle tail-formation stage when functional acetylcholinesterase mRNA accumulates, the results of our mRNA distribution experiments strongly suggest that the gene for ascidian acetylcholinesterase is active only in muscle and mesenchyme tissues. The histospecific occurrence of this enzyme apparently does not involve selective, cell-specific control of translation.     

Acetylcholinesterase activity in developing skeletal muscle cells

Acetylcholinesterase activity has been demonstrated biochemically and cytochemically in developing chick embryo skeletal muscle cells growing in culture. The enzyme shows the same pattern of drug sensitivity as that of adult skeletal muscle acetylcholinesterase and in present in cultured myogenic cells before the time of cell fusion, the formation of myotubes, and the subsequent increase in rate of myosin synthesis. Myogenic cell fusion is accompanied, however, by a large increase in activity of acetylcholinesterase. The enzyme activity is restricted in these cultures to myogenic cells. Neighboring fibroblasts show no cytochemical responses when challenged with techniques showing intense activity in myoblasts and myotubes. In addition, evidence is presented which strongly suggests that acetylcholinesterase activity in dividing myogenic cells is not constant over the cell cycle.     

Effects of Aldicarb and Fenamiphos on Acetycholinesterase and Motility of Caenorhabditis elegans

The ability of Caenorhabditis elegans to recover from exposure to high doses of aldicarb and fenamiphos was examined at the organismal and biochemical levels by determination of movement and acetylcholinesterase activity. Nematodes recovered rapidly from a 24-hour exposure to both compounds at concentrations that caused complete paralysis. Acetylcholinesterase regained nearly full activity after a 24-hour exposure to aldicarb but only 10% activity after exposure to fenamiphos. The nematodes were able to move normally, however, on the limited activity that was regained after fenamiphos treatment. Mutant C. elegans strains deficient in various molecular forms of acetylcholinesterase were utilized to demonstrate that the mechanism of recovery did not involve new synthesis of enzyme. This result was confirmed by experiments on acetylcholinesterase reactivation from live versus dead nematodes.     

Reinnervation of adult muscle in organ culture restores tetrodotoxin sensitivity in the absence of electrical activity.

Strips of denervated adult mouse diaphragm muscle maintained in organ culture were reinnervated by nerve processes growing out from explants of embryonic mouse spinal cord. In vivo, following denervation, the action potential loses its sensitivity to tetrodotoxin; this sensitivity is regained upon reinnervation. Similarly, action potentials in cultured muscle fibres were insensitive to tetrodotoxin, and sensitivity was restored in muscle fibres that became reinnervated in vitro. Tetrodotoxin sensitivity was also restored in cultured muscle fibres reinnervated in the continuous presence of d-tubocurarine, but it was not induced by 4 days of direct electrical stimulation of noninnervated muscles. We conclude that developing nerve terminals can exert a trophic action on adult muscle fibres that is independent of electrical activity in the muscle.     

Impact of malathion on acetylcholinesterase in the tissues of the fishTilapia mossambica (Peters)—a time course study

The sublethal toxic potency of malathion in inhibiting acetylcholinesterase activity of brain, muscle, gill and liver tissues of the fish,Tilapia mossambica was studied at 12 h intervals. Maximum in hibition at 36 and 48 h, and complete revival of acetylcholinesterase activity after 72 h was noticed, suggestive of the loss of inhibition of the enzyme activity was probably by suitable (acetylcholine) accumulation.     

In vivo and in vitro effects of cadmium on selected enzymes in different organs of the fish Barbus conchonius ham. (Rosy Barb)

1. Enzyme modulation by cadmium in selected organs of the fish, Barbus conchonius (rosy barb), was investigated in vivo (48 hr exposure to 12.6 mg/1 cadmium chloride) and in vitro (10⁻⁶M cadmium chloride).2. The acetylcholinesterase (AchE) activity was depressed in the gills but stimulated in the skeletal muscles and brain in vivo. The hepatic, branchial, and renal acid phosphatase (AcP) activity decreased marginally in vivo but it was significantly increased in the gut and ovary. In vitro, except for the liver, the AcP activity was depressed in the selected organs. Collaterally, gut alkaline phosphatase (A1P) was significantly inhibited but a pronounced stimulation was noted in the kidneys and ovary in vivo. In vitro, the AIP activity was conspicuously elevated in the kidneys and gut, and moderately in the gills.3. Cadmium inhibited the glutamate-oxaloacetate and glutamate-pyruvate transaminases (GOT and OPT) in the liver, gills and kidneys in vivo. In vitro, the GOT and GPT activities were decreased in the liver, gills and kidneys. The lactic dehydrogenase (LDH) was significantly stimulated by Cd in the heart in vivo but in vitro the metal inhibited the enzyme in the gills.4. Enzymes in the liver, followed by those in the kidneys and gills seem to be most seriously affected by Cd poisoning in this fish.     

The Arabidopsis thaliana ortholog of a purported maize cholinesterase gene encodes a GDSL-lipase

Acetylcholinesterase is an enzyme that is intimately associated with regulation of synaptic transmission in the cholinergic nervous system and in neuromuscular junctions of animals. However the presence of cholinesterase activity has been described also in non-metazoan organisms such as slime molds, fungi and plants. More recently, a gene purportedly encoding for acetylcholinesterase was cloned from maize. We have cloned the Arabidopsis thaliana homolog of the Zea mays gene, At3g26430, and studied its biochemical properties. Our results indicate that the protein encoded by the gene exhibited lipase activity with preference to long chain substrates but did not hydrolyze choline esters. The At3g26430 protein belongs to the SGNH clan of serine hydrolases, and more specifically to the GDS(L) lipase family.     

Muscle and nerve in muscular dysgenesis in the mouse at birth: Sprouting and multiple innervation

Muscular dysgenesis (mdg) in the mouse is a recessive autosomal mutation affecting the striated musculature: during the whole gestation period, the muscles never show any sign of contractile activity. They are cytologically immature at birth, although the diaphragm is more mature than limb muscles, as confirmed by the levels of creatine phosphokinase. In both limb muscles and diaphragm the cytochemical localization of acetylcholinesterase demonstrates focal accumulations on the entire surface of $\text{mdg}\text{mdg}$ muscles, whereas such foci of acetylcholinesterase activity are restricted to a narrow end plate-rich region in $\text{+}\text{mdg}$? diaphragms. Teased single $\text{mdg}\text{mdg}$ myofiber preparations show that one myofiber can possess several foci of acetylcholinesterase, generally presenting aspects of very immature motor end plates. A study of the motor innervation, after silver nitrate impregnation, provides evidence for a spectacular overgrowth and a generalized sprouting of $\text{mdg}\text{mdg}$ nerves and axons. The $\text{mdg}\text{mdg}$ nerve terminals are generally very immature-looking, with an intense ultraterminal sprouting. Aspects suggesting a denser multiple innervation of $\text{mdg}\text{mdg}$ than $\text{+}\text{mdg}$? myofibers have been observed and choline acetyltransferase activity is increased in $\text{mdg}\text{mdg}$ tissues. Acetylcholinesterase specific activity and the number of α-bungarotoxin binding sites per milligram protein increased in $\text{mdg}\text{mdg}$ compared to $\text{+}\text{mdg}$? diaphragms. The very low amount of 16 S (and 12 S) acetylcholinesterase is probably related to $\text{mdg}\text{mdg}$ muscle inactivity. If the cytological and biochemical data are compared, it seems possible to propose that $\text{mdg}\text{mdg}$ myofibers and axons are in contact in several regions of the same myofiber, in variably mature appositions, and with a very dense multi-innervation.     

CHOLINE ACETYLTRANSFERASE AND ACETYLCHOLINESTERASE IN CULTURED BRAIN CELLS FROM CHICK EMBRYOS

—Dissociated cells from brains of 7-day chick embryos were grown in primary culture for as long as 20 days. Many of the plated cells grew out long processes. Others, which proliferated rapidly, formed a confluent layer of flat cells after 4-6 days. Total DNA and protein increased five-fold, and activity of choline acetyltransferase (EC2.3.1.6) increased about 40-fold in 20 days. Acetylcholinesterase (EC3.1.1.7) increased three-fold by the fourth day of culture and then declined. The pattern of increase for choline acetyltransferase was similar to that for the in vivo development of the enzyme. l -Thyroxine, cyclic AMP (adenosine-3′,5′-monophosphate) or theophylline promoted increased levels of both enzymes by 30-200 per cent. l -Thyroxine also increased the activity of acetylcholinesterase in vivo by 40 per cent. When overgrowth by flat cells was prevented by the addition of 10^-3m -5-flourouracil, there was a decrease in the activity of choline acetyltransferase and an increase in the activity of acetylcholinesterase in comparison to control activities. The addition of 10^-3m -morphine or cocaine produced a 30 per cent elevation in the activity of choline acetyltransferase, but this effect could be mimicked with equimolar concentrations of ammonium ion.     

Fasciculin, a powerful anticholinesterase polypeptide from Dendroaspis angusticeps venom

A powerful inhibition of mammalian acetylcholinesterase was detected in the venom of the snake Dendroaspis angusticeps (green mamba). The substances responsible for such inhibition were isolated and purified by gel filtration on Sephadex G-50 and ion exchange chromatography on Bio-Rex 70 and SP Sephadex C-25. These substances were polypeptides and were named, fasciculins.Upon intraperitoneal injection into mice fasciculins elicited severe, generalized, long-lasting muscle fasciculations with complete clinical recovery.In vitro preincubation with fasciculins at concentrations of 0.01 μg ml^?1 inhibited brain and muscle acetylcholinesterases up to 80%. Histochemical assay for acetylcholinesterase showed an almost complete disappearance of the black-brown precipitate at the neuromuscular end-plate after in vitro incubation with fasciculins.Fasciculins represent a new type of acetylcholinesterase inhibitors provoking muscle fasciculations through a powerful inhibition of enzyme activity at the neuromuscular end-plate, interfering with the normal degradative activity of the acetylcholine molecule. Fasciculins are also powerful inhibitors of brain acetylcholinesterases.     

A novel muscle biopsy clamp yields accurate in vivo sarcomere length values

The measurement of in vivo muscle sarcomere length facilitates the definition of in vivo muscle functional properties and comparison of muscle designs amongst functional muscle groups. In vivo sarcomere lengths are available for just a handful of human muscles, largely due to the technical challenges associated with their measurement. The purpose of this report was to develop and test a muscle biopsy clamp that can quickly and accurately measure in vivo muscle sarcomere length. To test the device, muscle biopsies (n=23) were removed from the tibialis anterior muscles of New Zealand White rabbits immediately after sarcomere length measurements were made using laser diffraction. The muscle biopsy contained within the clamp was immediately fixed in Formalin for subsequent sarcomere length measurement. Comparisons of clamp-based and diffraction-based sarcomere lengths demonstrated excellent agreement between the two techniques, especially when the biopsy was obtained at relatively long lengths (above 2.6 μm). Given the intraoperative speed and simplicity of this technique and the relatively low-cost of the biopsy clamp, this method of measuring muscle sarcomere length should help investigators generate much-needed in vivo muscle structural and functional data.     

Human erythrocyte acetylcholinesterase in relation to cell age.

Acetylcholinesterase was studied in human red cells that had been fractionated on Ficoll/Triosil density gradients into classes representing different ages in vivo. Reticulocytes have negligible acetylcholinesterase activity; this is rapidly acquired on maturation to the erythrocyte. The activity per cell reaches a maximum and then, after a constant period, declines again towards the end of cell life. The maximum activity and the rates of activity gain and loss per cell are quantitatively different in adults and children. Kinetic studies showed that Vmax. follows the same age/activity profile but Km is unaffected by cell age. The acetylcholinesterase protein content, determined by quantitative crossed immunoelectrophoresis, also shows a profile of increase and then decrease with cell age but the specific activity calculated from the protein estimate shows a reverse picture in which there is a slight decrease from young to mid-age cells followed by an increase again in older cells. These results are interpreted to indicate a complex developmental picture in which the overall cell age against enzyme activity profile is determined partly by the amount of enzyme protein present and partly from the modifying effect on the enzyme activity, of interactions with an aging cell membrane.