Genetic and pharmacological models of muscle inactivity.
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Animals↗
Biomedical subjects
F Rieger
Publications and source records attributed to F Rieger.
The voltage-dependent sodium channel is co-localized with the acetylcholine receptor at the vertebrate neuromuscular junction.
Isolated motor endplates from mouse intercostal muscles can be obtained after subcellular fractionation. On these motor endplates, localization of the nicotinic receptor and of the voltage-dependent Na+ channel coincides as demonstrated by double labeling with rhodamine alpha-bungarotoxin and a specific anti-Na+ channel monoclonal antibody. High density of Na+ channel at the motor endplate is confirmed by the enrichment in TTX binding sites as compared to the crude homogenate. In contrast isolated motor endplates are almost completely devoid of Ca2+ channel antagonist binding sites.
Animals↗
Acetylcholinesterase is regulated by action potential generation and not by muscle contractile activity per se in mouse muscle in vitro.
Dysgenic (mdg/mdg) skeletal muscle of the mouse, grown in primary cell culture, fires action potentials in the absence of contractions, thus allowing analysis of the role of electrical activity (without contraction) on the specific activity and molecular forms of acetylcholinesterase (AChE). Specific activity of AChE was assessed by the spectrophometric method of Ellman (Biochem. Pharmacol., 7 (1961) 88-95) and found to increase by 2-5 times in the active myotubes (contraction and action potentials in normal and action potentials, alone in dysgenic muscle) compared to quiescent muscle. Sucrose density sedimentation analysis of muscle homogenates revealed an increase, by 2-3 times, in the proportion of the asymmetric (16S) molecular form of AChE in active muscle of both genotypes. Thus, electrical membrane activity, and not contraction per se, is directly involved in the regulation of levels of specific activity of and accumulation of the asymmetric (16S) form of AChE in muscle cells in culture.
Acetylcholinesterase↗
The electrophysiological expression of Ca2+ channels and of apamin sensitive Ca2+ activated K+ channels is abolished in skeletal muscle cells from mice with muscular dysgenesis.
Action potentials of myotubes in culture prepared from 18-19 day -old mouse embryos have a contractile activity and action potentials that are followed by a long lasting after hyperpolarization (ahp) which is blocked by apamin. Myotubes prepared from embryos of mice with muscular dysgenesis (mdg/mdg) did not contract and had action potentials which were never followed by a.h.p.'s. Voltage-clamp experiments have shown that Na+ channel activity was identical in mutant and control muscles and that the activity of fast and slow Ca2+ channels was nearly absent in the mutant.
Action Potentials↗
Acetylcholinesterase and butyrylcholinesterase in frontal cortex and cerebrospinal fluid of demented and non-demented patients with Parkinson's disease.
The molecular forms of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) were studied in frontal cortex (grey and white matter), postmortem, and in cerebrospinal fluid (CSF) of demented patients with Parkinson's disease compared to controls and non-demented parkinsonians. In the frontal cortex, AChE activity decreased significantly in both demented and non-demented parkinsonian subjects compared to controls; the 10S form of the enzyme was significantly lower in demented parkinsonians than in the non-demented subjects. The decrease in AChE activity was correlated with a decrease in choline acetyltransferase activity thought to reflect lesion of cholinergic neurones in the substantia innominata which innervate the cerebral cortex. BChE activity decreased only in the non-demented parkinsonians; in the demented subjects, BChE activity was at control levels. Similar results were obtained with grey and white matter, although absolute levels of the two enzymes were different in the two types of tissue, suggesting that the enzymes were affected in the cholinergic neurones before transport to cortical nerve terminals. No decreases in AChE or BChE activity were observed in the CSF of the patients studied. On the contrary, AChE and BChE levels were significantly higher in demented parkinsonian patients compared to the non-demented subjects.
Acetylcholinesterase↗
The development of motoneurons in the embryonic spinal cord of the mouse mutant, muscular dysgenesis (mdg/mdg): survival, morphology, and biochemical differentiation.
Motoneuron development was studied in the spinal cord of the mouse mutant, muscular dysgenesis, between embryonic days (E) 13 and 18. Dysgenic embryos are characterized by the absence of neuromuscular activity (motility) and exhibit a number of other striking changes in neuromuscular development. Many of these changes have also been observed in chick embryos chronically treated with neuromuscular blocking agents that suppress motility. Motoneuron survival, as well as several other aspects of neuronal development, was examined in the thoracic and lumbar spinal cords of mutant and control embryos. There was a significant decrease in motoneuron numbers in control embryos indicating the presence of naturally occurring cell death in the mouse spinal cord. At all ages examined, the dysgenic embryos had significantly more healthy and significantly fewer degenerating motoneurons than controls. There were no differences in the number of dorsal root ganglion neurons or in any of the other morphometric parameters examined between mutant and control embryos. Creatine kinase activity, a marker for myofiber maturation, was significantly reduced in the limb musculature of mutant embryos. Choline acetyltransferase activity was significantly increased in the spinal cord of mutant embryos. No significant differences were observed in spinal cord levels of acetylcholinesterase activity between control and mutant embryos. The absence of muscle contractions in the dysgenic mouse is associated with a number of changes in neuromuscular development, including a substantial reduction of naturally occurring motoneuron death.
Alleles↗
Difference in the ability of neonatal and adult denervated muscle to accumulate acetylcholinesterase at the old sites of innervation.
In adult rat sternocleidomastoid muscle, AChE is concentrated in the region rich in motor end-plates (MEP). All major AChE forms, "16 S," "10 S," and "4 S," are accumulated at high levels, and not only "16 S" AChE. After denervation, muscle AChE decreases; 2 weeks after denervation, low levels (20-40% of control) are reached for all forms. During the following weeks, a slow but steady increase in "10 S" and "16 S" AChE occurs in the denervated muscle. At this stage, all forms are again observed to be highly concentrated in the region containing the old sites of innervation. Thus, in adult rat muscle the structures able to accumulate "16 S," "10 S," and "4 S" AChE in the MEP-rich regions remain several months after denervation. In normal young rat sternocleidomastoid muscle at birth, all AChE forms are already accumulated in the MEP-rich region. After denervation at birth, the denervated muscle loses its ability to keep a high concentration of "4 S," "10 S," and "16 S" AChE in the old MEP-rich region. All AChE forms are still present 1 month after denervation, but they are decreased and diffusedly distributed over the whole length of the muscle. In particular, "16 S" AChE is detected in the same proportion (10-15%) all along the denervated muscle. Thus, the diffuse distribution of AChE, and especially "16 S" AChE, after neonatal denervation, contrasts with the maintained accumulation observed in adult denervated muscle. It seems that denervation of young muscle results in a specific loss of the muscle ability to concentrate high levels of all AChE forms at the old sites of innervation.
Acetylcholinesterase↗
Globular and asymmetric acetylcholinesterase in frog muscle basal lamina sheaths.
After denervation in vivo, the frog cutaneus pectoris muscle can be led to degenerate by sectioning the muscle fibers on both sides of the region rich in motor endplate, leaving, 2 wk later, a muscle bridge containing the basal lamina (BL) sheaths of the muscle fibers (28). This preparation still contains various tissue remnants and some acetylcholine receptor-containing membranes. A further mild extraction by Triton X-100, a nonionic detergent, gives a pure BL sheath preparation, devoid of acetylcholine receptors. At the electron microscope level, this latter preparation is essentially composed of the muscle BL with no attached plasmic membrane and cellular component originating from Schwann cells or macrophages. Acetylcholinesterase is still present in high amounts in this BL sheath preparation. In both preparations, five major molecular forms (18, 14, 11, 6, and 3.5 S) can be identified that have either an asymmetric or a globular character. Their relative amount is found to be very similar in the BL and in the motor endplate-rich region of control muscle. Thus, observations show that all acetylcholinesterase forms can be accumulated in frog muscle BL.
Acetylcholinesterase↗
Neuronal cell adhesion molecules and cytotactin are colocalized at the node of Ranvier.
Immunocytochemical methods were used to show that Ng-CAM (the neuron-glia cell adhesion molecule), N-CAM (the neural cell adhesion molecule), and the extracellular matrix protein cytotactin are highly concentrated at nodes of Ranvier of the adult chicken and mouse. In contrast, unmyelinated axonal fibers were uniformly stained by specific antibodies to both CAMs but not by antibodies to cytotactin. Ultrastructural immunogold techniques indicated that both N-CAM and Ng-CAM were enriched in the nodal axoplasm and axolemma of myelinated fibers as well as within the nodal regions of the myelinating Schwann cell. At embryonic day 14, before myelination had occurred, small-caliber fibers of chick embryos showed periodic coincident accumulations of the two CAMs but not of cytotactin, with faint labeling in the axonal regions between accumulations. Cytotactin was found on Schwann cells and in connective tissue. By embryonic day 18, nodal accumulations of CAMs were first observed in a few medium- and large-caliber fibers. Immunoblot analyses indicated that embryonic to adult conversion of N-CAM and a progressive decrease in the amount of Ng-CAM and N-CAM occurred while nodes were forming. Sciatic nerves of mouse mutants with defects in cell interactions showed abnormalities in the distribution patterns and amount of Ng-CAM, N-CAM, and cytotactin that were consistent with the known morphological nodal disorders. In trembler (+/Tr), intense staining for both CAMs appeared all along the fibers and the amounts of N-CAM in the sciatic nerve were found to be increased. In mice with motor endplate disease (med/med), Ng-CAM and N-CAM, but not cytotactin, were localized in the widened nodes. Both trembler and med/med Schwann cells stained intensely for cytotactin, in contrast to normal Schwann cells which stained only slightly. All of these findings are consistent with the hypothesis that surface modulation of neuronal CAMs mediated by signals shared between neurons and glia may be necessary for establishing and maintaining the nodes of Ranvier.
Animals↗
Altered expression of neuronal cell adhesion molecules induced by nerve injury and repair.
Peripheral nerve injury results in short-term and long-term changes in both neurons and glia. In the present study, immunohistological and immunoblot analyses were used to examine the expression of the neural cell adhesion molecule (N-CAM) and the neuron-glia cell adhesion molecule (Ng-CAM) within different parts of a functionally linked neuromuscular system extending from skeletal muscle to the spinal cord after peripheral nerve injury. Histological samples were taken from 3 to 150 d after crushing or transecting the sciatic nerve in adult chickens and mice. In unperturbed tissues, both N-CAM and Ng-CAM were found on nonmyelinated axons, and to a lesser extent on Schwann cells and myelinated axons. Only N-CAM was found on muscles. After denervation, the following changes were observed: The amount of N-CAM in muscle fibers increased transiently on the surface and in the cytoplasm, and in interstitial spaces between fibers. Restoration of normal N-CAM levels in muscle was dependent on reinnervation; in a chronically denervated state, N-CAM levels remained high. After crushing or cutting the nerve, the amount of both CAMs increased in the area surrounding the lesion, and the predominant form of N-CAM changed from a discrete Mr 140,000 component to the polydisperse high molecular weight embryonic form. Anti-N-CAM antibodies stained neurites, Schwann cells, and the perineurium of the regenerating sciatic nerve. Anti-Ng-CAM antibodies labeled neurites, Schwann cells and the endoneurial tubes in the distal stump. Changes in CAM distribution were observed in dorsal root ganglia and in the spinal cord only after the nerve was cut. The fibers within affected dorsal root ganglia were more intensely labeled for both CAMs, and the motor neurons in the ventral horn of the spinal cord of the affected segments were stained more intensely in a ring pattern by anti-N-CAM and anti-Ng-CAM than their counterparts on the side contralateral to the lesion. Taken together with the previous studies (Rieger, F., M. Grumet, and G. M. Edelman, J. Cell Biol. 101:285-293), these data suggest that local signals between neurons and glia may regulate CAM expression in the spinal cord and nerve during regeneration, and that activity may regulate N-CAM expression in muscle. Correlations of the present observations are made here with established events of nerve degeneration and suggest a number of roles for the CAMs in regenerative events.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Molecular forms of acetylcholinesterase in mammalian smooth muscles.
A comparative study of the molecular forms of acetylcholinesterase (AChE) was made in various smooth muscles (intestine, vas deferens, ciliary body, iris, nictitating membrane retractor, ureter, arteries, anococcygeus muscles) of some mammals (cat, guinea-pig, rat, rabbit, mouse), seeking for a correlation between the presence of 16 S (asymmetric, tailed) form of AChE in smooth muscles and their type of innervation defined by morphological criteria, as well as by the nature of the main neurotransmitters involved in their neuroeffector junctions. Contrary to previous assertions, many smooth muscles contain 16 S AChE, although all those examined here exhibited a proportion clearly less than that of striated muscles. There are large species-specific and individual variations in the percentage of 16 S AChE. The highest percentages of 16 S AChE were found in ciliary and iris muscles, which are provided with an individual (= multiunit) cholinergic innervation. The vas deferens muscles, which are also individually, but noradrenergically innervated contain practically no 16 S AChE. In the muscles having a fascicular (= unitary) innervation, the differences are striking: 16 S AChE is in rather high amount in intestine muscle layers, whereas it is very low or virtually absent in ureter or arterial muscles. Thus, the type of innervation is not clearly involved in the amount of 16 S AChE present in smooth muscles. As for the nature of neurotransmitter a clear correlation exists only in the case of individual innervation, in which only one neurotransmitter is involved or largely predominant.
Acetylcholinesterase↗
External and internal acetylcholinesterase in rat sympathetic neurones in vivo and in vitro.
The subcellular distribution of multiple molecular forms of acetylcholinesterase (AChE) in neurones of rat superior cervical ganglion (SCG) was determined both in vivo and in vitro by the use of selective lipid-soluble or -insoluble inhibitors. In vivo as well as in vitro, 10 S AChE is mainly outside the cell. In primary cultures of rat SCG neurones, both 4 S and 16 S AChE are mainly inside the cell. In near-term rat SCG, 4 S and 16 S are more external to the cell than in primary cultures. In adult rat SCG, 4 S AChE is equally distributed inside and outside and 16 S AChE is mainly outside the cell. Thus, specific AChE externalization probably occurs in neuronal cells as a developmentally regulated process.
Acetylcholinesterase↗
Abnormal transverse tubule system and abnormal amount of receptors for Ca2+ channel inhibitors of the dihydropyridine family in skeletal muscle from mice with embryonic muscular dysgenesis.
We have found two important sets of abnormalities in skeletal muscle from mice with embryonic muscular dysgenesis. These abnormalities involve the internal structural organization of the muscle fiber and its content of voltage-dependent Ca2+ channels. The first abnormality concerns the ultrastructural aspects of the membranous couplings between sarcoplasmic reticulum and the transverse tubules, known as triads. The triads are less numerous, are disorganized, and lack spaced densities (feet). The second abnormality is a significant decrease in specific binding sites for the dihydropyridine derivatives, (known as Ca2+ channel inhibitors) in striated skeletal muscle, but not in cardiac muscle. Both sets of abnormalities are potentially directly linked to the uncoupling of excitation and contraction.
Animals↗
N-CAM at the vertebrate neuromuscular junction.
We have detected the neural cell adhesion molecule, N-CAM, at nerve-muscle contacts in the developing and adult mouse diaphragm. Whereas we found N-CAM staining with fluorescent antibodies consistently to overlap with the pattern of alpha-bungarotoxin staining at nerve-muscle contacts both during development and in the adult, we observed N-CAM staining on the surfaces of developing myofibers and at much lower levels on adult myofibers. Consistent with its function, N-CAM was also detected on axons and axon terminals. Immunoblotting experiments with anti-N-CAM antibodies on detergent extracts of embryonic (E) diaphragm muscle revealed a polydisperse polysialylated N-CAM polypeptide, which in the adult (A) was converted to a discrete form of Mr 140,000; this change, called E-to-A conversion, was previously found to occur in different neural tissues at different rates. The Mr 140,000 component was not recognized by monoclonal antibody anti-N-CAM No. 5, which specifically recognizes antigenic determinants associated with N-linked oligosaccharide determinants on N-CAM from neural tissue. The relative concentration of the Mr 140,000 component prepared from diaphragm muscle increased during fetal development and then decreased sharply to reach adult values. Nevertheless, expression of N-CAM in muscle could be induced after denervation: one week after the sciatic nerve was severed, the relative amount of N-CAM increased dramatically as detected by immunoblots of extracts of whole muscle. Immunofluorescent staining confirmed that there was an increase in N-CAM, both in the cell and at the cell surface; at the same time, however, staining at the motor endplate was diminished. Our findings indicate that, in muscle, in addition to chemical modulation, cell-surface modulation of N-CAM occurs both in amount and distribution during embryogenesis and in response to denervation.
Age Factors↗
Rapid changes in levels of individual molecular forms of acetylcholinesterase after denervation of mouse sternocleidomastoid muscle.
Experimental denervation of adult mouse sternocleidomastoid muscle results in a decrease in total AChE. The most rapid change essentially affects the tailed, asymmetric 16 S AChE, since one day after nerve section, "16S" AChE is already significantly decreased to about 70% of its control value. We found that both background and junctional "16S" AChE are affected by this rapid decrease. Later, a sharp fall in "10S" and "4S" AChE occurs about seven days after denervation when muscle atrophy develops with loss of weight and proteins. A gaussian analysis of the sedimentation profiles of AChE extracted from denervated muscle shows that there is not only an early rapid decrease in 16 S AChE but also a decrease in the monomeric 3.3S AChE. This result suggests that there is a very rapid turn-over of two molecular forms of AChE, the supposedly monomeric precursor and the complex asymmetric 16S AChE.
Acetylcholinesterase↗
Distribution and quantification of ACh receptors and innervation in diaphragm muscle of normal and mdg mouse embryos.
Muscular dysgenesis (mdg) in the mouse is an autosomal recessive mutation, expressed in the homozygous state (in vivo and in vitro) as an absence of skeletal muscle contraction. The distribution of acetylcholine receptors (ACh R) in the diaphragms of phenotypically normal and dysgenic (mdg/mdg) embryos was studied from the 14th to 19th day of gestation by binding of 125I-alpha-bungarotoxin to the muscle, followed by autoradiography of longitudinally sectioned hemidiaphragms and/or of isolated muscle fibers. Localization of ACh R at putative motor end-plate regions begins 14 to 15 days in utero in both normal and dysgenic diaphragms. The distribution of high ACh R density patches is aberrantly scattered beyond the normal innervation pattern in dysgenic diaphragms. Isolated mutant fibers possess (1) multiple ACh R clusters, up to five per single fiber, (2) larger clusters of more variable morphology and variable receptor density than normal clusters, and (3) higher levels of extrajunctional receptors than normal fibers. These autoradiographic results correlate well with higher total level of toxin binding sites per diaphragm and per milligram protein in dysgenic vs normal muscle, as quantified from gamma counting of sucrose density gradient isolation of 125I-toxin-ACh R complexes. The dispersed distribution of ACh R patches on dysgenic muscle may be correlated with extensive phrenic nerve branching as demonstrated by silver impregnation technique. We suggest that the aberrant ACh R cluster distribution is a result of multiple innervation of single fibers from the branched nerve terminals. Possible causes of the excessive nerve branching in the mutant are discussed in light of generalized nerve sprouting found in paralyzed muscle.
Animals↗
Extensive nerve overgrowth and paucity of the tailed asymmetric form (16 S) of acetylcholinesterase in the developing skeletal neuromuscular system of the dysgenic (mdg/mdg) mouse.
In muscular dysgenesis (mdg) both skeletal muscle and motor innervation present an abnormal differentiation at birth (Rieger and Pinçon-Raymond. 1981). We establish in this report that the abnormalities of the motor innervation, extension of the innervation territory and nerve branching, and the low level of tailed asymmetric (16 S) acetylcholinesterase (AChE) are observed in the diaphragm as soon as the mutation is phenotypically expressed in the mdg/mdg embryo, at embryonic Day 13 1/2 or 14. The nerve-muscle interaction is thus abnormal at the very beginning of the formation of the neuromuscular system. The comparative electron microscopic study of normal and mutant muscle basal lamina shows that the newly formed mdg/mdg myotube lacks a basal lamina at Day 14. However, the biosynthesis of the basal lamina is not totally defective as, at Day 18, mdg/mdg myotubes possess basal lamina sheaths. Such delayed formation of extracellular material in the mutant may partly explain the abnormally low induction of 16 S AChE which is thought to be localized in the extracellular matrix and has potential important implications in nerve-muscle recognition and synapse stabilization.
Acetylcholinesterase↗
Paranodal dysmyelination and increase in tetrodotoxin binding sites in the sciatic nerve of the motor end-plate disease (med/med) mouse during postnatal development.
Motor end-plate disease (med), in the mouse, is a hereditary neuromuscular defect, caused by a single gene mutation and characterized by a progressive muscle weakness. +Med/+med mice die 21-23 days after birth and the neurobiological abnormalities already reported are nerve terminal sprouting and swelling and neurotransmission failures. We studied +med/+med mice at preclinical (9-11 days after birth) as well as at clinically recognized stages of the disease. The nonmyelinated gaps of the nodes of Ranvier in the +med/+med sciatic nerve are found to be significantly widened in +med/+med animals compared to control littermates, even in the preclinical stage, although the nodes of Ranvier are not yet ultrastructurally mature. The maximal binding capacity for [3H]ethylene-diamine tetrodotoxin, expressed in femtomoles per milligram of protein, is significantly increased in +med/+med sciatic nerves. Thus, Na+ channels, which are known to be located mainly at the nodes of Ranvier in normal myelinated axons, are increased in number in +med/+med mice even before the disease becomes clinically established. Both the ultrastructural and biochemical developmental abnormalities of the node of Ranvier rapidly approach their maximal expression as the behavioral signs develop. Such nerve abnormalities may be closely related to the physiological impairment of nerve impulse conduction which leads to the pathophysiological expression of motor end-plate disease.
Aging↗