Synapse-specific expression of acetylcholine receptor genes and their products at original synaptic sites in rat soleus muscle fibres regenerating in the absence of innervation

Development ◽  
1992 ◽  
Vol 116 (1) ◽  
pp. 41-53 ◽  
Author(s):  
H.R. Brenner ◽  
A. Herczeg ◽  
C.R. Slater
Keyword(s):  
Acetylcholine Receptor ◽  
Basal Lamina ◽  
Neuromuscular Junctions ◽  
Muscle Fibres ◽  
Northern Blots ◽  
Specific Expression ◽  
Epsilon Subunit ◽  
Synaptic Region ◽  
Rat Soleus Muscle

To test the hypothesis that synaptic basal lamina can induce synapse-specific expression of acetylcholine receptor (AChR) genes, we examined the levels mRNA for the alpha- and epsilon-subunits of the AChR in regenerating rat soleus muscles up to 17 days of regeneration. Following destruction of all muscle fibres and their nuclei by exposure to venom of the Australian tiger snake, new fibres regenerated within the original basal lamina sheaths. Northern blots showed that original mRNA was lost during degeneration. Early in regeneration, both alpha- and epsilon-subunit mRNAs were present throughout the muscle fibres but in situ hybridization showed them to be concentrated primarily at original synaptic sites, even when the nerve was absent during regeneration. A similar concentration was seen in denervated regenerating muscles kept active by electrical stimulation and in muscles frozen 41–44 hours after venom injection to destroy all cells in the synaptic region of the muscle. Acetylcholine-gated ion channels with properties similar to those at normal neuromuscular junctions were concentrated at original synaptic sites on denervated stimulated muscles. Taken together, these findings provide strong evidence that factors that induce the synapse-specific expression of AChR genes are stably bound to synaptic basal lamina.

Synaptic basal lamina contains a signal for synapse-specific transcription

Development ◽  
1992 ◽  
Vol 115 (3) ◽  
pp. 673-680 ◽  
Author(s):  
S.A. Jo ◽  
S.J. Burden
Keyword(s):  
Acetylcholine Receptor ◽  
Basal Lamina ◽  
Motor Nerve ◽  
Receptor Gene ◽  
High Rate ◽  
Denervated Muscle ◽  
Transcriptional Pattern ◽  
Synaptic Region ◽  
Acetylcholine Receptor Gene ◽  
Continuous Presence

Nuclei in the synaptic region of multinucleated skeletal myofibers are transcriptionally distinct, since acetylcholine receptor genes are transcribed at a high rate by these nuclei, but not by nuclei elsewhere in the myofiber. Although this spatially restricted transcription pattern is presumably imposed by the motor nerve, the continuous presence of the nerve is not required, since synapse-specific transcription persists after denervation. These results suggest either that a transcriptional signal persists at synaptic sites after nerve terminals have degenerated, or that a transcriptional pattern in the myofiber, once established, is stable in the absence of a nerve-derived signal. To distinguish between these possibilities, we denervated muscle and damaged the myofibers and specialized cells located near synaptic sites, and then studied transcription of an acetylcholine receptor gene in myofibers that regenerated in their original basal lamina sheaths, but remained denervated. We show that synapse-specific transcription is re-induced in these regenerated myofibers, and we conclude that a signal for synapse-specific transcription is stably maintained in the synaptic basal lamina.

scholarly journals Localization of nicotinic acetylcholine receptor alpha-subunit transcripts during myogenesis and motor endplate development in the chick.

1989 ◽  
Vol 108 (3) ◽  
pp. 1025-1037 ◽  
Author(s):  
B Fontaine ◽  
J P Changeux
Keyword(s):  
Acetylcholine Receptor ◽  
Nicotinic Acetylcholine Receptor ◽  
Muscle Development ◽  
Neuromuscular Junctions ◽  
Primary Cultures ◽  
Motor Endplate ◽  
Alpha Subunit ◽  
Subunit Gene ◽  
Nicotinic Acetylcholine

In 15-d-old chick latissimi dorsi muscles, the nicotinic acetylcholine receptor (AChR) alpha-subunit mRNA is densely accumulated at the level of subsynaptic nuclei of the motor endplate (Fontaine et al., 1988). In this paper, using in situ hybridization with genomic probes, we further show that the expression of the AChR alpha-subunit gene in the embryo, revealed by the accumulation of mature mRNAs, starts in myotomal cells and persists during the first stages of muscle development in a majority of muscle nuclei. Subsequently, the distribution of AChR alpha-subunit mRNAs becomes restricted to the newly formed motor endplates as neuromuscular junctions develop. To assess the transcriptional activity of individual nuclei in developing muscles, a strictly intronic fragment of the AChR alpha-subunit gene was used to probe in situ the level of unspliced transcripts. AChR alpha-subunit unspliced transcripts accumulate around a large number of sarcoplasmic nuclei at embryonic day 11, but can no longer be detected at their level after embryonic day 16 in the embryo. A similar decrease in the accumulation of AChR alpha-subunit transcripts is observed between day 4 and day 6 in primary cultures of muscle cells. On the other hand, in vivo denervation and in vitro blocking of muscle electrical activity by the sodium channel blocker tetrodotoxin results in an increase in the labeling of muscle nuclei. Yet, only 6% of the muscle nuclei appear labeled by the strictly intronic probes after denervation. The possible significance of such heterogeneity of muscle nuclei during motor endplate formation in AChR gene expression is discussed.

scholarly journals The influence of basal lamina on the accumulation of acetylcholine receptors at synaptic sites in regenerating muscle.

1984 ◽  
Vol 98 (4) ◽  
pp. 1453-1473 ◽  
Author(s):  
U J McMahan ◽  
C R Slater
Keyword(s):  
Plasma Membrane ◽  
Basal Lamina ◽  
Muscle Fiber ◽  
Acetylcholine Receptors ◽  
Skeletal Muscles ◽  
Neuromuscular Junctions ◽  
High Concentration ◽  
Direct Role ◽  
Synaptic Region ◽  
Membrane Infoldings

If skeletal muscles are damaged in ways that spare the basal lamina sheaths of the muscle fibers, new myofibers develop within the sheaths and neuromuscular junctions form at the original synaptic sites on them. At the regenerated neuromuscular junctions, as at the original ones, the muscle fiber plasma membrane is characterized by infoldings and a high concentration of acetylcholine receptors (AChRs). The aim of this study was to determine whether or not the synaptic portion of the myofiber basal lamina sheath plays a direct role in the formation of the subsynaptic apparatus on regenerating myofibers, a question raised by the results of earlier experiments. The junctional region of the frog cutaneous pectoris muscle was crushed or frozen, which resulted in disintegration and phagocytosis of all cells at the synapse but left intact much of the myofiber basal lamina. Reinnervation was prevented. When new myofibers developed within the basal lamina sheaths, patches of AChRs and infoldings formed preferentially at sites where the myofiber membrane was apposed to the synaptic region of the sheaths. Processes from unidentified cells gradually came to lie on the presynaptic side of the basal lamina at a small fraction of the synaptic sites, but there was no discernible correlation between their presence and the effectiveness of synaptic sites in accumulating AChRs. We therefore conclude that molecules stably attached to the myofiber basal lamina at synaptic sites direct the formation of subsynaptic apparatus in regenerating myofibers. An analysis of the distribution of AChR clusters at synaptic sites indicated that they formed as a result of myofiber-basal lamina interactions that occurred at numerous places along the synaptic basal lamina, that their presence was not dependent on the formation of plasma membrane infoldings, and that the concentration of receptors within clusters could be as great as the AChR concentration at normal neuromuscular junctions.

Effects of calcitonin gene-related peptide on synaptic acetylcholine receptor-channels in rat muscle fibres

1988 ◽  
Vol 234 (1276) ◽  
pp. 333-342 ◽  
Keyword(s):  
Acetylcholine Receptor ◽  
Amplitude Distribution ◽  
Calcitonin Gene Related Peptide ◽  
Muscle Fibres ◽  
Patch Clamp Recording ◽  
Adult Rats ◽  
Related Peptide ◽  
Calcitonin Gene ◽  
Synaptic Region ◽  
Denervated Muscles

Cultured myotubes and freshly dissociated muscle fibres from adult rats were exposed to calcitonin gene-related peptide (CGRP) and studied by patch-clamp recording during the peptide-induced maximal accumulation of cellular cyclic AMP (cAMP). Acetylcholine receptor- (AChR-) channel properties in myotubes were not modified by the presence of CGRP (10 -7 M). The peptide, applied to the non-patched membrane, significantly increased the variance of the AChR-channel amplitude distribution at the synaptic region of muscle fibres, and three classes of AChR-channels were resolved immediately after peptide application. AChR-channels at extrasynaptic regions of fibres from denervated muscles were unaffected by CGRP. It is suggested that CGRP may regulate the synaptic AChR-channel conductance through second messenger systems.

scholarly journals Neural regulation of muscle acetylcholine receptor epsilon- and alpha-subunit gene promoters in transgenic mice.

1993 ◽  
Vol 123 (6) ◽  
pp. 1535-1544 ◽  
Author(s):  
K Gundersen ◽  
J R Sanes ◽  
J P Merlie
Keyword(s):  
Acetylcholine Receptor ◽  
Fusion Gene ◽  
Transgenic Animals ◽  
Alpha Subunit ◽  
Gene Promoters ◽  
Promoter Sequences ◽  
Epsilon Subunit ◽  
Synaptic Region ◽  
Localization Signal ◽  
Activity Dependence

The effects of denervation were investigated in mice with transgenes containing promoter elements from the muscle acetylcholine receptor epsilon- and alpha-subunit genes. The promoter sequences were coupled to a nuclear localization signal-beta-galactosidase fusion gene (nlacZ) as a reporter. While many postsynaptic specializations form in the embryo, expression of the epsilon subunit is induced during the first two postnatal weeks. When muscles were denervated at birth, before the onset of epsilon expression, epsilon nlacZ still appeared at the former synaptic sites on schedule. This result suggests that the nerve leaves a localized "trace" in the muscle that can continue to regulate transcription. An additional finding was that epsilon nlacZ expression was much stronger in denervated than in intact muscles. This suggests that the epsilon promoter is similar to the other subunits in containing elements that are activated on cessation of neural activity. However, even after denervation, epsilon nlacZ expression was always confined to the synaptic region whereas alpha nlacZ expression increased in nuclei along the entire length of the fiber. This suggests that while the epsilon gene is similar in its activity dependence to other subunit genes, it is unique in that local nerve-derived signals are essential for its expression. Consequently, inactivity enhances epsilon expression only in synaptic nuclei where such signals are present, but enhances expression throughout the muscle fiber. Truncations and an internal deletion of the epsilon promoter indicate that cis-elements essential for the response to synaptic signals are contained within 280 bp of the transcription start site. In contrast to these results in young animals, denervation in older animals leads to an unexpected reduction in nlacZ activity. However, mRNA measurements indicated that transgene expression was increased in these animals. This discordance between nlacZ mRNA and enzyme activity, demonstrates a previously unknown limitation of nlacZ as a reporter gene in transgenic animals.

scholarly journals Myosin heavy-chain mRNA is present in both myofibrillar and subsarcolemmal regions of muscle fibres

1991 ◽  
Vol 279 (1) ◽  
pp. 309-310 ◽  
Author(s):  
J Hesketh ◽  
G Campbell ◽  
N Loveridge
Keyword(s):  
Myosin Heavy Chain ◽  
Soleus Muscle ◽  
Heavy Chain ◽  
Muscle Fibres ◽  
The Core ◽  
Rat Soleus Muscle ◽  
Hybridization In Situ

Hybridization in situ with riboprobes to the myosin heavy-chain slow isoform showed that, in the rat soleus muscle, the myosin heavy-chain mRNA was distributed throughout the myofibres. There was greater density of autoradiographic grains in the subsarcolemmal regions of the fibres, but there was also a considerable number of grains in the core myofibrillar region of the fibres. Microdensitometry showed that the grain density in the myofibrillar region was approximately half that in the subsarcolemmal rim; this would correspond to some 70% of the mRNA being present in the myofibrillar region. The results are consistent with the hypothesis that myosin is synthesized on polyribosomes present in the intermyofibrillar cytoplasm.

scholarly journals In situ hybridization and cytochemistry: localization of mRNA at stained neuromuscular junctions with 33P-labeled probes.

1994 ◽  
Vol 42 (10) ◽  
pp. 1407-1411 ◽  
Author(s):  
E Liu ◽  
M M Salpeter
Keyword(s):  
In Situ Hybridization ◽  
Ache Activity ◽  
Neuromuscular Junctions ◽  
Original Method ◽  
Sternocleidomastoid Muscle ◽  
Staining Reaction ◽  
Epsilon Subunit ◽  
Subunit Mrna ◽  
One Step

We modified the Karnovsky and Roots method of staining sites of acetylcholinesterase (AChE) activity at neuromuscular junctions (NMJs) to survive the lengthy, multiple steps of in situ hybridization and autoradiography. When the original method of Karnovsky and Roots is used to identify the muscle endplates, the stain does not survive the in situ hybridization procedures and association of mRNA to specific endplates can be inferred only indirectly. The successful modification involves secondary staining with diaminobenzidine (DAB) and H2O2 using the Karnovsky-Roots staining reaction product as a catalyst. Mounted longitudinal cryosections of mouse sternocleidomastoid muscle were fixed and stained in one step on the slide with paraformaldehyde plus the Karnovsky-Roots stain, followed by DAB-H2O2 secondary staining. The tissues were then processed for in situ hybridization and probed for the acetylcholine receptor (AChR) epsilon-subunit mRNA, known to be localized at the NMJ. The probe was labeled with 33P, which is ideal for in situ hybridization. By this procedure, the endplate stain was retained even after the hybridization and autoradiographic procedures, and the developed grains due to radiolabeling of the AChR epsilon-subunit mRNA were localized at readily identified endplates.

scholarly journals Identification of agrin in electric organ extracts and localization of agrin-like molecules in muscle and central nervous system

1987 ◽  
Vol 132 (1) ◽  
pp. 223-230 ◽  
Author(s):  
M. A. Smith ◽  
Y. M. Yao ◽  
N. E. Reist ◽  
C. Magill ◽  
B. G. Wallace ◽  
...  
Keyword(s):  
Neuromuscular Junction ◽  
Acetylcholine Receptor ◽  
Basal Lamina ◽  
Acetylcholine Receptors ◽  
Electric Organ ◽  
Synaptic Cleft ◽  
Muscle Fibres ◽  
Motor Neurones ◽  
Cultured Myotubes ◽  
Torpedo Electric Organ

The portion of the muscle fibre's basal lamina that occupies the synaptic cleft at the neuromuscular junction contains molecules that cause the aggregation of acetylcholine receptors and acetylcholinesterase on regenerating muscle fibres. Agrin, which is extracted from basal lamina-containing fractions of the Torpedo electric organ and causes the formation of acetylcholine receptor and acetylcholinesterase aggregates on cultured myotubes, may be similar, if not identical, to the acetylcholine receptor- and acetylcholinesterase-aggregating molecules at the neuro-muscular junction. Here we summarize experiments which led to the identification of agrin and established that the basal lamina at the neuromuscular junction contains molecules antigenically similar to agrin. We also discuss results which raise the possibility that agrin-like molecules at the neuromuscular junction are produced by motor neurones.

In vitro formation of neuromuscular junctions between adult Rana muscle fibres and embryonic Xenopus neurons

1987 ◽  
Vol 230 (1261) ◽  
pp. 425-441 ◽  
Keyword(s):  
Neuromuscular Junctions ◽  
Rana Temporaria ◽  
Amplitude Distribution ◽  
Mean Amplitude ◽  
Muscle Fibres ◽  
Electron Microscopical ◽  
Early Regeneration ◽  
Miniature Endplate Potentials

Adult muscle fibres of the frog Rana temporaria were cultured with neurons from embryos of the frog Xenopus laevis . Electron microscopical and electro-physiological examination of the cultures showed that hetero-specific ( Xenopus- Rana ) neuromuscular junctions were formed in vitro . Nerve processes, without any Schwann cell covering, made contacts anywhere along a muscle fibre, and the junctions resembled those seen during early regeneration of neuromuscular synapses in situ . Functional contacts, as inferred by the presence of spontaneous miniature endplate potentials, or currents, were more common if the muscle fibres were denervated prior to culturing with neurons. Miniature endplate currents (m. e. p. cs) had a skewed amplitude distribution, with many small events lost in the recording noise, and their mean amplitude was much smaller than that of m. e. p. cs in the original lumbricalis muscle. The time constant of decay of m. e. p. cs in the hetero-specific junctions formed in vitro was several times longer than the decay of m. e. p. cs in the original muscle. Analysis of membrane current noise elicited by ionophoretically applied acetylcholine (ACh) suggests that the slower decay of m. e. p. cs in the junctions formed in vitro is due to a prolonged lifetime of the channels opened by ACh and to repetitive activation of ACh-receptors, which becomes possible because of a comparative lack of cholinesterase in the junctions.

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