Endomicroscopy and electromyography of neuromuscular junctions in situ

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.

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Further Study of the Electrical and Mechanical Responses of Slow Fibers in Cat Extraocular Muscles

The Journal of General Physiology ◽

10.1085/jgp.50.9.2289 ◽

1967 ◽

Vol 50 (9) ◽

pp. 2289-2300 ◽

Cited By ~ 29

Author(s):

G. Pilar

Keyword(s):

Neuromuscular Junctions ◽

Muscle Fibers ◽

Slow Muscle ◽

Oblique Muscle ◽

Mechanical Responses ◽

Electrical Pulses ◽

Slow Type ◽

Types Of Muscle Fibers

Electrical and mechanical responses have been obtained in situ and in vitro from the superior oblique muscle stimulated by single and repetitive electrical pulses, applied to the trochlear nerve. Two different types of muscle fibers are described, the twitch and the slow. The slow type is characterized electrically by the presence of junctional potentials, which have reversal potentials between -10 and -20 mv, and do not show propagated responses or spikes, during nerve stimulation. When the slow muscle fibers are repetitively stimulated in situ, a prolonged contraction is maintained during stimulation. At the time, the recorded electrical activity is produced locally, at the level of the neuromuscular junctions of the slow fibers. These results indicate that the contractile mechanism of the slow muscle fibers is activated locally and segmentally.

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

Journal of Histochemistry & Cytochemistry ◽

10.1177/42.10.7523488 ◽

1994 ◽

Vol 42 (10) ◽

pp. 1407-1411 ◽

Cited By ~ 1

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.

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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 ◽

10.1242/dev.116.1.41 ◽

1992 ◽

Vol 116 (1) ◽

pp. 41-53 ◽

Cited By ~ 7

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.

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Confocal Endomicroscopy of Neuromuscular Junctions Stained with Physiologically Inert Protein Fragments of Tetanus Toxin

Biomolecules ◽

10.3390/biom11101499 ◽

2021 ◽

Vol 11 (10) ◽

pp. 1499

Author(s):

Cornelia Roesl ◽

Elizabeth R. Evans ◽

Kosala N. Dissanayake ◽

Veronika Boczonadi ◽

Ross A. Jones ◽

...

Keyword(s):

Tetanus Toxin ◽

Neuromuscular Junctions ◽

Motor Nerve ◽

Full Length ◽

Nerve Terminals ◽

Motor Nerve Terminals ◽

Mouse Muscle ◽

Confocal Endomicroscopy ◽

Derivatives Of

Live imaging of neuromuscular junctions (NMJs) in situ has been constrained by the suitability of ligands for inert vital staining of motor nerve terminals. Here, we constructed several truncated derivatives of the tetanus toxin C-fragment (TetC) fused with Emerald Fluorescent Protein (emGFP). Four constructs, namely full length emGFP-TetC (emGFP-865:TetC) or truncations comprising amino acids 1066–1315 (emGFP-1066:TetC), 1093–1315 (emGFP-1093:TetC) and 1109–1315 (emGFP-1109:TetC), produced selective, high-contrast staining of motor nerve terminals in rodent or human muscle explants. Isometric tension and intracellular recordings of endplate potentials from mouse muscles indicated that neither full-length nor truncated emGFP-TetC constructs significantly impaired NMJ function or transmission. Motor nerve terminals stained with emGFP-TetC constructs were readily visualised in situ or in isolated preparations using fibre-optic confocal endomicroscopy (CEM). emGFP-TetC derivatives and CEM also visualised regenerated NMJs. Dual-waveband CEM imaging of preparations co-stained with fluorescent emGFP-TetC constructs and Alexa647-α-bungarotoxin resolved innervated from denervated NMJs in axotomized WldS mouse muscle and degenerating NMJs in transgenic SOD1G93A mouse muscle. Our findings highlight the region of the TetC fragment required for selective binding and visualisation of motor nerve terminals and show that fluorescent derivatives of TetC are suitable for in situ morphological and physiological characterisation of healthy, injured and diseased NMJs.

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Formation of Neuromuscular Junctions in Transplanted Peroneus Longus Muscles in the Rat: A Quantitative Comparison with Reinnervation of the Muscle in situ

Scandinavian Journal of Plastic and Reconstructive Surgery ◽

10.3109/02844318309007174 ◽

1983 ◽

Vol 17 (1) ◽

pp. 7-18 ◽

Cited By ~ 3

Author(s):

Sigfrid Vedung ◽

Yngve Olsson

Keyword(s):

Neuromuscular Junctions ◽

Quantitative Comparison ◽

Peroneus Longus

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

Proceedings of the Royal Society of London Series B Biological Sciences ◽

10.1098/rspb.1987.0027 ◽

1987 ◽

Vol 230 (1261) ◽

pp. 425-441 ◽

Cited By ~ 3

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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Rendez vous with Saturn's rings

International Astronomical Union Colloquium ◽

10.1017/s0252921100101885 ◽

1984 ◽

Vol 75 ◽

pp. 743-759 ◽

Cited By ~ 2

Author(s):

Kerry T. Nock

Keyword(s):

Solar Energy ◽

Electric Propulsion ◽

Power Source ◽

Propulsion System ◽

Low Thrust ◽

Ring Plane ◽

The Earth ◽

Total Impulse ◽

Saturn's Rings

ABSTRACTA mission to rendezvous with the rings of Saturn is studied with regard to science rationale and instrumentation and engineering feasibility and design. Future detailedin situexploration of the rings of Saturn will require spacecraft systems with enormous propulsive capability. NASA is currently studying the critical technologies for just such a system, called Nuclear Electric Propulsion (NEP). Electric propulsion is the only technology which can effectively provide the required total impulse for this demanding mission. Furthermore, the power source must be nuclear because the solar energy reaching Saturn is only 1% of that at the Earth. An important aspect of this mission is the ability of the low thrust propulsion system to continuously boost the spacecraft above the ring plane as it spirals in toward Saturn, thus enabling scientific measurements of ring particles from only a few kilometers.

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