Name: measuring neuromuscular junction functionality
Uploaded: 2017-08-06T13:00:00
Description: Watch this Scientific Journal Video about Measuring Neuromuscular Junction Functionality at JoVE.com

Arbejde i forfatternes laboratorium blev støttet af Fondazione Roma og Telethon (give nr. GGP14066).

All the animal experiments were approved by the ethics committee of Sapienza University of Rome-Unit of Histology and Medical Embryology and were performed in accordance with the current version of the Italian Law on the Protection of Animals.
1. Experimental set-up
<ol>
	<li>Set-up the experimental system composed of 1 actuator/transducer, 2 stimulators, 1 in-vitro muscle apparatus, 1 preparatory tissue bath, 1 suction electrode, 1 digital oscilloscope, 1 stereomicroscope, 1 cold l...

<ol>
	<li>Dadon-Nachum, M., Melamed, E., Offen, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+&amp;#34;dying-back&amp;#34;+phenomenon+of+motor+neurons+in+ALS.">The &amp;#34;dying-back&amp;#34; phenomenon of motor neurons in ALS.</a> J Mol Neurosci. 43 (3), 470-477 (2011).</li><li>Dobrowolny, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+Muscle+Is+a+Primary+Target+of+SOD1G93A-Mediated+Toxicity.">Skeletal Muscle Is a Primary Target of SOD1G93A-Mediated Toxicity.</a> Cell Metab. 8 (5), 425-436 (2008).</li><li>Mantilla, C. B., Zhan, W. Z., Sieck, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotrophins+improve+neuromuscular+transmission+in+the+adult+rat+diaphragm.">Neurotrophins improve neuromuscular transmission in the adult rat diaphragm.</a> Muscle Nerve. 29 (3), 381-386 (2004).</li><li>Prakash, Y. S., Miyata, H., Zhan, W. Z., Sieck, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivity-induced+remodeling+of+neuromuscular+junctions+in+rat+diaphragmatic+muscle.">Inactivity-induced remodeling of neuromuscular junctions in rat diaphragmatic muscle.</a> Muscle Nerve. 22 (3), 307-319 (1999).</li><li>Sieck, D. C., Zhan, W. Z., Fang, Y. H., Ermilov, L. G., Sieck, G. C., Mantilla, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-activity+relationships+in+rodent+diaphragm+muscle+fibers+vs.+neuromuscular+junctions.">Structure-activity relationships in rodent diaphragm muscle fibers vs. neuromuscular junctions.</a> Respir Physiol Neurobiol. 180 (1), 88-96 (2012).</li><li>Van Lunteren, E., Moyer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+DAP+on+diaphragm+force+and+fatigue,+including+fatigue+due+to+neurotransmission+failure.">Effects of DAP on diaphragm force and fatigue, including fatigue due to neurotransmission failure.</a> J Appl Physiol. 81 (5), 2214-2220 (1996).</li><li>Van Lunteren, E., Moyer, M., Kaminski, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adverse+effects+of+myasthenia+gravis+on+rat+phrenic+diaphragm+contractile+performance.">Adverse effects of myasthenia gravis on rat phrenic diaphragm contractile performance.</a> J Appl Physiol. 97 (3), 895-901 (2004).</li><li>Lee, Y., Mikesh, M., Smith, I., Rimer, M., Thompson, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscles+in+a+mouse+model+of+spinal+muscular+atrophy+show+profound+defects+in+neuromuscular+development+even+in+the+absence+of+failure+in+neuromuscular+transmission+or+loss+of+motor+neurons.">Muscles in a mouse model of spinal muscular atrophy show profound defects in neuromuscular development even in the absence of failure in neuromuscular transmission or loss of motor neurons.</a> Dev Biol. 356 (2), 432-444 (2011).</li><li>Personius, K. E., Sawyer, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variability+and+failure+of+neurotransmission+in+the+diaphragm+of+mdx+mice.">Variability and failure of neurotransmission in the diaphragm of mdx mice.</a> Neuromuscular Disord. 16 (3), 168-177 (2006).</li><li>R&#246;der, I. V., Petersen, Y., Choi, K. R., Witzemann, V., Hammer, J. A., Rudolf, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+myosin+Va+in+the+plasticity+of+the+vertebrate+neuromuscular+junction+in+vivo.">Role of myosin Va in the plasticity of the vertebrate neuromuscular junction in vivo.</a> PLoS ONE. 3 (12), e3871 (2008).</li><li>Chevessier, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+for+congenital+myasthenic+syndrome+due+to+MuSK+mutations+reveals+defects+in+structure+and+function+of+neuromuscular+junctions.">A mouse model for congenital myasthenic syndrome due to MuSK mutations reveals defects in structure and function of neuromuscular junctions.</a> Hum Mol Genet. 17 (22), 3577-3595 (2008).</li><li>Farchi, N., Soreq, H., Hochner, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+acetylcholinesterase+overexpression+induces+multilevelled+aberrations+in+mouse+neuromuscular+physiology.">Chronic acetylcholinesterase overexpression induces multilevelled aberrations in mouse neuromuscular physiology.</a> J Physiol. 546 (1), 165-173 (2002).</li><li>Kuei, J. H., Shadmehr, R., Sieck, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relative+contribution+of+neurotransmission+failure+to+diaphragm+fatigue.">Relative contribution of neurotransmission failure to diaphragm fatigue.</a> J Appl Physiol (Bethesda, Md: 1985). 68 (1), 174-180 (1990).</li><li>Del Prete, Z., Musar&#242;, A., Rizzuto, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+mechanical+properties,+including+isotonic+fatigue,+of+fast+and+slow+MLC/mIgf-1+transgenic+skeletal+muscle.">Measuring mechanical properties, including isotonic fatigue, of fast and slow MLC/mIgf-1 transgenic skeletal muscle.</a> Ann Biomed Eng. 36 (7), 1281-1290 (2008).</li><li>Lynch, G. S., Hinkle, R. T., Chamberlain, J. S., Brooks, S. V., Faulkner, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force+and+power+output+of+fast+and+slow+skeletal+muscles+from+mdx+mice+6-28+months+old.">Force and power output of fast and slow skeletal muscles from mdx mice 6-28 months old.</a> J Physiol. 535 (2), 591-600 (2001).</li><li>Brooks, S. V., Faulkner, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contractile+properties+of+skeletal+muscles+from+young,+adult+and+aged+mice.">Contractile properties of skeletal muscles from young, adult and aged mice.</a> J Physiol. 404, 71-82 (1988).</li><li>Lynch, G. S., Hinkle, R. T., Faulkner, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force+and+power+output+of+diaphragm+muscle+strips+from+mdx+and+control+mice+after+clenbuterol+treatment.">Force and power output of diaphragm muscle strips from mdx and control mice after clenbuterol treatment.</a> Neuromuscul Disord. 11 (2), 192-196 (2001).</li><li>Rizzuto, E., Pisu, S., Musar, A., Del Prete, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+Neuromuscular+Junction+Functionality+in+the+SOD1+G93A+Animal+Model+of+Amyotrophic+Lateral+Sclerosis.">Measuring Neuromuscular Junction Functionality in the SOD1 G93A Animal Model of Amyotrophic Lateral Sclerosis.</a> Ann Biomed Eng . 43, (2015).</li><li>Soliani, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manuale di statistica per la ricerca e la professione statistica univariata e bivariata parametrica e non-parametrica per le discipline ambientali e biologiche. , (2004).</li><li>Gurney, M. E., Pu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+neuron+degeneration+in+mice+that+express+a+human+Cu,Zn+superoxide+dismutase+mutation.">Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation.</a> Science (New York, N.Y). 264 (5166), 1772-1775 (1994).</li></ol>

Forsøgsplan beskrevet ovenfor giver en ideel måde at måle og diskriminere nogen funktionelle ændringer, der er opstået direkte i musklen eller indirekte neuromuskulære junction plan. Da denne teknik er baseret på en indirekte måling af NMJ funktionalitet, kan det bruges til at etablere hvis nogen defekt er relateret til morfologiske ændringer eller biokemiske ændringer. Derimod giver det en effektiv metode til at afgøre, om morfologiske eller biokemiske ændringer har reduceret neurotransmission signalering fu...

Den neuromuskulære junction (NMJ) er en kemisk synapse dannet af forbindelsen mellem den motor endplate af muskelfiber og motoriske neuron axon terminal. NMJ har vist sig at spille en afgørende rolle, når kommunikationen mellem muskel og nerve er nedsat, som opstår i aldring eller Amyotrofisk lateral sklerose (ALS). Som muskel og nerve kommunikere i en tovejs måde1,2, kan være i stand til at måle NMJ defekter adskilt fra muskel defekter give ny indsigt i deres physiopathological samspil. Ja, denne funktionelle evaluering kan bidrage til at vurdere, om morfologiske eller biokemiske ændringer reducere neurotransmission signalering funktionalitet.
Sammenligning af muskel kontraktile reaktion fremkaldt af nervestimulation og svar af den samme muskel fremkaldt af direkte stimulering af sin membran er blevet foreslået som en indirekte måling af NMJ funktionalitet. Faktisk, siden membran stimulation af passerer neurotransmission signalering, eventuelle forskelle i de to kontraktile svar kan tilskrives ændringer i NMJ. Denne tilgang har været flittigt foreslået for rotter3,4,5,6,7, og også brugt til at indsamle oplysninger om mus modeller8,9,10,11,12.
Her, beskriver vi i detaljer en procedure for punktafgifter og teste to muskel-nerve præparater, i. e. soleus-ischiadicus og membran-phrenic forberedelserne. Ved hjælp af en skræddersyet software, vi designet en kontinuerlig prøvningsprotokol, der kombinerer måling af flere parametre, der karakteriserer både NMJ og muskel funktioner, derved giver en omfattende vurdering af NMJ skader separat fra de muskel. Navnlig måler protokollen spjæt kraft, muskel kinetik, kraft-frekvens kurve for direkte og nerve stimulering, neurotransmission fiasko13 for både en fyring og de tetanic frekvenser og intratetanic træthed7.

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			<td>pulse stimulator (nerve)</td>
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			<td>pulse stimulator (muscle)</td>
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			<td style="width:135px;">158400</td>
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			<td style="width:161px;">Sigma-Aldrich</td>
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			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:135px;">C4901</td>
			<td>anhydrous, powder, &#8805;97%</td>
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measuring neuromuscular junction functionality

Neuromuskulære junction (NMJ) funktionalitet spiller en central rolle, når man studerer sygdomme, hvor kommunikationen mellem motorneuron og muskel er svækket, såsom aldring og Amyotrofisk lateral sklerose (ALS). Her beskriver vi en forsøgsplan, der kan bruges til at måle NMJ funktionalitet ved at kombinere to typer af elektrisk stimulation: direkte membran muskelstimulation og stimulering gennem nerve. Sammenligning af muskel svar på disse to forskellige stimulering kan bidrage til at definere, på funktionsniveau, potentielle ændringer i NMJ, der fører til funktionelle nedgang i muskelmasse.
Ex vivo præparater er velegnede til velkontrollerede undersøgelser. Her beskriver vi en intensiv protokol for at måle flere parametre af muskel og NMJ funktionalitet for soleus-ischiadicus nerve forberedelse og membran-phrenic nerve forberedelse. Protokollen varer ca. 60 min. og foregår uafbrudt ved hjælp af en skræddersyet software, der måler spjæt kinetik egenskaber, kraft-frekvens-forholdet for både muskel og nerve stimulering og to parametre specifikt for NMJ funktionalitet, i.e. neurotransmission fiasko og intratetanic træthed. Denne metode blev brugt til at opdage skader i soleus og mellemgulv muskel-nerve præparater ved hjælp af SOD1G93A Transgene mus, en eksperimentel model af ALS, allestedsnærværende overexpresses mutant antioxidant enzymet superoxiddismutase 1 (SOD1).

Protokollen beskrev vi giver oplysninger om funktionelle denervering i flere neuromuskulære sygdomme eller aldring-sarcopenia. Denne protokol kan bruges til at afgøre, om (og, i bekræftende fald, på hvilket niveau) muskel ændringer skyldes selektiv forandringer, der sker i musklen selv eller i neuromuskulære transmission. De data, der er vist nedenfor er resultaterne af et tidligere værk af vores gruppe18, udført på SOD1G93A transgene musemodel ...

Watch this Scientific Journal Video about Measuring Neuromuscular Junction Functionality at JoVE.com

Measuring Neuromuscular Junction Functionality

En funktionel vurdering af den neuromuskulære junction (NMJ) kan give vigtige oplysninger om kommunikation mellem muskel og nerve. Her beskriver vi en protokol for at omfattende evaluere både NMJ og muskel funktionalitet ved hjælp af to forskellige muskel-nerve præparater, dvs soleus-ischiadicus og membran-phrenic.

Måling af neuromuskulære Junction funktionalitet

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is ...

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Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The Drosophila  larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila  is well known for ...

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

Sekventiel fotoblegende at afgrænse Single Schwann-celler ved den neuromuskulære overgang

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

Dissektion af Transversus Abdominis Muscle for Hele mount Neuromuskulære Junction Analyse

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat ...

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Påvisning af<em&gt; In Situ</em&gt; Protein-protein-komplekser på<em&gt; Drosophila</em&gt; Larve Neuromuskulær Junction Brug Proximity ligeringsassayet

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval ...

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

Den neuromuskulære forbindelse: Måling Synapse Størrelse, fragmentering og ændringer i Synaptic Protein Density Brug Konfokal Fluorescens mikroskopi

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Hvorfor Kvantificering Matters: Karakterisering af fænotyper på<em&gt; Drosophila</em&gt; Larve Neuromuskulær Junction

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic ...

Loading a Calcium Dye into Frog Nerve Endings Through the Nerve Stump: Calcium Transient Registration in the Frog Neuromuscular Junction

Indlæsning af et calciumfarvestof i frøens nerveender gennem nervestumpen: Kalktransient registrering i Frog Neuromuscular Junction

One of the most feasible methods of measuring presynaptic calcium levels in presynaptic nerve terminals is optical recording. It is based on using ...

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

To Algoritmer for High-throughput og Multi-parametrisk Kvantificering af<em&gt; Drosophila</em&gt; Neuromuskulære forbindelse Morfologi

Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The ...

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Focal Macropatch optagelser af Synaptic strømninger fra Drosophila larve neuromuskulære Junction

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...

Preparation of the Rat Vocal Fold for Neuromuscular Analyses

Forberedelse af Rat Vocal Fold til neuromuskulære analyser

The purpose of this tutorial is to describe the preparation of the rat vocal fold for histochemical neuromuscular study. This protocol outlines procedures ...