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

Arbeid i forfatternes laboratorium ble støttet av Fondazione Roma og Telethon (gi nei. 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>

Eksperimentell protokollen beskrevet ovenfor gir en ideell måte å måle og diskriminerende alle funksjonelle endringer som har skjedd i muskelen eller indirekte på nevromuskulær krysset nivå. Siden denne teknikken er basert på en indirekte måling av NMJ funksjonalitet, kan det brukes til å opprette hvis noen feil knyttet til morfologiske endringer eller biokjemiske endringer. Derimot, gir den en effektiv måte å avgjøre om noen morfologiske eller biokjemiske endringer har redusert neurotransmission signalering ...

Nevromuskulær krysset (NMJ) er en kjemisk synapse dannet av forbindelsen mellom den motor endplate av muskel fiber og motor neuron axon terminal. NMJ har vist seg å spille en avgjørende rolle når kommunikasjonen mellom muskler og nerve er svekket, som forekommer i aldring eller amyotrofisk lateral sklerose (ALS). Som muskel- og kommunisere på et toveis måte1,2, kan å kunne måle NMJ feil separat fra muskel defekter gi ny innsikt i deres physiopathological samspill. Faktisk kan denne funksjonelle evalueringen hjelpe for å vurdere om morfologiske eller biokjemiske endringer redusere neurotransmission signalering funksjonalitet.
Sammenligning av muskel kontraktile respons skapte nervestimulering og responsen fra det likt muskelen fremkalt av direkte stimulering av dets membran foreslått som en indirekte måling av NMJ funksjonalitet. Faktisk siden membranen stimulering by-pass neurotransmission signalering, kan eventuelle forskjeller i de to kontraktile svarene tilskrives endringer i NMJ. Denne tilnærmingen har vært mye foreslått for rotter,3,,4,,5,,6,,7, og også brukes til å samle informasjon om musen modeller8,9,10,11,12.
Her beskrive vi i detalj en prosedyre for å avgiftsdirektoratet og teste to muskel-nerve preparater, IE soleus-ischias og membran-phrenic forberedelsene. Ved hjelp av en skreddersydd programvare, vi utviklet en kontinuerlig testing protokoll som kombinerer måling av flere parametere som preger både NMJ og muskel-funksjonalitet, og dermed gir en omfattende vurdering av NMJ skade separat fra det muskler. Spesielt måler protokollen twitch styrken, muskel kinetikk, kraft-frekvens kurve for direkte og nerve stimulations, den neurotransmission feil13 for både en avfyring og tetanic frekvenser og intratetanic trøtthet7.

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			<td>pulse stimulator (nerve)</td>
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			<td style="width:135px;">158400</td>
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			<td>573000</td>
			<td>with a home-made reference&#160;</td>
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			<td>AMD Phenom II x4 970</td>
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			<td height="21" style="height:21px;width:283px;">Krebs-Ringer Bicarbonate Buffer&#160;</td>
			<td>Sigma-Aldrich</td>
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			<td>physiological buffer</td>
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			<td height="21" style="height:21px;width:283px;">Sodium bicarbonate</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:135px;">S5761&#160;</td>
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			<td height="25" style="height:25px;width:283px;">Calcium chloride CaCl2</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:135px;">C4901</td>
			<td>anhydrous, powder, &#8805;97%</td>
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			<td height="21" style="height:21px;">Potassium dihydrogen phosphate KH&#8322;PO&#8324;</td>
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			<td>AnalaR</td>
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			<td>&#8805;99.5% (titration)</td>
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			<td>for aquarium</td>
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measuring neuromuscular junction functionality

Nevromuskulær krysset (NMJ) funksjonaliteten spiller en avgjørende rolle når studere sykdommer der kommunikasjonen mellom motor neuron og muskel er svekket, som aldring og amyotrofisk lateral sklerose (ALS). Her beskriver vi en eksperimentell protokoll som kan brukes til å måle NMJ funksjonalitet ved å kombinere to typer elektrisk stimulering: direkte membran muskelstimulasjon og stimulering gjennom nerve. Sammenligning av muskel respons på disse to forskjellige stimulations kan bidra til å definere, på funksjonell nivå, mulige endringer i NMJ som fører til funksjonelle nedgang i muskel.
Ex vivo forberedelser er velegnet for godt kontrollerte studier. Her beskriver vi en intensiv protokoll for å måle flere parametere av muskel- og NMJ funksjonalitet for soleus-sciatic nerve utarbeidelse og membran-phrenic nerve forberedelse. Protokollen varer ca 60 min og utføres uavbrutt gjennom et skreddersydd programvare som måler egenskapene twitch kinetikk, kraft-frekvens forholdet for både muskel- og stimulations og to parametere gjelder for NMJ funksjonaliteten, i.e. neurotransmission svikt og intratetanic trøtthet. Denne metoden ble brukt til å oppdage skader i soleus og membran muskel-nerve forberedelser ved hjelp SOD1G93A transgene musen, en eksperimentell modell av ALS som overalt overexpresses mutant antioksidant enzymet superoxide dismutase 1 (SOD1).

Protokollen vi beskrev gir informasjon om funksjonelle denervation i flere neuromuscular sykdommen eller aldring-sarcopenia. Denne protokollen kan brukes til å fastslå om (og hvis så, på hvilket nivå) muskel endringer leies selektiv endringer som skjer i muskel selv eller nevromuskulære overføring. Dataene nedenfor er resultatet av et tidligere verk av vår gruppe18, gjennomført på SOD1G93A transgene musemodell av amyotrofisk lateral sklerose p?...

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

Measuring Neuromuscular Junction Functionality

En funksjonell vurdering av nevromuskulær krysset (NMJ) kan gi viktig informasjon om kommunikasjonen mellom muskler og nerve. Her beskriver vi en protokoll for grundig vurdere både NMJ og muskel funksjonaliteten med to forskjellige muskel-nerve preparater, i.e. soleus-ischias og membran-phrenic.

Måle nevromuskulær Junction funksjonalitet

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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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

Sekvensiell Foto bleking å avgrense Enkelt Schwann celler på nevromuskulær Junction

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

Disseksjon av Transversus Abdominis Muscle for hel-mount Neuromuscular 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 av<em&gt; In Situ</em&gt; Protein-protein komplekser på<em&gt; Drosophila</em&gt; Larve Nevromuskulær Junction hjelp Proximity Ligation analysen

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

Nevromuskulære krysset: Måling Synapse Størrelse, fragmentering og endringer i Synaptic Protein Tetthet Bruke Confocal Fluorescensmikroskopi

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 Kvantifisering Matters: Karakterisering av fenotyper på<em&gt; Drosophila</em&gt; Larve Nevromuskulær 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

Laster et kalsiumfargestoff inn i froskens nerveender gjennom nervestubben: Kalsiumtransientregistrering 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 høy gjennomstrømming og Multi-parametrisk Kvantifisering av<em&gt; Drosophila</em&gt; Neuromuskulære 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

Fokal Macropatch opptak av Synaptic strøm fra Drosophila larver nevromuskulær krysset

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 av Rat Vocal Fold for nevromuskulære analyser

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