Name: levator auris longus preparation for examination of mammalian neuromuscular transmission under voltage clamp conditions
Uploaded: 2018-05-05T14:00:00
Description: Watch this Scientific Journal Video about Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions at JoVE.com

Vi takker Dr. Mark M. Rich og Daniel Miranda for redaksjonelle kommentarer, Ahmad Khedraki for å hjelpe etablere denne teknikken, og Wright State University for økonomisk støtte (oppstart fondet til A.A.V.).

Alle dyr prosedyrer ble utført i henhold til Animal Care og bruk komiteen av Wright State University.
1. musen Eutanasi
<ol><li>Plass musen i en lufttett glass anesthetizing kammer i avtrekksvifte.</li>
	<li>Utsett musen via innånding til en dødelig dose isoflurane (Mette, eller ~ 25%). Drar musen i kammeret til ikke puste kan observeres.</li>
	<li>Fjern musen fra kammeret og utføre en cervical forvridning som sekundær av euthanasia.</li>
</ol>2. fjerning av hår f...

<ol>
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L., Gomez-Sanchez, L., Tomas-Zapico, C., Lucas, J. J., Fernandez-Chacon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+neurotransmitter+release+at+the+neuromuscular+junction+in+a+mouse+model+of+polyglutamine+disease.">Increased neurotransmitter release at the neuromuscular junction in a mouse model of polyglutamine disease.</a> J Neurosci. 31 (3), 1106-1113 (2011).</li><li>Takeuchi, A., Takeuchi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Further+analysis+of+relationship+between+end-plate+potential+and+end-plate+current.">Further analysis of relationship between end-plate potential and end-plate current.</a> J Neurophysiol. 23, 397-402 (1960).</li><li>McLachlan, E. M., Martin, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non+linear+summation+of+end+plate+potentials+in+the+frog+and+mouse.">Non linear summation of end plate potentials in the frog and mouse.</a> The Journal of Physiology. 311 (1), 307-324 (1981).</li><li>Obis, T., 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=The+novel+protein+kinase+C+epsilon+isoform+modulates+acetylcholine+release+in+the+rat+neuromuscular+junction.">The novel protein kinase C epsilon isoform modulates acetylcholine release in the rat neuromuscular junction.</a> Mol Brain. 8 (1), 80 (2015).</li><li>Silveira, P. E., 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=Ryanodine+and+inositol+triphosphate+receptors+modulate+facilitation+and+tetanic+depression+at+the+frog+neuromuscular+junction.">Ryanodine and inositol triphosphate receptors modulate facilitation and tetanic depression at the frog neuromuscular junction.</a> Muscle Nerve. 52 (4), 623-630 (2015).</li><li>Wood, S. J., Slater, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+factor+at+the+neuromuscular+junction.">Safety factor at the neuromuscular junction.</a> Prog Neurobiol. 64 (4), 393-429 (2001).</li><li>Miranda, D. R., 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=Progressive+Cl-+channel+defects+reveal+disrupted+skeletal+muscle+maturation+in+R6/2+Huntington's+mice.">Progressive Cl- channel defects reveal disrupted skeletal muscle maturation in R6/2 Huntington's mice.</a> J Gen Physiol. 149 (1), 55-74 (2017).</li><li>Waters, C. W., Varuzhanyan, G., Talmadge, R. J., Voss, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Huntington+disease+skeletal+muscle+is+hyperexcitable+owing+to+chloride+and+potassium+channel+dysfunction.">Huntington disease skeletal muscle is hyperexcitable owing to chloride and potassium channel dysfunction.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (22), 9160-9165 (2013).</li><li>Khedraki, A., 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=Depressed+Synaptic+Transmission+and+Reduced+Vesicle+Release+Sites+in+Huntington's+Disease+Neuromuscular+Junctions.">Depressed Synaptic Transmission and Reduced Vesicle Release Sites in Huntington's Disease Neuromuscular Junctions.</a> Journal of Neuroscience. 37 (34), 8077-8091 (2017).</li><li>Greene, E. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+ATP+inhibits+chloride+channels+in+mature+mammalian+skeletal+muscle+by+activating+P2Y(1)+receptors.">Extracellular ATP inhibits chloride channels in mature mammalian skeletal muscle by activating P2Y(1) receptors.</a> Journal of Physiology-London. 587 (23), 5739-5752 (2009).</li><li>Albuquerque, E. X., McIsaac, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+and+slow+mammalian+muscles+after+denervation.">Fast and slow mammalian muscles after denervation.</a> Experimental Neurology. 26 (1), 183-202 (1970).</li><li>Santafe, M. M., Urbano, F. J., Lanuza, M. A., Uchitel, O. 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Beskrevet her er forberedelse og bruk av musen LAL muskel for måling av nevromuskulær overføring under gjeldende - eller spenning-klemme forhold. Det er flere viktige punkter å vurdere for dissekere ut av LAL. Rengjøring overflødig bindevev muskel AIDS i elektrode impalement, som elektrodene kan ulempe bindevevet når posisjonering dem for impalement. Imidlertid bare fjerne bindevev som kan tas bort lett å begrense sjansene for skade muskelen. Isolasjon av nerve bør utføres med forsiktighet, fordi det er veldig ...

Studere synaptic overføring i nevromuskulær krysset gir innsikt i dynamiske forholdet mellom nervøs og skjelettlidelser muskel-systemer og er en utmerket modell for å undersøke synaptic fysiologi. Levator auris longus (LAL) er en tynn muskel, slik at de nevromuskulær Forbindelsesstykke å være lett å visualisere. Tidligere rapporter har beskrevet bruke LAL undersøke synaptic narkotika og giftstoffer og har preget de skjelettlidelser muskel fiber egenskapene av LAL1,2. Tallrike studier har brukt LAL undersøke nevromuskulær fysiologi3,4,5,6,7,8. For elektrofysiologi, muligheten til å observere LAL nevromuskulær veikryss gir nøyaktig plassering av microelectrodes på motor endplate og reduserer klemme plassproblemer i innspillingen synaptic overføring. Gjeldende-klemme opptak av muskel membran egenskapene som membran tidskonstant (τm) og inngang motstand (Ri) er lett oppnådd. Disse egenskapene kan videre måles fra samme muskelfibre brukes til å registrere nevromuskulær overføring, slik at for en direkte sammenligning av synaptiske funksjon i muskel membran egenskapene. Analyse av disse dataene gir innblikk i de fysiske mekanismene av mange neuromuscular sykdommen og USA endret aktivitet.
Et viktig aspekt av teknikken beskrevet her er bruk av spenning-klemme for synaptic innspillinger, som er ikke underlagt de ikke-lineære funksjonene i gjeldende-klemme og er uavhengig av egenskapene muskel membran. Fordeler ved spenning-klemme i motsetning til gjeldende-klemme undersøke nevromuskulær overføring ble etablert av banebrytende innsats i 1950-tallet9. Under gjeldende-klemme er EPPs som overskrider 10-15 mV i amplitude ikke en lineær produkt av mEPP amplitude9. For eksempel, hvis den gjennomsnittlige mEPP 1 mV, en EPP 5 mV kan antas for å være av 5 mEPPs (QC 5); mens en EPP 40 mV blir produktet av mer enn 40 mEPPs. Denne ikke-linearitet på større EPPs oppstår fordi drivkraft for EPP, som er forskjellen mellom membran potensialet og likevekt potensial for acetylcholin reseptor (~ -10 mV), vesentlig synker under store EPPs. Dette problemet unngås spenning-klemme eksperimenter fordi muskel membran potensialet ikke endres under spenning-klemme eksperimenter. En ulempe er at spenningen-klemme eksperimenter er teknisk vanskelig å fullføre enn gjeldende-klemme opptak. Med dette i bakhodet utviklet McLachlan og Martin en enkel matematisk korreksjon som står for ikke-lineære funksjonene i gjeldende-klemme innspillinger av EPPs10. Rettelsene fungerer bra11,12,13, men viktigere, antar at muskel membran egenskapene ikke er avbrutt.
Muskel membran egenskapene er spesielt viktig å vurdere om studere forhold eller sykdom stater som forstyrrer muskelen. For eksempel er Skjelettmuskel fra R6/2 transgene modellen av Huntingtons sykdom hyperexcitable på grunn av en gradvis reduksjon i hvile klorid og kalium strøm14,15. Som en konsekvens, er mEPPs og EPPs forsterket i R6/2 skjelettmuskelen. Flere faktorer kan sikkert, endre mEPPs og EPPs. Arbeide med en annen modell av Huntingtons sykdom mus (R6/1) fant endringer i EPPs som syntes å være relatert til SNARE-proteiner8. For å vurdere mekanismer forårsaker endret nevromuskulær overføring, ville det være gunstig å eliminere effektene av endrede muskel membran egenskaper ved hjelp av en spenning-klemme. I en fersk studie, ble R6/2 nevromuskulær overføringen studert under både gjeldende - og spenning-klemme forhold ved hjelp av teknikken beskrevet heri. Helheten av motor endplates var spenning-festet med mindre enn 1% feilen ved å plassere to microelectrodes i lengde konstant endplate16. Det ble vist at spenningen-klemme og korrigert gjeldende-klemme poster gitt kontrasterende målinger av nevromuskulær overføring i R6/2 muskel. Dette understreker at det kan være vanskelig å korrigere EPPs for ikke-lineære funksjonene Hvis egenskapene muskel membran har endret og viser fordelene ved å få spenning-klemme poster som er uavhengige av muskel membran egenskapene. Protokollen presenteres her er ideelt for å undersøke forhold eller sykdom stater som påvirker synaptic overføring og egenskapene postsynaptic membran.

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			<td height="21" style="height:21px;width:216px;">40x Objective</td>
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			<td height="44" style="height:44px;width:216px;">Right-handed Micromanipulator</td>
			<td style="width:183px;">Siskiyou Corp.</td>
			<td style="width:119px;">MX1641/45DR</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">Single Motion Controler</td>
			<td style="width:183px;">Siskiyou Corp.</td>
			<td style="width:119px;">MC100e</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="161">
			<td height="161" style="height:161px;width:216px;">Crossed Roller Micromanipulator</td>
			<td style="width:183px;">Siskiyou Corp.</td>
			<td style="width:119px;">MX1641R</td>
			<td style="width:288px;">This was added to the Z-axis of the Left and Right-handed micromanipulators to allow the z axis to be motorized. This custom set-up is cheaper and less bulky than buying a 4-axis motorized micromanipulator. It also allows us to control both micromanipulators with one controller</td>
		</tr>
		<tr height="21">
			<td colspan="3" height="21" style="height:21px;width:517px;">All chemicals were orded from Fisher except,</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">BTS</td>
			<td style="width:183px;">Toronto Research Chemicals</td>
			<td style="width:119px;">B315190</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CTX</td>
			<td style="width:183px;">Alomone Labs</td>
			<td style="width:119px;">C-270</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">4-Di-2-Asp</td>
			<td style="width:183px;">Molecular Probes</td>
			<td style="width:119px;"></td>
			<td style="width:288px;">Molecular probes is no longer a company. Now ordered through Fisher</td>
		</tr>
	</tbody>
</table>

levator auris longus preparation for examination of mammalian neuromuscular transmission under voltage clamp conditions

Denne protokollen beskriver en teknikk posten synaptic overføring fra nevromuskulær krysset under gjeldende-klemme og spenning-klemme forhold. En ex vivo utarbeidelse av levator auris longus (LAL) brukes fordi det er en tynn muskel som gir enkel visualisering av nevromuskulær krysset microelectrode impalement på motor endplate. Denne metoden gjør opptak av spontane miniatyr endplate potensialer og strøm (mEPPs og mEPCs), nerve-utløste endplate potensialer og strøm (EPPs og EPCs), samt egenskapene membran motor endplate. Resultatene fra denne metoden inkluderer det quantal innholdet (QC), antall vesicle release nettsteder (n), sannsynligheten for vesicle utgivelsen (prel), synaptic tilrettelegging og depresjon, samt muskel membran tiden konstant (τ m) og input motstand. Bruk av denne teknikken til musen modeller for menneskelig sykdom kan markere viktige patologi i sykdom stater og identifisere romanen behandling strategier. Fullt spenning-klemanordning en enkelt synapse gir denne metoden en av de mest detaljerte analysene av synaptic overføring tilgjengelig.

Figur 8 viser et eksempel på de nåværende pulser (figur 8A) og spenning svar (figur 8B) fra en LAL fiber under gjeldende-klemme fra en 12-uke-gamle wild type R6/2-mus. Tilstedeværelsen av mEPPs indikerer at disse postene ble tatt fra motor endplate. Postene ble innhentet i normal fysiologisk saltløsning. Disse gjeldende-klemme poster kan bli analysert for å fastslå Ri og τm</s...

Watch this Scientific Journal Video about Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions at JoVE.com

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

Levator Auris Longus Forberedelse for undersøkelse av pattedyr nevromuskulær overføring Under spenning klemme forhold

Protokollen beskrevet i dette dokumentet bruker musen levator auris longus (LAL) muskler registrere spontan og nerve-utløste postsynaptic potensialer (gjeldende-klemme) og strøm (spenning-klemme) i nevromuskulær krysset. Bruk av denne teknikken kan gi viktig innsikt i mekanismer av synaptic overføring under normal og sykdom.

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ...

Research

JoVE Journal

Neuroscience

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

Subcutaneous Administration of Muscarinic Antagonists and Triple-Immunostaining of the Levator Auris Longus Muscle in Mice

Hind limb muscles of rodents, such as gastrocnemius and tibialis anterior, are frequently used for in vivo  pharmacological studies of the signals ...

Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is ...

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

<sub&gt; 1</sub&gt; F<sub&gt; O</sub&gt; ATPase Vesikkel Forberedelse og teknikk for utøvende Patch Clamp Innspillinger av Submitochondrial vesikkelmikrosomer Membraner

Mitochondria are involved in many important cellular  functions including metabolism, survival1, development and, calcium signaling2. Two of the most ...

Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina

Patch Clamp Opptak av Starburst Amacrine celler i en Flat-mount Utarbeidelse av Deafferentated Mouse Retina

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic ...

Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions

Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions

Overvåking astrocytter reaktivitet og spredning in Vitro Under iskemiske-lignende forhold

Ischemic stroke is a complex brain injury caused by a thrombus or embolus obstructing blood flow to parts of the brain. This leads to deprivation of ...

Measuring Neuromuscular Junction Functionality

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 ...

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Elektrofysiologisk metode for helcelle spenningsklemmeopptak fra<em&gt; Drosophila</em&gt; Fotoreseptorer

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, ...

Slice Patch Clamp Technique for Analyzing Learning-Induced Plasticity

Skive Patch klemme teknikk for å analysere læring-indusert plastisitet

The slice patch clamp technique is a powerful tool for investigating learning-induced neural plasticity in specific brain regions. To analyze ...

Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons

Utarbeidelse av akutt ryggmargen skiver for hele celle Patch-klemme opptak i Substantia Gelatinosa nerveceller

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms ...