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 redaktionelle kommentarer, Ahmad Khedraki for støtte til oprettelse af denne teknik, og Wright State University for finansiel støtte (Start fond til A.A.V.).

Alle dyr procedurer blev udført i overensstemmelse med Animal Care og brug Udvalget af Wright State University.
1. Mouse dødshjælp
<ol><li>Placere musen i et lufttæt glas bedøve kammer i et stinkskab.</li>
	<li>Afsløre musen via indånding til en dødelig dosis af isofluran (mætte, eller ~ 25%). Bevæger musen i salen, indtil ingen vejrtrækning kan observeres.</li>
	<li>Fjerne musen fra salen og udføre en cervikal dislokation som en sekundær metode af aktiv dødshjælp.</li>
</ol><...

<ol>
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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. 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Beskrevet her er forberedelse og brug af musen LAL muskel til måling af neuromuskulære transmission strøm eller spænding klemme betingelser. Der er flere vigtige punkter at overveje for dissekere ud LAL. Rengøring overskydende bindevæv fra muskler aids i elektrode Spidning på pæl, som elektroderne kan snag bindevæv, når positionering dem til Spidning på pæl. Dog kun fjerne bindevæv, der kan blive taget væk nemt at begrænse chancer for at beskadige musklen. Isolation af nerve bør udføres med omhu, fordi d...

Studerer synaptisk transmission på den neuromuskulære junction giver et indblik i det dynamiske forholdet mellem de nervøse og skelet muskuløs systemer og er en fremragende model for behandlingen af synaptic fysiologi. Levator auris longus (LAL) er en tynd muskel, giver mulighed for de neuromuskulære knudepunkter til visualiseres nemt. Tidligere rapporter har beskrevet bekvemmeligheden for at bruge LAL at undersøge synaptic stoffer og toksiner og har karakteriseret skelet muskel fiber type Karakteristik af LAL1,2. Talrige undersøgelser har brugt LAL for at undersøge neuromuskulære fysiologi3,4,5,6,7,8. Elektrofysiologi, mulighed for at nemt observere LAL neuromuskulære knudepunkter giver mulighed for den nøjagtige placering af microelectrodes på de motoriske endplate og reducerer klemme rumspørgsmål i optagelse synaptisk transmission. Nuværende-clamp optagelser af egenskaberne muskel membran, membran tid konstant (τm) og input modstand (Ri) er let opnås. Desuden kan disse egenskaber måles fra de samme muskel fibre bruges til at registrere neuromuskulære transmission, giver mulighed for en direkte sammenligning af synaptiske funktion til muskel membran egenskaber. Analyse af disse data kan give indsigt i de fysiske mekanismer af mange neuromuskulære sygdomme og tilstande af ændret aktivitet.
Et centralt aspekt af den teknik beskrevet her er brugen af spænding-klemme til synaptic optagelser, som ikke er underlagt de ikke-linearities opstod i strøm-clamp og er uafhængige af muskel membran egenskaber. Fordele ved at bruge spænding-klemme i modsætning til aktuelle-klemme til at undersøge neuromuskulære transmission blev etableret af banebrydende indsats i 1950s9. Under aktuelle-clamp er EPPs, der overstiger 10-15 mV i amplitude ikke en lineær produkt af Mellemøsten amplitude9. For eksempel, hvis den gennemsnitlige Mellemøsten er 1 mV, en EPP 5 mV kan antages for at være produktet af 5 mEPPs (QC 5); der henviser til, et EPP 40 mV vil være et produkt af mere end 40 mEPPs. Denne ikke-linearitet på større EPPs opstår fordi drivkraft for PPE-gruppen, som er forskellen mellem membran potentiale og ligevægt potentiale for acetylcholin receptor (~ -10 mV), væsentligt falder under store EPPs. Dette problem undgås i spænding-clamp eksperimenter, fordi muskel membran potentiale ikke ændrer under spænding-clamp eksperimenter. En ulempe er, at spændingen-clamp eksperimenter er teknisk sværere at gennemføre end nuværende-clamp optagelse. Med dette i tankerne udviklet McLachlan og Martin en ligetil matematiske korrektion, der tegner sig for ikke-linearities i nuværende-clamp optagelser af EPPs10. Korrektionerne, der fungerer godt11,12,13, men vigtigere, antage, at muskel membran egenskaber ikke er blevet afbrudt.
Muskel membran egenskaber er især vigtigt at overveje, om at studere betingelser eller sygdomstilstande, der forstyrrer musklen. Skeletmuskulatur fra R6/2 transgene model af Huntington's chorea er f.eks hyperexcitable på grund af en progressiv reduktion i hvilepuls chloride og kalium strømme14,15. Som en konsekvens, forstærkes mEPPs og EPPs i R6/2 skeletmuskulatur. Bestemt, yderligere faktorer kan ændre mEPPs og EPPs. Arbejde med en anden model af Huntington's chorea-mus (R6/1) fandt ændringer i EPPs, der syntes at være relateret til SNARE-proteiner8. For at vurdere de mekanismer, der forårsager ændrede neuromuskulære transmission, ville det være gavnligt at fjerne virkningerne af ændrede muskel membran egenskaber ved hjælp af en spænding-klemme. I en nylig undersøgelse, var R6/2 neuromuskulære transmission studerede på både strøm og spænding klemme betingelser ved hjælp af den teknik beskrevet heri. Helhed af den motoriske endplates var spænding-fastspændt med mindre end 1% fejl ved at placere to microelectrodes inden for konstanten længde af endplate16. Det var vist spænding-clamp og korrigeret aktuelle-clamp poster givet kontrasterende målinger af neuromuskulære transmission i R6/2 muskel. Dette understreger, at det kan være svært at rette EPPs for ikke-linearities, hvis muskel membran egenskaber er blevet ændret og viser fordelene ved at opnå spænding-clamp poster, der er uafhængige af muskel membran egenskaber. Protokollen præsenteres heri er ideelle til at undersøge forhold eller sygdomstilstande, der påvirker synaptisk transmission og postsynaptiske membran egenskaber.

<table border="0" cellpadding="0" cellspacing="0" width="806">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Olympus Compound Microscope</td>
			<td style="width:183px;">Olympus</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10x Objective</td>
			<td style="width:183px;">Olympus</td>
			<td style="width:119px;">UMPLFLN10XW</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">40x Objective</td>
			<td style="width:183px;">Olympus</td>
			<td style="width:119px;">LUMPLFLN40XW</td>
			<td style="width:288px;"></td>
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			<td style="width:119px;">BF150-86-7.5</td>
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			<td style="width:119px;">2500&#8208;0179&#160;&#160;</td>
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			<td style="width:119px;">1-2500-0180</td>
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			<td height="52" style="height:52px;width:216px;">Non-metalic Syringes 34 Gauge</td>
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			<td height="21" style="height:21px;width:216px;">Nikon Stereomicroscope</td>
			<td style="width:183px;">Nikon</td>
			<td style="width:119px;">SMZ800N</td>
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			<td height="21" style="height:21px;width:216px;">No. 5 Forceps</td>
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			<td style="width:119px;">15006-09</td>
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			<td height="21" style="height:21px;width:216px;">No. 2 Forceps</td>
			<td style="width:183px;">Roboz</td>
			<td style="width:119px;">RS-5Q41</td>
			<td style="width:288px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microdissecting Scissors</td>
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			<td height="42" style="height:42px;width:216px;">Sylgard 184 Silicone Elastomer Kit</td>
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			<td height="27" style="height:27px;width:216px;">Grass SD9 Stimulator</td>
			<td style="width:183px;">Grass Medical</td>
			<td style="width:119px;"></td>
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			<td height="46" style="height:46px;width:216px;">Model P-1000 Micropipette Puller</td>
			<td style="width:183px;">Sutter Instruments</td>
			<td style="width:119px;">P-1000</td>
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			<td height="37" style="height:37px;width:216px;">Axon Digidata 1550 Low-noise Data Acuisition System</td>
			<td style="width:183px;">Molecular Devices</td>
			<td style="width:119px;"></td>
			<td style="width:288px;"></td>
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			<td height="44" style="height:44px;width:216px;">Low Pass Bessell Filter</td>
			<td style="width:183px;">Warner Instrument Corp.</td>
			<td style="width:119px;">LPF-8</td>
			<td style="width:288px;"></td>
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			<td height="44" style="height:44px;width:216px;">Left-handed Micromanipulator</td>
			<td style="width:183px;">Siskiyou Corp.</td>
			<td style="width:119px;">MX1641/45DL</td>
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		</tr>
		<tr height="44">
			<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 protokol beskriver en teknik til at optage synaptisk transmission fra den neuromuskulære junction aktuelle-clamp og spænding-clamp betingelser. En ex vivo forberedelse af levator auris longus (LAL) bruges, fordi det er en tynd muskel, der giver nem visualisering af den neuromuskulære junction for mikroelektrode Spidning på pæl på den motor endplate. Denne metode giver mulighed for optagelse af spontan miniature endplate potentialer og strømme (mEPPs og mEPCs), nerve-evoked endplate potentialer og strømforhold (EPPs og EPCs), samt egenskaberne membran i den motor endplate. Resultaterne fra denne metode omfatter den quantal indhold (QC), antallet af vesikel frigivelse sites (n), sandsynligheden for vesikel release (prel), synaptic lettelse og depression samt muskel membran tid konstant (τ m) og input modstand. Anvendelse af denne teknik til musemodeller af sygdomme hos mennesker kan fremhæve vigtige patologier i sygdomstilstande og hjælpe med at identificere nye behandling strategier. Af fuldt spænding fastspænding en enkelt synapse, giver denne metode en af de mest detaljerede analyser af synaptisk transmission i øjeblikket tilgængelige.

Figur 8 viser et eksempel på de nuværende pulser (figur 8A) og spænding svar (fig. 8B) fra en LAL fiber under aktuelle-klemme fra et 12-uge-forhenværende vilde type R6/2-mus. Tilstedeværelsen af mEPPs angiver, at disse poster blev taget fra den motor endplate. Posterne var opnået i normal fysiologisk saltvand løsning. Disse aktuelle-clamp poster kan analyseres for at bestemme Ri og ...

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 til eksamen mammale neuromuskulære transmission spænding klemme betingelser

Den protokol, der er beskrevet i denne hvidbog bruger musen levator auris longus (LAL) muskel til at optage spontane og nerve-fremkaldte postsynaptiske potentialer (nuværende-klemme) og strøm (spænding-klemme) på den neuromuskulære junction. Brug af denne teknik kan give vigtig indsigt i mekanismer af synaptisk transmission under normal og sygdom.

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 Vehikelpræparationen og Teknik for Performing patchclamp Optagelser af Submitochondrial vesikelmembraner

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 Registrering af Starburst amacrine Celler i en væg-mount Udarbejdelse af 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

Overvågning Astrocyte reaktivitet og spredning in Vitro iskæmisk-lignende betingelser

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

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 ...

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

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

Elektrofysiologisk metode til hele celle spænding clamp optagelser fra<em&gt; Drosophila</em&gt; Photoreceptors

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 teknik til at analysere læring-induceret plasticitet

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

Forberedelse af akut rygmarven skiver til hele-celle Patch-klemme optagelse i Substantia Gelatinosa neuroner

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