Name: patch clamp recordings on intact dorsal root ganglia from adult rats
Uploaded: 2016-09-29T13:00:00
Description: Watch this Scientific Journal Video about Patch Clamp Recordings on Intact Dorsal Root Ganglia from Adult Rats at JoVE.com

The authors would like to acknowledge the Painless Research Foundation for support of the work. This work was also supported by the NIH grants R01 NS080921-01 and R21 NS079897-01A1.

Etik Statement: Alle procedurer for vedligeholdelse og brug af forsøgsdyrene var i overensstemmelse med reglerne i UCSF Udvalgene vedrørende Animal Research og blev udført i overensstemmelse med de retningslinjer for NIH regler om brug af dyr og pleje (publikation 85-23, Revideret 1996 ). Den UCSF Institutional Animal Care og brug Udvalg godkendte protokoller, der bruges i denne undersøgelse. 1. Udarbejdelse af Instrumenter, løsninger og fade <ol><li> Forbered Kunstig cerebrospinalvæ...

<ol>
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Vi rapporterer en metode til at forberede hele DRG til patch clamp studier. Der er flere vigtige elementer for at forberede en ideel eksemplar. For det første er det vigtigt at dissekere DRG med dorsale rødder fastgjort. Efter dette, epineurium skal fjernes omhyggeligt og samtidig undgå beskadigelse af neuroner. Endelig, for at blotlægge de neuroner og de omgivende satellit gliaceller, er det nødvendigt at fordøje det resterende bindevæv. Intakte DRG fra voksne rotter tilberedt med den her beskrevne metode vil op...

Sensation er afgørende for en organismes overlevelse og trivsel. Transmissionen af ​​stimuli er afhængig af de sensoriske veje, der starter ved perifere endelser axoner fra primære sensoriske neuroner. Primære sensoriske neuroner, med undtagelse af mesencephale kerne af trigeminusnerven, er placeret i den trigeminale ganglier og dorsalrodsganglier (DRG). De tjener som dørvogtere af den sensoriske information 1. På perikarial membran, ligesom på centrale og perifere terminaler, DRG-neuroner udtrykker receptorer og ionkanaler, såsom glutamatreceptorer, TNF alfa-receptorer, forbigående receptor potentielle kation kanal underfamilie V medlem 1 (TRPV1), natriumkanaler mv 2 -7. Patch clamp optagelser af perikarial membran tillader forstå funktionelle ændringer af mange af disse receptorer og kanaler i hele neuron. Patch-clamp optagelse teknik er et stærkt værktøj til STUdøende aktiviteter kanaler eller receptorer og en lang række undersøgelser er blevet udført ved at anvende denne teknik på DRG-neuroner 8-10. I de fleste undersøgelser DRG fjernes ved at skære den dorsale rodspirerne og spinal nerve tæt på ganglion. Efter hakning, er ganglion derefter placeret i fordøjelsesenzymer, der resulterer i dissociation af DRG-neuroner, som derefter kan registreres straks eller dyrket i flere dage forud for optagelse. Desværre dissociation af DRG-neuroner involverer en nødvendig axotomi tæt på perikarya. Når dissocieret og axotomiserede, DRG-neuroner undergår fænotypiske ændringer samt ændringer i membran ophidselse 11,12. Tabet af kontakt mellem perikarya af individuelle neuroner og satellit gliaceller, der normalt omgiver dem kan bidrage til disse ændringer 13. Den krydstale mellem neuroner og satellit gliaceller er både afgørende fysiologiske forhold og i tilpasning til pathologiCal betingelser som dem, der fører til intraktabel smerte 14,15. Det ville være en udfordring at studere interaktionen mellem neuroner og satellit gliaceller bruger dissocieret DRG præparat. Intakte DRG, på den anden side giver tættere på in vivo betingelser. I de sidste mange år, har vores laboratorium, samt nogle andre grupper, brugt intakte DRG fra voksne rotter til at undersøge ændringer af primære sensoriske neuroner i forskellige forhold, der er forbundet med kroniske smerter 3-5,11,15-17. Selvom de teknikker, der anvendes i disse undersøgelser noget er etableret, en trin-for-trin beskrivelse er endnu ikke blevet offentliggjort. I den foreliggende manuskript, beskriver vi en bekvem og hurtig måde at fremstille intakte DRG og deres anvendelse til patch clamp optagelser.

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			<td height="20" style="height:20px;width:252px;">Pentobarbital sodium</td>
			<td style="width:172px;">vortech Pharmaceuticals</td>
			<td style="width:108px;"></td>
			<td style="width:484px;"></td>
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			<td height="20" style="height:20px;">syringe</td>
			<td>BD</td>
			<td>309659</td>
			<td>1 ml, 5 ml.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">scalpel</td>
			<td>BD</td>
			<td></td>
			<td>size: 15</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mayo straight scissor</td>
			<td>Fine Science Tools</td>
			<td>14010-15</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mayo curved scissor</td>
			<td>Fine Science Tools</td>
			<td>14011-15</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rongeur</td>
			<td>Fine Science Tools</td>
			<td>16021-14</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Adson toothed forceps</td>
			<td>Fine Science Tools</td>
			<td>11027-12</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Iris Scissor</td>
			<td>Fine Science Tools</td>
			<td>14084-08</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Noyes spring scissor</td>
			<td>Fine Science Tools</td>
			<td>15124-12</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bone scissors</td>
			<td>Fine Science Tools</td>
			<td>16044-10</td>
			<td>Special for cutting the bones.&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Forceps: Dumont, Dumoxel Biologie #5</td>
			<td>Fine Science Tools</td>
			<td>11252-30</td>
			<td>These have the fine tips that do not need sharpening when first purchased.</td>
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			<td height="21" style="height:21px;">periosteal elevator</td>
			<td>Sklar</td>
			<td>97-0530</td>
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			<td height="20" style="height:20px;">Dissection microscope</td>
			<td>WILD</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Transfer pipette</td>
			<td>Fisher brand</td>
			<td>13-711-5AM</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Petri dish (10 cm)</td>
			<td>Pyrex</td>
			<td></td>
			<td>Glass petri dish can avoid damaging the tips of fine forceps</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Collagenase (Liberase TM)</td>
			<td>Roche</td>
			<td>05-401-119-001</td>
			<td>dissolve at the concentration of 13 u/ml, aliquot into glass pipette. Avoid repeated freeze and thaw.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">filter</td>
			<td>Thermo scientific</td>
			<td>7232520</td>
			<td>Filter the internal solutions for patch clamp recording to avoid clog.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass pipette</td>
			<td>Sutter</td>
			<td>BF150-110-7.5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anchor</td>
			<td>Havard apparatus</td>
			<td>64-0250</td>
			<td>stabilize the DRG to avoid drift.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Peristaltic pump</td>
			<td>WPI</td>
			<td></td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Pipette puller</td>
			<td>Sutter</td>
			<td>P97</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Amplifier</td>
			<td>Molecular devices</td>
			<td>Axopatch 200B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Digitizer</td>
			<td>Molecular devices</td>
			<td>1440D</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope</td>
			<td>NIKON</td>
			<td>FN600</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Micro-manipulator</td>
			<td>Sutter</td>
			<td>MPC200</td>
			<td></td>
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			<td height="20" style="height:20px;">microinjection dispense system</td>
			<td>General Valve</td>
			<td>Picrospitzer II</td>
			<td>fast drug application system</td>
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			<td height="20" style="height:20px;">Carbogen (95% O2, 5% CO2)</td>
			<td>Local Medical Gas supplier</td>
			<td></td>
			<td></td>
		</tr>
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patch clamp recordings on intact dorsal root ganglia from adult rats

Patch clamp undersøgelser fra dorsalrodsganglier (DRG) neuroner har øget vores forståelse af det perifere nervesystem. I øjeblikket er de fleste optagelser foretaget på dissocierede DRG-neuroner, hvilket er en standard forberedelse til de fleste laboratorier. Neuronale egenskaber, men kan ændres ved axonal skade som følge af enzym fordøjelse bruges i at erhverve dissocierede neuroner. Endvidere kan dissocierede neuron præparater, der ikke fuldt ud repræsentere mikromiljø af DRG da tab af kontakt med satellit gliaceller, der omgiver de primære sensoriske neuroner er en uundgåelig konsekvens af denne metode. For at overvinde de begrænsninger i at bruge konventionelle dissocierede DRG-neuroner til patch clamp optagelser, i denne rapport beskriver vi en metode til at forberede intakte DRG og gennemføre patch clamp optagelser på individuelle primære sensoriske neuroner ex vivo. Denne fremgangsmåde tillader hurtig og enkel tilberedning af intakte DRG, efterligner ivivo betingelser ved at holde DRG-neuroner forbundet med deres omgivende satellit gliaceller og basalmembranen. Endvidere fremgangsmåden undgår axonal skade mod manipulation og enzym fordøjelse såsom når dissociering DRG. Denne ex vivo præparat kan yderligere anvendes til at studere interaktionen mellem primære sensoriske neuroner og satellit gliaceller.

Figur 1 viser processen med at forberede intakt DRG for patch optagelse. Figur 1A viser eksponeringen og placering af ganglier efter laminektomi. Figure1B viser L3, L4 og L5 DRG med nerve rødder knyttet efter fjernelse af rygmarven. Derefter L4 og 5 DRG er omhyggeligt dissekeret og befriet fra ryghvirvlerne. Dernæst epineurium, en transparent membran omkring DRG, fjernes (gul pil, figur 1D). Den bedste placering for at...

Watch this Scientific Journal Video about Patch Clamp Recordings on Intact Dorsal Root Ganglia from Adult Rats at JoVE.com

Patch Clamp Recordings on Intact Dorsal Root Ganglia from Adult Rats

This manuscript describes how to prepare intact dorsal root ganglia for patch clamp recordings. This preparation maintains the microenvironment for neurons and satellite glial cells, thus avoiding the phenotypic and functional changes seen using dissociated DRG neurons.

Patch Clamp optagelser på Intact dorsalrodsganglier fra voksne rotter

Patch clamp studies from dorsal root ganglia (DRGs) neurons have increased our understanding of the peripheral nervous system. Currently, the majority of ...

Research

JoVE Journal

Biochemistry

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Time-løst elektrosprayioniserings Brint-deuterium Exchange-massespektrometri til at studere proteiners struktur og Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

Spændingsklemme Fluorometri i<em&gt; Xenopus</em&gt; Oocytter ved hjælp af fluorescerende unaturlige aminosyrer

Voltage-Clamp Fluorometry (VCF) has been the technique of choice to investigate the structure and function of electrogenic membrane proteins where ...

Glycoproteomics of the Extracellular Matrix: A Method for Intact Glycopeptide Analysis Using Mass Spectrometry

Glycoproteomics af den ekstracellulære matrix: En metode til Intakt glycopeptid analyse ved hjælp massespektrometri

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). ...

In Vitro Polymerization of F-actin on Early Endosomes

In Vitro Polymerization of F-actin on Early Endosomes

In Vitro Polymerisering af F-actin på tidlig Endosomes

Many early endosome functions, particularly cargo protein sorting and membrane deformation, depend on patches of short F-actin filaments nucleated onto ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

En simpel metode til High Throughput kemisk Screening i Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Elektrokemisk detektion af Deuterium Kinetic isotop effekt på ekstracellulære elektron Transport i Shewanella oneidensis hr.-1

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM ...

Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy

Analysere dynamisk proteinkomplekser samles på og frigivet fra Biolayer interferometri Biosensor ved hjælp af massespektrometri og elektronmikroskopi

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this ...

Measurement of Ion Concentration in the Unstirred Boundary Layer with Open Patch-Clamp Pipette: Implications in Control of Ion Channels by Fluid Flow

Måling af Ion koncentration i Unstirred grænselag med åben Patch-Clamp Pipette: konsekvenser i kontrol af Ionkanaler af væske Flow

Fluid flow is an important environmental stimulus that controls many physiological and pathological processes, such as fluid flow-induced vasodilation. ...

Identification of Orexin and Endocannabinoid Receptors in Adult Zebrafish Using Immunoperoxidase and Immunofluorescence Methods

Identifikation af orexin og Endocannabinoide receptorer hos voksne zebrafisk ved hjælp af Immunoperoxidase og immunofluorescens metoder

Immunohistochemistry (IHC) is a highly sensitive and specific technique involved in the detection of target antigens in tissue sections with labeled ...

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

Undersøgelse af metabolismen af seks systemiske insekticider i en nyetableret celle affjedrings kultur afledt af te (Camellia sinensis L.) Blade

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...