Name: measuring spinal presynaptic inhibition in mice by dorsal root potential recording in vivo
Uploaded: 2014-03-29T14:00:00
Description: Watch this Scientific Journal Video about Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo at JoVE.com

Vi takker Manfred Heckmann for nyttige diskussioner under oprettelse af metoden. Endvidere takker vi Claudia Sommer til teknisk bistand og Frank Schubert støtte producere videoen. Arbejdet blev støttet af Forbundsministeriet for Uddannelse og Forskning (BMBF), Tyskland, FKZ: 01EO1002 og Interdisciplinært Center for Klinisk Forskning (IZKF) i Jena Universitetshospital.

Alle eksperimentelle procedurer, der er nævnt i den følgende protokol blev godkendt af Thüringer statslige myndigheder (Thüringer Landesamt für Verbraucherschutz, Reg.-Nr. 02-044/12). 1.. Forberedelserne til eksperiment <ol><li> Fabrikation af sugeelektroder <ol><li> Træk en mikropipette hjælp af en standard borsilikatglas kapillar med en mikropipette aftrækker, fx en standard patch elektrode. </li><li> Brake elektroden tip diameter på 0,5-1 mm (lidt større end diametere...

<ol>
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Biol. 87, 33-46 (2005).</li><li>Hochman, S., Shreckengost, J., Kimura, H., Quevedo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+inhibition+of+primary+afferents+by+depolarization:+observations+supporting+nontraditional+mechanisms.">Presynaptic inhibition of primary afferents by depolarization: observations supporting nontraditional mechanisms.</a> Ann. N.Y. Acad. Sci. 1198, 140-152 (2010).</li><li>Thompson, S. W., Wall, P. 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+effect+of+GABA+and+5-HT+receptor+antagonists+on+rat+dorsal+root+potentials.">The effect of GABA and 5-HT receptor antagonists on rat dorsal root potentials.</a> Neurosci. 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D., Devor, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+peripheral+nerve+injury+on+dorsal+root+potentials+and+on+transmission+of+afferent+signals+into+the+spinal+cord.">The effect of peripheral nerve injury on dorsal root potentials and on transmission of afferent signals into the spinal cord.</a> Brain Res. 209, 95-111 (1981).</li><li>Witschi, 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=Presynaptic+&#945;2-GABAA+Receptors+in+Primary+Afferent+Depolarization+and+Spinal+Pain+Control.">Presynaptic &#945;2-GABAA Receptors in Primary Afferent Depolarization and Spinal Pain Control.</a> J. 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Neural Transm. 119 (8), 981-985 (2012).</li><li>Barron, D. H., Matthews, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interpretation+of+potential+changes+in+the+spinal+cord.">The interpretation of potential changes in the spinal cord.</a> J. Physiol. 92, 276-321 (1938).</li><li>Schmidt, R. F., Trautwein, W., Zimmermann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsal+root+potentials+evoked+by+natural+stimulation+of+cutaneous+afferents.">Dorsal root potentials evoked by natural stimulation of cutaneous afferents.</a> Nature. 212, 522-523 (1966).</li><li>Eccles, J. C., Schmidt, R. F., Willis, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+inhibition+of+the+spinal+monosynaptic+reflex+pathway.">Presynaptic inhibition of the spinal monosynaptic reflex pathway.</a> J. Physiol. 161, 282-297 (1962).</li><li>Manjarrez, E., Rojas-Piloni, J. G., Jimenez, I., Rudomin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+synaptic+transmission+from+segmental+afferents+by+spontaneous+activity+of+dorsal+horn+spinal+neurones+in+the+cat.">Modulation of synaptic transmission from segmental afferents by spontaneous activity of dorsal horn spinal neurones in the cat.</a> J. 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Ekstra-og intracellulære elektrofysiologiske optagelser af neuronal aktivitet og synaptiske potentialer in vivo er state of the art teknikker i at undersøge CNS neuronale funktioner og patofysiologi. Spinal integration er afgørende for motorisk funktion, f.eks bevægeapparatet og til multimodal sansning. Præsynaptiske hæmning er et afgørende mekanisme i denne beregningsmæssige proces, der sikrer hensigtsmæssige reaktioner på sensoriske input. GABAergic synapser på Ia afferente fibre hæmmer e...

Præsynaptiske hæmning er en af ​​de mest magtfulde hæmmende mekanismer i rygmarven. Det hæmmer excitatoriske postsynaptiske potentialer (EPSPS) i monosynaptically ophidset motoneurons uden at ændre den postsynaptiske membran potentiale og ophidselse af motoneurons 1-3. Primary afferent depolarisering (PAD) induceret af GABAerge axo-axonale synapser på sensoriske præsynaptiske fibre er den underliggende mekanisme 4-7 (se også Figure1a). Disse synapser indeholder GABA A-og GABA B-receptorer (GABA A R og GABA B R). GABAA R-aktivitet fører til en stigning i chloridkonduktans, som fremkalder PAD skyldes den lokale ion distribution. Denne depolarisering blokerer udbredelsen af virkningspotentialer ind i axonterminaler og reducerer deres styrke fører til en nedsat Ca2 +-tilstrømning og en reduktion af transmitter udgivelse. Aktivering af GABAB-receptorer gør ingent bidrage til PAD, men fører til en reduktion af Ca 2 +-tilstrømning og dermed forbedre præsynaptiske hæmning. Mens aktivering af GABA A R synes at være involveret i kortvarig hæmning er GABA B R involveret i langsigtet modulation 8-10. I tillæg til GABA, der tegner sig for den største del af PAD og præsynaptiske hæmning, kan andre sendere systemer også modulere og bidrage til denne mekanisme 11,12. Patologiske ændringer i præsynaptisk inhibering synes at være afgørende i flere sygdomstilstande, fx perifer inflammation og neuropatisk smerte 13,14 samt unormal central smerte behandling 15, rygmarvsskade 16, og CNS-sygdomme med motor hyperexcitabilitet medieret af defekte GABAergic transmission 17 18.. Således estimering præsynaptiske hæmning er værd at undersøge eksperimentelle patologiske tilstande på rygmarven niveau in vivo </em&gt;. PAD giver anledning til foretaget volumen potentialer giver et direkte mål for den præsynaptiske inhibering i rygmarven. Disse potentialer kaldes dorsalrods potentialer (DRP) og kan måles fra rygmarven dorsale rødder efter stimulering af tilstødende dorsale rødder 7. Er blevet rapporteret første målinger af DRP hos katte og frøer 19 og blev intensivt studeret hos katte ved Eccles, Schmidt, og andre i de tidlige 1970&#39;ere 3,4,20,21. Mens in vivo optagelser af DRP hos katte 22 og rotter 23 er ofte blevet brugt, målinger i mus er næsten udelukkende udført i ex vivo isolerede rygmarv præparater 15,24. Her beskriver vi en metode til at optage DRP i bedøvede mus in vivo giver et direkte mål for præsynaptisk inhibering i den intakte organisme.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Glass tubing (inner diameter 1.16 mm)</td>
			<td>Science Products (Hofheim, Germany)</td>
			<td>GB200F-10</td>
			<td>Other glass tubing might also be suitable</td>
		</tr>
		<tr>
			<td>Superfusion solution (sterile, 0,9% NaCl)</td>
			<td>Braun Melsungen AG&#160;</td>
			<td>3570350</td>
			<td></td>
		</tr>
		<tr>
			<td>(Melsungen, Germany)</td>
		</tr>
		<tr>
			<td>Rompun 2% (Xylazine)</td>
			<td>Bayer Animal Health GmbH (Leverkusen, Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamin 10%</td>
			<td>Medistar GmbH (Ascheberg, Germany)</td>
			<td>KETAMIN 10%</td>
			<td></td>
		</tr>
		<tr>
			<td>30G micro needle/ Sterican</td>
			<td>Braun Melsungen AG&#160;</td>
			<td>4656300</td>
			<td></td>
		</tr>
		<tr>
			<td>(Melsungen, Geramny)</td>
		</tr>
		<tr>
			<td>Salts for aCSF</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>Diverse</td>
			<td></td>
		</tr>
		<tr>
			<td>S88 Dual Output Square Pulse</td>
			<td>Grass Technologies (Warwick, USA)</td>
			<td>S88X</td>
			<td></td>
		</tr>
		<tr>
			<td>Stimulator</td>
		</tr>
		<tr>
			<td>SIU5 RF Transformer Isolation Unit</td>
			<td>Grass Technologies (Warwick, USA)</td>
			<td>SIU-V</td>
			<td></td>
		</tr>
		<tr>
			<td>InstruTECH LIH 8+8</td>
			<td>HEKA (Lambrecht, Deutschland)</td>
			<td>LIH 8+8 + Patchmaster software</td>
			<td></td>
		</tr>
		<tr>
			<td>Data acquisition&#160;</td>
		</tr>
		<tr>
			<td>Universal amplifier</td>
			<td>npi (Tamm, Deutschland)</td>
			<td>ELC-03X</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter Instruments (Novato, USA)</td>
			<td>P-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Olympus (Tokyo, Japan)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Sutter Instruments (Novato, USA)</td>
			<td>MPC-200/MPC-325</td>
			<td>Mechanical micromanipulators also possible</td>
		</tr>
		<tr>
			<td>Homeothermic Blanket System</td>
			<td>Stoelting (Wood Dale, USA)</td>
			<td>50300V</td>
			<td></td>
		</tr>
		<tr>
			<td>Intra-/extracellular recording electrode holder</td>
			<td>Harvard Apparatus (Holliston, USA)</td>
			<td>641227</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring spinal presynaptic inhibition in mice by dorsal root potential recording in vivo

Præsynaptiske hæmning er en af ​​de mest magtfulde hæmmende mekanismer i rygmarven. Den underliggende fysiologiske mekanisme er en depolarisering af primære afferente fibre medieret af GABAerge AXO-axonale synapser (primær afferent depolarisering). Styrken af ​​primære afferente depolarisering kan måles ved registrering af volumen-udført potentialer på den dorsale rod (dorsale potentialer DRP). Patologiske ændringer af præsynaptiske hæmning er afgørende i den unormale central processing af visse smertetilstande og i nogle forstyrrelser af motorisk hyperexcitabilitet. Her beskriver vi en metode til registrering af DRP in vivo i mus. Udarbejdelsen af ​​rygmarv dorsale rødder i den bedøvede dyr, og optagelsen procedure ved hjælp sugeelektroder forklares. Denne metode giver måling GABAergic DRP og dermed anslå spinal præsynaptiske hæmning i levende mus. I kombination med transgene musemodeller, kan DRP optagelse serve som et effektivt redskab til at undersøge sygdom-associeret spinal patofysiologi. In vivo optagelse har flere fordele i forhold til ex vivo isolerede præparater rygmarv, fx muligheden for samtidig optagelse eller manipulation af supraspinale netværk og induktion af DRP ved stimulation af perifere nerver.

Typiske DRP spor er vist i figur 3.. Den fremtrædende stimulation artefakt er normalt efterfulgt af en kort nedadgående afbøjning. Derefter en langsom, langvarig opad afbøjning, der repræsenterer DRP kan tydeligt skelnes. I en undergruppe af optagelser, dorsale rod reflekser er synlige som små pigge på toppen af ​​DRP. I normale wild-type mus, synes dorsalrods reflekser oftest, når stimulation spænding er for høj. Da dorsalrods reflekser ikke kan fremkaldes med en høj reproducerbarhed i d...

Watch this Scientific Journal Video about Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo at JoVE.com

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

Måling Spinal Præsynaptiske Hæmning i mus ved dorsale Potentiel optagelse<em&gt; In Vivo</em

GABAerge præsynaptisk inhibering er en stærk hæmmende mekanisme i rygmarven vigtigt for motorisk og sensorisk integration signal i rygmarv netværk. Underliggende primære afferente depolarisering kan måles ved registrering af dorsale potentialer (DRP). Her vi demonstrere en fremgangsmåde til in vivo-optagelse af DRP i mus.

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of ...

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Neuroscience

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

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Samtidige<em&gt; Ex vivo</em&gt; Functional Test af to nethinder af<em&gt; In vivo</em&gt; Elektroretinogrammet System

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Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...

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Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for ...

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Hurtig isolering af dorsale Rodganglion makrofager

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after ...