Dieses Protokoll wurde durch Forschung Zuschüsse aus dem National Natural Science Foundation of China (#31171066, #81270464) und das Sino-German Science Center (GZ919) unterstützt.

der experimentelle Protokoll berichtet hier genehmigt wurde, indem das Tier ethischen Ausschuss der Shanghai Jiaotong University School of Medicine (# SYXK2013-0050). das Sezieren von Colorectum mit intakten Ganglion und Nerven Stamm dauert mindestens 15 Minuten für eine Person in dieser Technik erfahren. Es ist daher notwendig, um das Tier lebendig aber Tiefe Narkose während Sezierungen, um die Lebensfähigkeit des Gewebes für nachfolgende elektrophysiologische Aufnahme gewährleistet.
 1. Vorbereitung der Perfusion Lösung und Testverbindungen <ol><li>bereiten 5 L Krebs Lösung: 113 mM NaCl, 5,9 mM KCl, 1,2 mM NaH 2 PO 4, 1,2 mM MgSO 4, 25 mM Nahco3 3, 1,2 mM CaCl 2 , und 11,5 mM Glukose. Die Lösung mit 95 % O 2 + 5 % CO 2-Gas-Gemisch zu sättigen. ~ 500 mL Sauerstoff Krebs im Kühlschrank Vorkühlen.</li>
	<li>Vorbereiten Aliquote des Bestandes testen Verbindungen (1 mM Capsaicin in Ethanol und 10 mM 5-Hydroxytryptamine (5-HT) in Kochsalzlösung) nach Bedarf. Den Bestand im Krebs (für Bad Anwendung) und in Kochsalzlösung (für Intraluminal Verwaltung) auf die Endkonzentration nur vor Gebrauch verdünnen.</li>
</ol> 2. Vorbereitung der Aufnahme Elektrode <ol><li>ziehen die Aufnahme Elektrode aus Standardglas ohne innere Filamente (1,5 mm Außendurchmesser Rohre) mit einem herkömmlichen Elektrode Abzieher. Passen Sie die Einstellungen der Abzieher für Wärme und ziehen, so dass der Schaft der gezogenen Elektroden zwischen 20 und 25 mm.</li>
</ol> 3. Gewebe-Sammlung <ol><li>betäuben die Ratte tief mit Natrium-Pentobarbital (80 mg/kg, i.p.). </li> <li>Während der Sterilität zu gewährleisten, setzen die Bauchhöhle durch ausführen einen Mittellinie Einschnitt auf der Bauchdecke mit einem Skalpell. Ziehen Sie das Mesenterium und anderen Geweben beiseite, die Colorectum verfügbar zu machen.</li>
	<li>Legen Sie das Tier unter dem sezierenden Mikroskop. Lokalisieren Sie durch sorgfältige Präparation der linken PG und zu identifizieren Sie, die PN und die LSN beitreten. Schneiden Sie diese Nerven ein paar Millimeter vom PG 
	Hinweis: Die PG liegt in der Nähe der kolorektalen Kreuzung. In der Regel 3-4 PN aus der lumbosakralen Rückenmark und ein LSN-Projekt in den PG ( Abbildung 1).</li>
	<li>Schneiden die After Knochen um den Mastdarm freizulegen. Entfernen Sie das Gewebe (z.B. Harnblase, etc.) über die Colorectum; die PG intakt lassen.</li>
	<li>Die Ratte durch die intravenöse Injektion einer Überdosierung von Pentobarbital Opfern. Transekt Dickdarm ca. 3 cm oberhalb der PG und dem Colorectum aus das Tier mit einer Pinzette entfernen.</li>
	<li>Übertragen die Colorectum auf einer Petrischale mit vorgekühlten Krebs-Lösung gefüllt. Entfernen Sie den Kot von sanft Spülung des Dickdarms. Entfernen Sie die Überbleibsel der Harnblase und anderen Geweben vorsichtig, ohne Kompromisse bei dem PG.</li>
</ol> 4. Dissektion der Kolon afferenten Nerven <ol><li>legen Sie den Doppelpunkt in einer Aufnahme Kammer (20 mL) und das Gewebe ständig mit Carbogenated Krebs Lösung durchspülen. Die Perfusion Rate auf 15 mL/min eingestellt</li>
	<li>Cannulate die Colorectum an den oral- und Analsex enden. Beginnen Sie die Intraluminal Infusion des Dickdarms mit Kochsalzlösung mit einer Rate von 10 mL/h in der Oral bis anal Richtung.</li>
	<li>Finden Sie die wichtigsten PG unter dem sezierenden Mikroskop. Verwenden Sie Insekten Stifte, um das Ganglion verfügbar zu machen. Finden Sie mit sorgfältiger Präparation eine feine Verzweigung der Nerven, die aus dem Ganglion und läuft auf den Dickdarm ( Abbildung 1). Schneiden Sie die Nerven in der Nähe des Ganglion. 
	Hinweis: Abbildung 1 zeigt eine Aufnahme von einem distalen Nerv Zweig auf dem PG. Der Nerv enthält vermutlich eine Mischung aus Becken- und Lendenbereich splanchnic afferenten Fasern. Alternativ kann eine Aufnahme aus der PN und/oder die LSN proximal zu PG hergestellt werden</li>
	<li>Schalten Sie die Heizung Bad und Warm die Krebs-Lösung, die Temperatur bei 34 ± 0,5 ° c zu halten</li>
</ol> 5. Vorbereitung der Saug-Elektroden <ol><li>nehmen Sie eine Pre-gezogenem Glas-Pipette (Schritt 2) und unter dem sezierenden Mikroskop zu inspizieren. Die Spitze der Elektrode mit einer Zange zu brechen, so dass es kompatibel mit dem Durchmesser des Nervus aufzuzeichnende Größe.</li>
	<li>Abschrägung die Spitze indem man es in der Nähe von leichter Blendenflecken.</li>
</ol> 6. Elektrophysiologische Aufnahme <ol><li>Connect die abgeschrägte Elektrode der Elektrodenhalter. Verbinden Sie eine 10-mL-Spritze mit der Seitenanschluss auf dem Halter für negativen oder positiven Druck auf die Elektrode.</li>
	<li>Den Inhaber an der Headstage von den Bioamplifier anschließen und montieren Sie die Headstage auf einem Manipulator.</li>
	<li>Verschieben die Elektrode in das Gewebe Bad und füllen Sie die Elektrode mit der Krebs-Lösung durch sanfte Saugwirkung anwenden, bis es den Silberdraht des Inhabers in Kontakt. Legen Sie die Elektrodenspitze in der Nähe das abgeschnittene Ende des Nervs und negativen Druck, den Nerv in der Elektrode zu saugen. Negativeren Druck ausüben, so dass ~ 1 mm des Nervs in der Elektrode gezogen wird und einen dichten Abschluss bildet.</li>
	<li>Turn auf der Bioamplifier und der Filter auf 300-3.000 Hz. das Signal auf dem Oszilloskop aufzeichnen und das Nervensignal (20 kHz Sampling-Rate) und das Intraluminal Drucksignal (100-Hz-Sampling-Rate) von einem Computer mit eine Spike-Datenverarbeitung Software.</li>
</ol> 7. Die Darmspülung afferenten Empfindlichkeit testen <ol><li>anwenden Rampe Distension des Dickdarms durch den Dreiwege Hahn an der Steckdose Kanüle während der Infusion kontinuierlich Intraluminally schließen. Überwachung des Intraluminal Drucks bis 60 MmHg, zu welchem Zeitpunkt offen den Dreiwege Hahn an der Entwässerung Kanüle erreicht.</li>
	<li>Wiederholen Sie diesen Vorgang in regelmäßigen Abständen von 15 min. anwenden Drogen extra- oder Intraluminally, die chemische Empfindlichkeit der afferenten Nerven zu testen.</li>
</ol>

<ol>
	<li>Al-Chaer, E. D., Traub, 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=Biological+basis+of+visceral+pain:+recent+developments.">Biological basis of visceral pain: recent developments.</a> Pain. 96 (3), 221-225 (2002).</li><li>Christianson, J. A., Traub, R. J., Davis, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+spinal+distribution+and+neurochemical+phenotype+of+colonic+afferents+in+mouse+and+rat.">Differences in spinal distribution and neurochemical phenotype of colonic afferents in mouse and rat.</a> J Comp Neurol. 494 (2), 246-259 (2006).</li><li>Wynn, G., Rong, W., Xiang, Z., Burnstock, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purinergic+mechanisms+contribute+to+mechanosensory+transduction+in+the+rat+colorectum.">Purinergic mechanisms contribute to mechanosensory transduction in the rat colorectum.</a> Gastroenterology. 125 (5), 1398-1409 (2003).</li><li>Dong, L., 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=Impairments+of+the+Primary+Afferent+Nerves+in+a+Rat+Model+of+Diabetic+Visceral+Hyposensitivity.">Impairments of the Primary Afferent Nerves in a Rat Model of Diabetic Visceral Hyposensitivity.</a> Mol Pain. 11, (2016).</li><li>Lynn, P. A., Blackshaw, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+recordings+of+afferent+fibres+with+receptive+fields+in+the+serosa,+muscle+and+mucosa+of+rat+colon.">In vitro recordings of afferent fibres with receptive fields in the serosa, muscle and mucosa of rat colon.</a> J Physiol. 518 (Pt 1), 271-282 (1999).</li><li>Page, A. J., 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=Different+contributions+of+ASIC+channels+1a,+2,+and+3+in+gastrointestinal+mechanosensory+function.">Different contributions of ASIC channels 1a, 2, and 3 in gastrointestinal mechanosensory function.</a> Gut. 54 (10), 1408-1415 (2005).</li><li>Hockley, J. 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=P2Y+Receptors+Sensitize+Mouse+and+Human+Colonic+Nociceptors.">P2Y Receptors Sensitize Mouse and Human Colonic Nociceptors.</a> J Neurosci. 36 (8), 2364-2376 (2016).</li><li>Peiris, M., 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=Human+visceral+afferent+recordings:+preliminary+report.">Human visceral afferent recordings: preliminary report.</a> Gut. 60 (2), 204-208 (2011).</li><li>Feng, B., Gebhart, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+silent+afferents+in+the+pelvic+and+splanchnic+innervations+of+the+mouse+colorectum.">Characterization of silent afferents in the pelvic and splanchnic innervations of the mouse colorectum.</a> Am J Physiol Gastrointest Liver Physiol. 300 (1), G170-G180 (2011).</li><li>Feng, B., 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=Activation+of+guanylate+cyclase-C+attenuates+stretch+responses+and+sensitization+of+mouse+colorectal+afferents.">Activation of guanylate cyclase-C attenuates stretch responses and sensitization of mouse colorectal afferents.</a> J Neurosci. 33 (23), 9831-9839 (2013).</li><li>Feng, B., Gebhart, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+Functional+Characterization+of+Mouse+Colorectal+Afferent+Endings.">In vitro Functional Characterization of Mouse Colorectal Afferent Endings.</a> J Vis Exp. (95), e52310 (2015).</li><li>Nullens, S., 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=In+Vitro+Recording+of+Mesenteric+Afferent+Nerve+Activity+in+Mouse+Jejunal+and+Colonic+Segments.">In Vitro Recording of Mesenteric Afferent Nerve Activity in Mouse Jejunal and Colonic Segments.</a> J Vis Exp. (116), (2016).</li><li>Rong, W., Hillsley, K., Davis, J. B., Hicks, G., Winchester, W. J., Grundy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Jejunalafferent+nerve+sensitivity+in+wild-type+and+TRPV1+knockout+mice.">Jejunalafferent nerve sensitivity in wild-type and TRPV1 knockout mice.</a> J Physiol. 560 (Pt 3), 867-881 (2004).</li><li>Brierley, S. M., 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=Differential+chemosensory+function+and+receptor+expression+of+splanchnic+and+pelvic+colonic+afferents+in+mice).">Differential chemosensory function and receptor expression of splanchnic and pelvic colonic afferents in mice).</a> J Physiol. 567 (Pt 1), 267-281 (2005).</li><li>Brierley, S. M., Jones, R. C. 3. r. d., Gebhart, G. F., Blackshaw, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Splanchnic+and+pelvic+mechanosensory+afferents+signal+different+qualities+of+colonic+stimuli+in+mice.">Splanchnic and pelvic mechanosensory afferents signal different qualities of colonic stimuli in mice.</a> Gastroenterology. 127 (1), 166-178 (2004).</li><li>La, J. H., Schwartz, E. S., Gebhart, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+the+expression+of+transient+receptor+potential+channel+V1,+transient+receptor+potential+channel+A1+and+mechanosensitive+two+pore-domain+K++channels+between+the+lumbar+splanchnic+and+pelvic+nerve+innervations+of+mouse+urinary+bladder+and+colon.">Differences in the expression of transient receptor potential channel V1, transient receptor potential channel A1 and mechanosensitive two pore-domain K+ channels between the lumbar splanchnic and pelvic nerve innervations of mouse urinary bladder and colon.</a> Neuroscience. 186, 179-187 (2001).</li><li>Wang, G., Tang, B., Traub, 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=Differential+processing+of+noxious+colonic+input+by+thoracolumbar+and+lumbosacral+dorsal+horn+neurons+in+the+rat.">Differential processing of noxious colonic input by thoracolumbar and lumbosacral dorsal horn neurons in the rat.</a> J Neurophysiol. 94 (6), 3788-3794 (2005).</li></ol>

Die Autoren erklären keinen Interessenskonflikt.

Die hier vorgestellten Protokoll ist ein relativ einfach experimentelle Methode, um die elektrophysiologischen Eigenschaften des Kolon Afferenzen von Ratten zu beurteilen. Das Protokoll (von Gewebe Dissektion zum Einrichten der Nerv-Aufnahme) dauert in der Regel ca. 2 Stunden in Anspruch. Gewebe-Sammlung (Schritt 3) und Vorbereitung der Saug Elektrode (Schritt 5) sind die entscheidenden Schritte. Es ist entscheidend, PG, LSN und die PN zu finden und kümmern sich nicht um das Ganglion und Nerven während Gewebe Dissektio...

Der Magen-Darm-Trakt (GIT) ist reich mit extrinsischen afferenten Nerven innerviert, zu vermitteln, dass sensorische Signale aus dem Bauch heraus auf das zentrale Nervensystem und der Darm-Hirn-Interaktion beitragen. Veränderte Erregbarkeit des diese extrinsischen Afferenzen sowie veränderte zentrale Verarbeitung der afferenten Eingänge zugrunde liegt, viszerale Schmerzen und andere Symptome der GI-Bedingungen, einschließlich der Funktions- und entzündliche Darm-Erkrankungen-1. Sensorischer Informationen aus dem Colorectum vermittelt in erster Linie durch die Kopf/Unterbauch und lumbosakralen/Beckennerven (PN)2. Es wurde ein erhöhtes Interesse an einem Studium der elektrophysiologischen Eigenschaften dieser primären afferenten Fasern in Nagetier Krankheitsmodelle. Jedoch in Vivo elektrophysiologische Aufnahmen von Kolon Afferenzen bei Nagern ist eine technische Herausforderung und erfordert erhebliche chirurgische Fähigkeiten. Darüber hinaus können hämodynamischen Veränderungen, Gewebe Bewegung und Anästhetika auch Nerven-Aktivität und die Sensibilität für Reize in Vivozu testen auswirken. Daher haben in den letzten Jahren eine wachsende Zahl von Studien in Vitro (ex Vivo) Vorbereitungen der verschiedenen Arten, darunter Mäuse, Ratten, Meerschweinchen und Menschen, zu prüfen, die Mechanismen der sensorischen Transduktion in Colon eingesetzt Afferenzen und die veränderte Erregbarkeit in Krankheitsbildern. 3 , 4 , 5 , 6 , 7 , 8
In erster Linie zwei Arten von ex-Vivo Kolon Vorbereitung gemeldet wurden: die "Flat-Blatt" Vorbereitung5,9,10 und die "Tube" Vorbereitung3,4. Ein video Protokoll für die "Flat-Blatt" murinen Colorectum Vorbereitung wurde bisher erschienen11. In diesem Protokoll, die Maus Colorectum mit der PN) oder lumbale splanchnic Nerven (LSN) angeschlossen, wird geerntet und unterkühlte in einer Gewebe-Kammer. Die Colorectum ist längs aufschneiden und das Nervenbündel erstreckt sich in einem Aufnahme-Fach mit Paraffin-Öl gefüllt. Nerven-Aktivität ist eine monopolare Elektrode Platin-Iridium mit aufgenommen. Das Protokoll ermöglicht die Identifikation der rezeptiven Felder der einzelnen afferenten Fasern mittels unvoreingenommenen elektrischen Stimulation. Es lokalisiert, die Anwendung von chemischen reizen, sowie die Anwendung der verschiedenen mechanischen Stimulation Paradigmen (z.B. fokale Schleimhaut sondieren und umlaufenden Stretch), zu den afferenten Nervenendigungen. Weil der Nerv zu einer separaten Kammer aus der Gewebe-Kammer erweitert werden muss, ist es wichtig, den beigefügten Nerv relativ lange zu halten; die erfolgreiche Dissektion der Nerven stellt eine Herausforderung für die neue dieser Methodik. In jüngerer Zeit, Nullens Et Al. ein video Protokoll für die in-vitro- Aufnahme des mesenterialen Afferenzen in murinen Jejunal und Kolon Segmente12veröffentlicht. In dieser Zubereitung "Röhre" wird der Darm-Segment mit dem Mesenterium befestigt intakt, wodurch für abgestufte Blähungen und der Intra- und Extra-luminalen Verwaltung verschiedener chemischer Stoffe gehalten. Da die Mesenterium Nerven aufgezeichnet wird mit einer Absaugung Elektrode, die in der Nähe des Gewebes positioniert werden kann, kann afferenter Aktivität erfasst werden, obwohl der Mesenterium Nerv relativ kurz ist. Jedoch der Mesenterium Nerv besteht aus gemischten Populationen von vagalen und spinalen afferenten Fasern, die das Jejunum innervieren oder Kopf Unterbauch. Lumbosakralen Becken-Afferenzen innervieren die Colorectum, die in diesem Protokoll kann nicht diskriminiert zu werden. Hier präsentieren wir Ihnen ein detailliertes Protokoll zur elektrophysiologischen Erfassung der Ratte Kolon Afferenzen mit der "Tube" Colorectum Vorbereitung mit einer intakten PG. Diese Methode kann für die Charakterisierung der funktionalen Eigenschaften der Lendenwirbelsäule splanchnic (Unterbauch) und lumbosakralen Becken-Afferenzen.

<table width="889">
	<tbody>
		<tr height="40">
			<td height="40" style="width:215px;height:40px;">Sodium Pentobarbital</td>
			<td style="width:123px;">Shanghai Westang Bio-Tech</td>
			<td style="width:344px;">B558</td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Capsaicin</td>
			<td>Sigma</td>
			<td>M2028</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Electrode puller</td>
			<td>MicroData Instrument Inc</td>
			<td>PMP107</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Neurolog System (Bioamplifier)</td>
			<td>Digitimer, Ltd</td>
			<td>Neurolog System</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">A/D converter</td>
			<td>Cambridge Electronic Design</td>
			<td>Micro1401</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Data processing software</td>
			<td>Cambridge Electronic Design</td>
			<td>Spike2 version 6</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Silver wire</td>
			<td>World Precision Instruments</td>
			<td>EP12</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glass tubes</td>
			<td>World Precision Instruments</td>
			<td>1B150-4</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Electrode holder</td>
			<td>World Precision Instruments</td>
			<td>MEH3SBW</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Heating bath</td>
			<td>Grant</td>
			<td>GR150</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dissecting microscope</td>
			<td>Leica</td>
			<td>Zoom2000</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dissecting microscope</td>
			<td>World Precision Instruments</td>
			<td>PZMIII-BS</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cigarette lighter</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Surgical tools</td>
			<td>World Precision Instruments</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Insect pins</td>
			<td>home-made from 0.1 mm stainless steel wire</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Three way manipulator</td>
			<td>World Precision Instruments</td>
			<td>KITF-R</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Rats</td>
			<td>Any</td>
			<td>NA</td>
			<td>Any strain/sex can be used.</td>
		</tr>
	</tbody>
</table>

in vitro characterization of the electrophysiological properties of colonic afferent fibers in rats

Dysfunktion des Kolon Sinnesnerven hat in der Pathophysiologie von mehreren gemeinsamen Bedingungen, einschließlich funktionelle und entzündliche Darmerkrankungen und Diabetes verwickelt. Hier beschreiben wir ein Protokoll für die in Vitro Charakterisierung der elektrophysiologischen Eigenschaften des Kolon Afferenzen in Ratten. Die Colorectum mit der intakten Becken Ganglion (PG) angeschlossen, wird aus der Ratte entfernt; unterkühlte mit Carbogenated Krebs-Lösung in der Aufnahme-Kammer; und Kontrolle an den oral- und Analsex enden, um Blähungen zu ermöglichen. Eine feine Nervenbündel aus PG wird identifiziert, und die multiunit afferenten Nerven-Aktivität ist mit einer Saug-Elektrode aufgenommen. Distension des Segments Kolon entlockt allmählichen Anstieg multiunit Entlastung. Eine Hauptkomponentenanalyse wird durchgeführt, um die niedrigschwellige, die hohe Schwelle und die weiten Dynamikbereich afferenten Fasern zu unterscheiden. Chemischen Empfindlichkeit des Kolon Afferenzen kann durch die Badewanne oder Intraluminal Gabe von Testverbindungen untersucht werden. Dieses Protokoll kann geändert werden, für die Anwendung auf andere Arten, wie Mäuse und Meerschweinchen und die Unterschiede in den elektrophysiologischen Eigenschaften des Kopf/Unterbauch und lumbosakralen/Becken-Afferenzen des absteigenden Dickdarms in Normal zu studieren und pathologischen Zuständen.

Abbildung 1 zeigt die schematische Darstellung des experimentellen Aufbaus Vorbereitung Colorectum ex Vivo "Tube" mit einem Vertreter, die Aufnahme von einem distalen Nerv auf dem PG. Der Nerv enthalten vermutlich eine Mischung aus Becken- und Lendenbereich splanchnic Afferenzen. In Zubereitungen aus normalen Ratten haben Kolon afferenten Nerven in der Regel eine geringe unregelmäßige spontane Aktivität. Rampe Distension des Darms bewirkt einen al...

Watch this Scientific Journal Video about In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats at JoVE.com

In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats

Anormale sensorische Funktion zugrunde liegt, viszerale Schmerzen und andere Symptome von funktionellen und entzündlichen Darmerkrankungen. Ein Protokoll für die elektrophysiologische Aufzeichnung der Kolon afferenten Nerven in eine ex Vivo Ratte Colorectum Vorbereitung wird hier vorgestellt.

In-vitro- Charakterisierung der elektrophysiologischen Eigenschaften des Kolon afferenten Fasern in Ratten

Dysfunction of the colonic sensory nerves has been implicated in the pathophysiology of several common conditions, including functional and inflammatory ...

in-vitro-characterization-electrophysiological-properties-colonic

Research

JoVE Journal

Neuroscience

In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats

6.8K Aufrufe.  Shanghai Jiaotong University School of Medicine. Anormale sensorische Funktion zugrunde liegt, viszerale Schmerzen und andere Symptome von funktionellen und entzündlichen Darmerkrankungen. Ein Protokoll für die elektrophysiologische Aufzeichnung der Kolon afferenten Nerven in eine ex Vivo Ratte Colorectum Vorbereitung wird hier vorgestellt.

Serie: In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

Eine allgemeine Methode zur Bewertung der Inkubation von Saccharose Craving in Ratten

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Forschung

Neurowissenschaften

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals1. Though being a crucial part of the visual processing by shaping the retinal output2, most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event.
Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue3,4. These pre-labeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage5. Also, it leaves the retina responsive to visual stimuli since infrared light (&gt;850 nm) is only poorly absorbed by photopigments6.
In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels.

This article depicts the recording of individual cells from fluorescently tagged neuronal populations in the intact mouse retina. By using two-photon infrared excitation transgenetically labeled cells were targeted for patch-clamp recording to study their light responses, receptive field properties, and morphology.

Elektrophysiologische Charakterisierung von GFP-exprimierenden Zellpopulationen in der intakte Retina

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

Visualisierung von Propriozeptoren in<em&gt; Drosophila</em&gt; Larven und Puppen

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Dual-Elektrophysiologische Recordings synaptisch-evozierte astrogliale und neuronale Antworten in Acute Hippocampusschnitte

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling of auditory axons within the VIIIth cranial nerve would enhance the study of central auditory circuit development. This approach is challenging because multiple sensory organs of the inner ear contribute to the VIIIth nerve 1. Moreover, markers that reliably distinguish auditory versus vestibular groups of axons within the avian VIIIth nerve have yet to be identified. Auditory and vestibular pathways cannot be distinguished functionally in early embryos, as sensory-evoked responses are not present before the circuits are formed. Centrally projecting VIIIth nerve axons have been traced in some studies, but auditory axon labeling was accompanied by labeling from other VIIIth nerve components 2,3. Here, we describe a method for anterograde tracing from the acoustic ganglion to selectively label auditory axons within the developing VIIIth nerve. First, after partial dissection of the anterior cephalic region of an 8-day chick embryo immersed in oxygenated artificial cerebrospinal fluid, the cochlear duct is identified by anatomical landmarks. Next, a fine pulled glass micropipette is positioned to inject a small amount of rhodamine dextran amine into the duct and adjacent deep region where the acoustic ganglion cells are located. Within thirty minutes following the injection, auditory axons are traced centrally into the hindbrain and can later be visualized following histologic preparation. This method provides a useful tool for developmental studies of peripheral to central auditory circuit formation.

Here we describe a microdissection technique followed by fluorescent dye injection into the acoustic ganglion of early chick embryos for selective tracing of auditory axon fibers in the nerve and hindbrain.

Selektive Tracing von Auditory Fibers im Avian Embryonale vestibulocochlearis

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling ...

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram recordings. This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments, allowing recording of neural activity. Immobilization of the adult requires a mechanism to deliver dissolved oxygen to the gills in lieu of buccal and opercular movement. With our technique, animals are immobilized and perfused with habitat water to fulfill this requirement. A craniotomy is performed under tricaine methanesulfonate (MS-222; tricaine) anesthesia to provide access to the brain. The primary electrode is then positioned within the craniotomy window to record extracellular brain activity. Through the use of a multitube perfusion system, a variety of pharmacological compounds can be administered to the adult fish and any alterations in the neural activity can be observed. The methodology not only allows for observations to be made regarding changes in neurological activity, but it also allows for comparisons to be made between larval and adult zebrafish. This gives researchers the ability to identify the alterations in neurological activity due to the introduction of various compounds at different life stages.

This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

Elektrophysiologische Aufnahme im Gehirn von erwachsenen Zebrafisch Intact

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram ...

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional afferent fibers monitor other characteristics of the muscle environment, including metabolite buildup, temperature, and nociceptive stimuli. Overall, abnormal activation of sensory neurons can lead to movement disorders or chronic pain syndromes. We describe the isolation of the extensor digitorum longus (EDL) muscle and nerve for in vitro study of stretch-evoked afferent responses in the adult mouse. Sensory activity is recorded from the nerve with a suction electrode and individual afferents can be analyzed using spike sorting software. In vitro preparations allow for well controlled studies on sensory afferents without the potential confounds of anesthesia or altered muscle perfusion. Here we describe a protocol to identify and test the response of muscle spindle afferents to stretch. Importantly, this preparation also supports the study of other subtypes of muscle afferents, response properties following drug application and the incorporation of powerful genetic approaches and disease models in mice.

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

Muscle sensory neurons are involved in proprioceptor signaling and also report on metabolic state and injury related events. We describe an adult mouse in vitro muscle-nerve preparation for studies on stretch-activated muscle afferents.

Ein<em&gt; In-vitro-</em&gt; Adult Maus Muskel-Nerven Vorbereitung für das Studium der Brenneigenschaften des Muskel-Afferenzen

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional ...

In Vitro Recording of Mesenteric Afferent Nerve Activity in Mouse Jejunal and Colonic Segments

Afferent nerves not only convey information concerning normal physiology, but also signal disturbed homeostasis and pathophysiological processes of the different organ systems from the periphery towards the central nervous system. As such, the increased activity or &#39;sensitization&#39; of mesenteric afferent nerves has been allocated an important role in the pathophysiology of visceral hypersensitivity and abdominal pain syndromes.
Mesenteric afferent nerve activity can be measured in vitro in an isolated intestinal segment that is mounted in a purpose-built organ bath and from which the splanchnic nerve is isolated, allowing researchers to directly assess nerve activity adjacent to the gastrointestinal segment. Activity can be recorded at baseline in standardized conditions, during distension of the segment or following the addition of pharmacological compounds delivered intraluminally or serosally. This technique allows the researcher to easily study the effect of drugs targeting the peripheral nervous system in control specimens; besides, it provides crucial information on how neuronal activity is altered during disease. It should be noted however that measuring afferent neuronal firing activity only constitutes one relay station in the complex neuronal signaling cascade, and researchers should bear in mind not to overlook neuronal activity at other levels (e.g., dorsal root ganglia, spinal cord or central nervous system) in order to fully elucidate the complex neuronal physiology in health and disease.
Commonly used applications include the study of neuronal activity in response to the administration of lipopolysaccharide, and the study of afferent nerve activity in animal models of irritable bowel syndrome. In a more translational approach, the isolated mouse intestinal segment can be exposed to colonic supernatants from IBS patients. Furthermore, a modification of this technique has been recently shown to be applicable in human colonic specimens.

In Vitro Recording of Mesenteric Afferent Nerve Activity in Mouse Jejunal and Colonic Segments

Mesenteric afferent nerves convey information from the gastrointestinal tract towards the brain regarding normal homeostasis as well as pathophysiology. Gastrointestinal afferent nerve activity can be assessed by mounting isolated intestinal segments with attached afferent nerves into an organ bath, isolating the nerve, and assessing basal as well as stimulated activity.

In - vitro - Aufnahme von Mesenteriale afferenten Nervenaktivität in Maus - Jejunal und Kolon - Segmente

Afferent nerves not only convey information concerning normal physiology, but also signal disturbed homeostasis and pathophysiological processes of the ...

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the pathophysiological basis of these diseases, it is necessary to precisely characterize in which way the processing of information is disturbed between the different neuronal parts of the circuit. Using extracellular in vivo electrophysiological recordings, it is possible to accurately delineate neuronal activity within a neuronal network. The application of this method has several advantages over alternative techniques, e.g., functional magnetic resonance imaging and calcium imaging, as it allows a unique temporal and spatial resolution and does not rely on genetically engineered organisms. However, the use of extracellular in vivo recordings is limited since it is an invasive technique that cannot be universally applied. In this article, a simple and easy to use method is presented with which it is possible to simultaneously record extracellular potentials such as local field potentials and multiunit activity at multiple sites of a network. It is detailed how a precise targeting of subcortical nuclei can be achieved using a combination of stereotactic surgery and online analysis of multi-unit recordings. Thus, it is demonstrated, how a complete network such as the hyperdirect cortico-basal ganglia loop can be studied in anesthetized animals in vivo.

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

In this study, the methodology is presented on how to perform multi-site in vivo electrophysiological recordings from the hyperdirect pathway under urethane anesthesia.

Akut<em&gt; In vivo</em&gt; Elektrophysiologische Aufzeichnungen von lokalen Feldpotentialen und Multi-Unit-Aktivitäten von der Hyperdirect-Pathway in anästhesierten Ratten

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the ...

Wireless Electrophysiological Recording of Neurons by Movable Tetrodes in Freely Swimming Fish

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain activity from freely moving fish would advance research on the neural basis of fish behavior considerably. Moreover, precise control of the recording location in the brain is critical to studying coordinated neural activity across regions in fish brain. Here, we present a technique that records wirelessly from the brain of freely swimming fish while controlling for the depth of the recording location. The system is based on a neural logger associated with a novel water-compatible implant that can adjust the recording location by microdrive-controlled tetrodes. The capabilities of the system are illustrated through recordings from the telencephalon of goldfish.

A novel wireless technique for recording extracellular neural signals from the brain of freely swimming goldfish is presented. The recording device is composed of two tetrodes, a microdrive, a neural data logger, and a waterproof case. All parts are custom-made except for the data logger and its connector.

Drahtlose elektrophysiologische Aufzeichnung von Neuronen durch bewegliche Tetrodes in frei schwimmenden Fischen

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain ...