The authors sincerely thank the Canadian Institutes of Health Research MOP 130258 and the Star Foundation for Children&#8217;s Health Research, along with the Molly Towell Foundation, for the provision of the research facility and financial support. The authors also sincerely thank Dr. Kinkead Richard for manuscript proofreading and advice.

Diese Methode erforderte den Einsatz von Versuchstieren, die von der Universität Laval Tierethikkommission (Protokoll # 2012-170) erlaubt. 1. Aufbau und Vorbereitung <ol><li> Lösungen <ol><li> Bereiten aCSF Stammlösungen gemäß den folgenden Rezepturen 7,17. Weitere Rezepte mit Konzentrationsschwankungen sind in der Literatur. Shop-Stammlösungen bei 4 ° C für bis zu einem Monat. <ol><li> Salzlösung: add 75,39 g NaCl (129 mM final); 2,5 g KCl (3,35 mM final); 0,81 g NaH2PO4 (0,58 mM final); 2,33 g MgCl &amp; sub2; (1,15 mM final); 1,85 g CaCl2 (1,26 mM). Man löst in 800 ml destilliertem Wasser, dann mit 1 l mit destilliertem Wasser zu füllen. </li><li> Bicarbonat-Lösung: fügen 17,65 g NaHCO 3 (21 mM final). Man löst in 900 ml destilliertem Wasser, dann auf 1 Liter mit destilliertem Wasser zu füllen. Variationen der Bicarbonat-Konzentration wird im pH-Schwankungen führen. </li><li> Glucose-Lösung: fügen 54,06g Glucose (30 mM Endkonzentration). Man löst in 400 ml destilliertem Wasser und dann auf 500 ml mit destilliertem Wasser zu füllen. </li></ol></li><li> Vorbereitung aCSF durch Verdünnen mit 100 ml Salzlösung, 100 ml Natriumbicarbonat-Lösung und 50 ml Glukoselösung in 1 l destilliertem Wasser. Lagerung bei 4 ° C bis zur Verwendung. </li><li> Bereiten Sie eine Glaselektrode durch Erwärmen und Strecken einer Glasröhre, bis er bricht. Sand auf der Spitze vor dem Gebrauch. Die Elektrode kann viele Zeit wiederverwendet werden, solange es sauber bleibt. </li></ol></li><li> Versuchsaufbau Hinweis: Die Setup-Details sind in Abbildung 2 dargestellt. <ol><li> Einschalten des Verstärkers, Bewegen Mittelwertbildner, Datenerfassungssystem, einem Temperaturregler und der Pumpe. Sie können die Signalverstärkung (Verstärkung = 10.000), die Filtration (niedrige Schwelle, 10 Hz; hohe Schwelle, 5 kHz) und die Abtastrate des Analog-Digital-Umwandlung des Rohsignals (2,5 kHz). </li><li> Füllen Sie eine Flasche mit aCSF und blasen es mit Carbogen (95% O 2 </sub&gt;, 5% CO 2). Induzieren eine bubbled- und erwärmten aCSF Fluss in der Aufzeichnungskammer (Volumen 5 ml) bei 4 ml / min. Halten der Temperatur in dem Aufnahmeraum auf 26 (± 1) ° C. pH-Wert sollte 7,4 in diesen Standardbedingungen ist. </li><li> Halten Sie 50 ml RT und Carbogen blasigen aCSF in einem 50-ml-Spritze in der Nähe der Dissektion Kammer. </li><li> Schalten Sie den Computer und starten Sie die Aufnahme-Software. </li></ol></li></ol> 2. Dissection <ol><li> Wiegen und das Geschlecht des Tieres visuell zu bestimmen (Männer haben eine längere anogenitalen Abstand, während Frauen haben eine kurze anogenitalen Abstand; Männchen Genitalien sind oft dunkel, während weiblichen Genitalien sind rosa). </li><li> Betäuben die neugeborenen Nagern durch eine der folgenden Optionen: cryoanesthesia (das Tier im Eis vollständig einzutauchen für 4 - 5 min 18), Injektion (equitensine bei 4 ml / kg) 19 oder Einatmen von volatilen Anästhetika (Isofluran 20 oder Ether-21). Konfirm ein ausreichender Ebene Anästhesie durch das Fehlen einer Pfotenrückzugsreflex. </li><li> Legen Sie das Tier auf der Bank, Bauchseite nach unten. Abschnitt der rostralen Teil des Kopfes koronal mit einem Skalpell am Bregma Ebene (durch die Haut und Schädel sichtbar). Führen Sie diesen Schritt unmittelbar nach dem das Tier narkotisiert. </li><li> Abschnitt der Körper koronal mit einem Skalpell unter den vorderen Mitglieder. </li><li> Entfernen Sie Haut, Muskeln, Fettgewebe und Eingeweide mit chirurgischen und dünne Scheren und Zangen. Da die Knochen sind weich in diesem Alter, seien Sie vorsichtig, um das Nervensystem nicht beschädigen. Führen Sie diesen Schritt auf der Bank. </li><li> Legen Sie die Zubereitung in der Dissektion Kammer. Verwenden Sie die aCSF in der Spritze aufbewahrt, um die Herstellung mit Sauerstoff zu versorgen. Von diesem Punkt an das Ende der Aufzeichnung, mit einem Mikroskop. </li><li> Auf der Rückenseite der Vorbereitung, schneiden Sie den Schädel und Wirbel von der rostral zur Schwanzteil entlang der mittleren Achse mit einer chirurgischen und dünne Scheren und Zangen. Öffnen Sie die Schnitt Schädelund Wirbeln, um das Nervengewebe freizulegen. Verwenden Sie die aCSF in der Spritze strored, um die Herstellung mit Sauerstoff zu versorgen. </li><li> Mit der Zange, entfernen Sie die Arachnoidea, eine, die die Oberfläche des Nervengewebes dünne Gewebe. Bewahren Sie die Pia mater und der Blutgefäße gegenüber dem Nervengewebe. Verwenden Sie die aCSF in der Spritze aufbewahrt, um die Herstellung mit Sauerstoff zu versorgen. </li><li> Platzieren Sie das Präparat mit der Rückenseite nach unten, und sorgfältig bringen den Hirnstamm und Rückenmark, indem die Nerven und Bindegewebe mit der Schere, während Sie die Zubereitung vorhanden. Ein dorsalen Zugang ist ebenfalls möglich. Halten Sie die Wurzeln und Nerven so lange wie möglich. Entfernen Sie die Knochen, um den Hirnstamm und Rückenmark zu isolieren. Verwenden Sie die aCSF in der Spritze aufbewahrt, um die Herstellung mit Sauerstoff zu versorgen. </li><li> Entfernen Sie das Kleinhirn und Rest rostral Strukturen durch Schneiden sie mit dem Skalpell. Verwenden Sie die aCSF im Nadel gespeichert werden, um die Herstellung mit Sauerstoff zu versorgen. </li><li> Gegebenenfalls in Abhängigkeit experimental Design, entfernen Sie die Brücke mit dem Skalpell durch einen Frontalschnitt ventral des unteren Kleinhirnarterie 22. Beachten Sie, dass das Halten der gesamten Brücke wird den Rhythmus bei Ratten zu verlangsamen und vollständig zu hemmen sie in Mäusen. Präparate, die nur das Schwanzteil der Brücke sind zeigen eine Atem-ähnliche Aktivität mit einer stabilen Frequenz am C4 Wurzel. </li></ol> 3. Aufnahme <ol><li> Legen Sie die Zubereitung in der Aufnahme Kammer, Bauchseite nach oben. Befestigen Sie die Vorbereitung mit Stiften im untersten Teil des Rückenmarks und rostral-größten Teil des Hirnstamms. </li><li> Unter Verwendung einer Spritze zu der Elektrode durch die Nadel verbunden ist, induzieren eine Vertiefung in der Elektrode (Glas Spitze einen Durchmesser von 150 bis 225 um) durch Herausziehen des Spritzenkolbens, um die Elektrode teilweise zu füllen mit aCSF. </li><li> Mit einem Mikromanipulator, vorsichtig die Elektrode in der Nähe von einer der vierten Vorderwurzeln. Andere ventralen Wurzeln könnte auch präsentieren eine Atem-ähnliche Aktivität (z.g., XII Hirnnerv, C1 ventralen Wurzel). </li><li> Induzieren eine Vertiefung in der Elektrodentank über der Spritze durch Herausziehen des Kolbens, um sanft saugen Sie den Nerven rootlet. Dann vorsichtig die Elektrode zu bewegen, um es gegen das Rückenmark an. Anmerkung: Idealerweise wird die Größe des rootlet passt die Größe der Elektrodenöffnung und schafft so eine Dichtung zwischen den inneren und äußeren Kammern der Elektrode. Da Differenzverstärker werden üblicherweise für solche Aufzeichnungen verwendet, jede Öffnung zwischen der Innenseite der Elektrode und dem Aufzeichnungsraum reduziert die Qualität des Signals und macht es schwierig, Hintergrundrauschen zu beseitigen. </li><li> Starten Sie die Aufnahme. </li><li> Aufzeichnung der durch die Herstellung unter normoxischen Bedingungen hergestellt Rhythmus (dh geperlt aCSF mit Carbogen: 95% O 2 und 5% CO 2) für mindestens 20 min, um die Basislinienparameter der Zubereitung bestimmen. </li><li> Schalten Sie die Perfusion von Carbogen blasigen aCSF zuaCSF Stimulus (dh mit 95% N 2 und 5% CO 2 für Hypoxie Stimulus perlt) für 15 min. Alter Expositionsdauer in Abhängigkeit der Versuchsprotokoll. Notieren Sie sich die Expositionsdauer auf das Aufzeichnungs und Tier Datenblatt. Verwenden Edelstahlrohre zwischen aCSF Flasche und der Kammer, wenn möglich, Gasdiffusion zu der Außenseite des Rohres zu vermeiden. </li><li> Schalten Sie die Durchblutung wieder in Standard Carbogen blasigen aCSF für mindestens 15 Minuten auf eine Erholung Aufnahme. Nehmen Sie diese auf dem Aufzeichnungs und Tier Datenblatt. </li><li> Beenden Sie die Aufnahme. </li></ol> 4. Statistische Analyse <ol><li> Aus dem integrierten Signal, die Berechnung der Frequenz wie die Anzahl der Bursts pro Minute (in Bursts / min ausgedrückt wird), die Amplitude der Differenz zwischen dem Ausgangswert und der Spitze des Bursts (in mV), die Burstdauer als die Dauer von Anfang bis zum Ende des Bursts (ausgedrückt in s), die Burst-Fläche wie die Fläche unter der curve des Bursts auf dem integrierten Signal (in mV · s) (Abbildung 3). </li><li> Berechnen Sie die Frequenz, Amplitude, Burstdauer und Burst-Bereich unter normoxischen (Baseline), hypoxische (Stimulus) und Post-hypoxischen (post-Stimulus) Erholung Bedingungen wie der Durchschnitt der letzten 5 Minuten der Aufnahmen für jede Bedingung. Analyse nicht den ersten Abschnitt einer jeden Zustand, da es eine Verzögerung zwischen dem Einschalten der Perfusion und aCSF Homogenisierung im Aufnahmeraum. </li><li> Express dann Stimulus (dh Hypoxie) und Post-Stimulus (dh post-hypoxischen) Recovery-Werte als Prozentsatz der Baseline-Werte für die entsprechende Aufnahme. Die Ergebnisse sind als Mittelwert ± SD </li></ol>

<ol>
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M., Paton, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizations+of+eupnea,+apneusis+and+gasping+in+a+perfused+rat+preparation.">Characterizations of eupnea, apneusis and gasping in a perfused rat preparation.</a> Respir Physiol. 123, 201-213 (2000).</li></ol>

The authors have no competing financial interests or conflicts of interests to disclose.

Genaue Quantifizierung der Atmungsaktivität kann eine Herausforderung sein. In der Tat ist die Atmung eine Funktion, die automatische und freiwillig sein kann, und das ist entsprechend der Umgebung, die Bedürfnisse des Körpers, den emotionalen Zustand und das Verhalten moduliert. Der Vorteil dieser Technik ist die Isolierung von den neuronalen Elementen zur Erzeugung des Atembefehl verantwortlich. Somit elektrophysiologische Aufzeichnungen von Hirnstamm-Rückenmarkspräparaten und Plethysmographie komplementäre Tech...

Die Atmung ist eine komplexe und Lebenstätigkeit vom Gehirn gesteuert, so dass Disauerstoff (O 2) Aufnahme und Kohlendioxid (CO 2) Beseitigung. Der zentraler Atemantrieb wird durch ein komplexes Netzwerk im Hirnstamm sowohl in Säugetieren als 1 liegt, Amphibien 2, Reptilien 3, 4 Vögel und Fische 5 generiert. Auch wenn die Untersuchung der Atmung kann in vivo verarbeitet werden, präzise mechanistische Untersuchungen erfordern einen direkten Zugang zu dem Atemsteuerungsnetz. Hierzu Adrian und Buytendijk entwickelt eine reduzierte Goldfischzubereitung, bei dem Elektroden auf der Hirnoberfläche aktenkundig den erzeugten Rhythmuskiemen Belüftung 5 verbunden. Dieser Ansatz wurde später von Suzue 1984 6 zur Verwendung bei neugeborenen Nagern angepasst. Das Aufkommen dieser Zubereitung zu erheblichen Fortschritten in der Atem Neurobiologie geführt. Da es relativ einfach ist, hat die Technik here zugänglich ist eine breite Palette von Grundlagenuntersuchungen der rhythmische motorische Verhaltensweisen und ihren Ursprung in der neugeborenen Nagern. Das allgemeine Ziel dieser Methode ist es, das neuronale Korrelat inspiratorischen Aktivität aufzuzeichnen, eine Atemwegsartigen Rhythmus genannte fiktive Atmung, ausgelöst durch den Atem Netzwerk hergestellt. Dieses Verfahren kann in einem breiten Spektrum von Forschungsziele eingesetzt werden, Targeting Inspirations antwortet Atemschwankungen oder Pharmakologie in beiden Wildtyp 7 und 8 transgenen Tieren. Da die Versuche werden bei einer niedrigen Temperatur durchgeführt wird, ohne afferenten Neuronen, und unter Bedingungen, bei denen die Konzentrationen von Glucose und O 2 in der aCSF hoch sind Fragen aufgeworfen über die physiologische Bedeutung des aufgezeichneten Signals. Zwar gibt es deutliche Unterschiede zwischen den in-vivo- und in-vitro-Bedingungen (z. B. die Frequenz des inspiratorischen Bursts) bleibt die Tatsache, dass das Vorhandensein vondie Kernelemente der Atemnetz 6 ist es möglich, eine robuste Rhythmus zu einer lebenswichtigen homöostatischen Funktion 9,10 assoziiert zu studieren. Das Grundprinzip hinter der Entwicklung und der Einsatz dieser Technik ist es, einen direkten Zugang zum Hirnstamm Elemente des Atem Netzwerk, das in vivo schwer zugänglich sind, insbesondere bei Neugeborenen zu erleichtern. Der Hirnstamm unter streng kontrollierten Bedingungen ausgesetzt: das aufgezeichnete Rhythmus nicht von peripheren afferenten Inputs der Lunge oder der Halsschlagkörpern moduliert, so dass die Untersuchung auf den zentralen Atemantrieb selbst 11 konzentrieren. Somit ist dieser Zugang genutzt, um Reize gelten und notieren Sie die Ausgangssignal. Im Gegensatz zu Aufzeichnungen Plethysmographie wird der Atemrhythmus durch alle ihre Komponenten im ganzen Körper moduliert (z. B. Lungendehnung, periphere Chemosensoren) und macht es schwierig, eine genaue Stimuli anzuwenden. In einem (newborn Ratte, das Protokoll aus der Aufzeichnung der vierten ventralen Wurzel Signal auf einem isolierten Hirnstamm und ein verkürztes Rückenmark, in künstlichen Cerebrospinalflüssigkeit (aCSF) gehalten. Die von Hirnstamm-Rückenmarkspräparationen erzeugten Rhythmus der einzelnen langsamen Bursts, die auf der Inspirationssignal 9 verknüpft sind. Isolated Hirnstamm-Rückenmarkspräparationen sind leicht bei Ratten bespielbar von postnatalen Tag 0-4 (P0 - P4) 7. Dieser Ansatz wird häufig verwendet, um die hypoxische Reaktion der Atem Netzwerk und auch die Antwort auf Hyperkapnie, Azidose oder Arzneimittel zu bewerten. Eine akute Hypoxie Protokoll wird hier vorgestellt. Diese Stimulation wird durch Entnahme von O 2 in der aCSF erhalten; Dieser Ansatz wird häufig verwendet, um die Toleranz und Ansprechbarkeit auf hypoxische Beleidigungen beurteilen. Das Protokoll verursacht eine Rhythmus Vertiefung von der ersten Minute, bis zum Ende der Belichtung Hypoxie (1) 12. Diese Vertiefung wird umgekehrtwährend der post-hypoxischen Recovery 12. Bezüglich der Versuchsanordnung ist es wichtig, zu bemerken, daß die Brücke, an der rostralen Teil der Hirnstamm, eine hemmende Wirkung auf den Rhythmusgenerator 8. So Zubereitungen der volle Hirnstamm und Rückenmark rostral zeigen eine geringere Rhythmus. Einbeziehung der Brücke in der isolierten Probe auf die Aufzeichnung wird entsprechend dem Ziel des Experiments 13 bestimmt wird; das Studium der pontine Einfluss auf das verlängerte Mark Netz erfordern würde Aufnahmen mit und ohne die Brücke, um die Ergebnisse 14 zu vergleichen. Außerdem ist einer der Vorteile dieser Technik die Möglichkeit einer Ausdehnung des rostralen Teil der Vorbereitung auf Mesencephalon-und / oder Zwischenhirnregionen 15,16 umfassen, die es ermöglicht, die Wirkung dieser Regionen auf dem ponto-medullären Atemnetz bewerten.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Sylgard</td>
			<td>Sigma Aldrich</td>
			<td>761036-5EA</td>
			<td>Use under hood</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Bioshop</td>
			<td>SOD002</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Bioshop</td>
			<td>POC888</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Bioshop</td>
			<td>CCL444</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Bioshop</td>
			<td>MAG510</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Bioshop</td>
			<td>SOB999</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Bioshop</td>
			<td>SPM306</td>
			<td></td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Bioshop</td>
			<td>GLU501</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbogen</td>
			<td>Linde</td>
			<td>343-02-0006&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature Controller</td>
			<td>Warner Instruments, Hamden, CT, USA</td>
			<td>TC-324B</td>
			<td></td>
		</tr>
		<tr>
			<td>Suction electrode</td>
			<td>A-M Systems, Everett, WA, USA</td>
			<td>model 573000</td>
			<td></td>
		</tr>
		<tr>
			<td>Differential AC amplifier</td>
			<td>A-M Systems, Everett, WA, USA</td>
			<td>model 1700</td>
			<td></td>
		</tr>
		<tr>
			<td>Moving averager</td>
			<td>CWE, Ardmore, PA, USA</td>
			<td>model MA-821</td>
			<td></td>
		</tr>
		<tr>
			<td>Data acquisition system</td>
			<td>Dataq Instruments, Akron, OH, USA</td>
			<td>model DI-720</td>
			<td></td>
		</tr>
		<tr>
			<td>LabChart software</td>
			<td>ADInstruments, Colorado Springs, CO, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Prism sofware</td>
			<td>Graphpad, La Jolla, CA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection chamber</td>
			<td>Plastic box (e.g. petri box) will do</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Recording chamber</td>
			<td>Home made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Base</td>
			<td>Kanetec, Bensenville, IL, USA</td>
			<td>MB</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>World Precision Instrument Inc, Sarasota, FL, USA</td>
			<td>KITE-R</td>
			<td></td>
		</tr>
		<tr>
			<td>Base</td>
			<td>Kanetec, Bensenville, IL, USA</td>
			<td>MB</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Gilson, Middleton, WI, USA</td>
			<td>MINIPULS 3</td>
			<td></td>
		</tr>
		<tr>
			<td>Faraday Cage</td>
			<td>Home made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Computer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

electrophysiology on isolated brainstem-spinal cord preparations from newborn rodents allows neural respiratory network output recording

While it is well known that the central respiratory drive is located in the brainstem, several aspects of its basic function, development, and response to stimuli remain to be fully understood. To overcome the difficulty of accessing the brainstem in the whole animal, isolation of the brainstem and part of the spinal cord is performed. This preparation is maintained in artificial cerebro-spinal fluid where gases, concentrations, and temperature are controlled and monitored. The output signal from the respiratory network is recorded by a suction electrode placed on the fourth ventral root. In this manner, stimuli can be directly applied onto the brainstem, and the effect can be recorded directly. The signal recorded is linked to the inspiratory signal sent to the diaphragm via the phrenic nerve, and can be described as bursts (around 8 bursts per minute). Analysis of these bursts (frequency, amplitude, length, and area under the curve) allows precise characterization of the stimulus effect on the respiratory network. The main limitation of this method is the viability of the preparation beyond the early post-natal stages. Thus, this method greatly focuses on the study of the whole network without the peripheral inputs in the newborn rat.

Wie in der Einleitung erwähnt, ist einer der wichtigsten Vorteile dieser Technik die direkten Zugang zum Hirnstamm verschiedene Stimuli anzuwenden. Als Beispiel wurde hier Hypoxie aufgetragen. Abbildung 1. AB zeigt eine vollständige Protokollaufzeichnung, mit beiden normoxischen und hypoxischen Bedingungen. Abbildung 1.CE zeigt den Rhythmus in normoxischen Bedingungen (dh aufgezeichnet, sprudelte aCSF mit 95% O 2 und 5% CO 2 bei 26 ° C). Wie zuvor in diesem genaue...

Watch this Scientific Journal Video about Electrophysiology on Isolated Brainstem-spinal Cord Preparations from Newborn Rodents Allows Neural Respiratory Network Output Recording at JoVE.com

Electrophysiology on Isolated Brainstem-spinal Cord Preparations from Newborn Rodents Allows Neural Respiratory Network Output Recording

The central respiratory drive is located in the brainstem. Spontaneous respiratory motor output from an isolated brainstem-spinal cord is recorded by placing an electrode on the fourth ventral root. This experimental approach is valuable for pharmacological investigations or the assessment of respiratory challenges and genetic manipulations on rhythmic motor behavior.

Elektrophysiologie auf isolierte Hirnstamm-Rückenmarks Zubereitungen aus Newborn Nagetiere Ermöglicht Neural Atem Netzwerk Output Recording

While it is well known that the central respiratory drive is located in the brainstem, several aspects of its basic function, development, and response to ...

electrophysiology-on-isolated-brainstem-spinal-cord-preparations-from

Research

JoVE Journal

Neuroscience

8.5K Aufrufe.  Laval University. The central respiratory drive is located in the brainstem. Spontaneous respiratory motor output from an isolated brainstem-spinal cord is recorded by placing an electrode on the fourth ventral root. This experimental approach is valuable for pharmacological investigations or the assessment of respiratory challenges and genetic manipulations on rhythmic motor behavior.

Serie: Electrophysiology on Isolated Brainstem-spinal Cord Preparations from Newborn Rodents Allows Neural Respiratory Network Output Recording

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Dissection und Kultur kommissuralen Nervenzellen aus embryonalen Rückenmark

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Forschung

Neurowissenschaften

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

Spinal Cord Elektrophysiologie II: Extrazelluläre Saugelektrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Examining Local Network Processing using Multi-contact Laminar Electrode Recording

Cortical layers are ubiquitous structures throughout neocortex1-4 that consist of highly recurrent local networks. In recent years, significant progress has been made in our understanding of differences in response properties of neurons in different cortical layers5-8, yet there is still a great deal left to learn about whether and how neuronal populations encode information in a laminar-specific manner.
Existing multi-electrode array techniques, although informative for measuring responses across many millimeters of cortical space along the cortical surface, are unsuitable to approach the issue of laminar cortical circuits. Here, we present our method for setting up and recording individual neurons and local field potentials (LFPs) across cortical layers of primary visual cortex (V1) utilizing multi-contact laminar electrodes (Figure 1; Plextrode U-Probe, Plexon Inc).
The methods included are recording device construction, identification of cortical layers, and identification of receptive fields of individual neurons. To identify cortical layers, we measure the evoked response potentials (ERPs) of the LFP time-series using full-field flashed stimuli. We then perform current-source density (CSD) analysis to identify the polarity inversion accompanied by the sink-source configuration at the base of layer 4 (the sink is inside layer 4, subsequently referred to as granular layer9-12). Current-source density is useful because it provides an index of the location, direction, and density of transmembrane current flow, allowing us to accurately position electrodes to record from all layers in a single penetration6, 11, 12.

A fundamental issue in our understanding of cortical circuitry is how networks in different cortical layers encode sensory information. Here we describe electrophysiological techniques utilizing multi-contact laminar electrodes to record single-units and local field potentials and present analyses to identify cortical layers.

Untersuchen Local Network Processing mit Multi-Contact-Laminar Electrode Recording

Cortical layers are ubiquitous structures throughout neocortex1-4 that consist of highly recurrent local networks. In recent years, significant progress ...

Large-scale Recording of Neurons by Movable Silicon Probes in Behaving Rodents

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of neurons and circuits requires methods with the spatial selectivity and temporal resolution appropriate for mechanistic analysis of neural ensembles in the behaving animal, i.e. recording of representatively large samples of isolated single neurons. Ensemble monitoring of neuronal activity has progressed remarkably in the past decade in both small and large-brained animals, including human subjects1-11. Multiple-site recording with silicon-based devices are particularly effective because of their scalability, small volume and geometric design.
Here, we describe methods for recording multiple single neurons and local field potential in behaving rodents, using commercially available micro-machined silicon probes with custom-made accessory components. There are two basic options for interfacing silicon probes to preamplifiers: printed circuit boards and flexible cables. Probe supplying companies (<a href="http://www.neuronexustech.com/" >http://www.neuronexustech.com/</a>; <a href="http://www.sbmicrosystems.com/">http://www.sbmicrosystems.com/</a>; <a href="http://www.acreo.se/">http://www.acreo.se/</a>) usually provide the bonding service and deliver probes bonded to printed circuit boards or flexible cables. Here, we describe the implantation of a 4-shank, 32-site probe attached to flexible polyimide cable, and mounted on a movable microdrive. Each step of the probe preparation, microdrive construction and surgery is illustrated so that the end user can easily replicate the process.

We describe methods for large-scale recording of multiple single units and local field potential in behaving rodents with silicon probes. Drive fabrication, probe attachment to the drive and probe implantation processes are illustrated in sufficient details for easy replication.

Großflächige Aufnahme von Neuronen durch Movable Silicon Probes in Behaving Nagetiere

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

Retrograde Fluoreszenzmarkierung Ermöglicht Gezielte Extracellular Einzel-Einheit Aufnahme von identifizierten Neuronen<em&gt; In vivo</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

In Vivo Zwei-Photonen-Imaging Of Experience-abhängigen molekularen Veränderungen in kortikalen Neuronen

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Neural Circuit Recording from an Intact Cockroach Nervous System

The cockroach ventral nerve cord preparation is a tractable system for neuroethology experiments, neural network modeling, and testing the physiological effects of insecticides. This article describes the scope of cockroach sensory modalities that can be used to assay how an insect nervous system responds to environmental perturbations. Emphasis here is on the escape behavior mediated by cerci to giant fiber transmission in Periplaneta americana. This in situ preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of neuronal activity. Peptides or other chemical reagents can then be applied directly to the nervous system in solution with the physiological saline. Insecticides could also be administered prior to dissection and the escape circuit can serve as a proxy for the excitable state of the central nervous system. In this context the assays described herein would also be useful to researchers interested in limb regeneration and the evolution of nervous system development for which P. americana is an established model organism.

This article describes the cockroach ventral nerve cord dissection and extracellular recordings from the cercal nerve and connectives. Evoked responses are generated by electrical stimulation of the cercal nerve or direct mechanical stimulation of the cerci.

Neuronalen Schaltkreis-Aufnahme von einem intakten Nervensystem Schabe

The cockroach ventral nerve cord preparation is a tractable system for neuroethology experiments, neural network modeling, and testing the physiological ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

Zeitabhängige Erhöhung der Netzwerkreaktion auf die Stimulation von neuronalen Zellkulturen auf Mikroelektroden-Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

Neuronalen Aktivität im primären somatosensorischen Cortex mit Thy1Imaging-GCaMP6s transgene Mäuse

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...