Unterstützt von der NIH Grant R01 MH 099557

1. Herstellung der Aufnahme Elektroden <ol><li><ol><li>Gebrauch der folgenden ziehen die Glaselektroden Protokoll für die Mikroelektrode Puller (siehe Tabelle der Materialien): <ol><li>Linie 1: Wärme 510 ziehen - Geschwindigkeit 30 mal 250; Linie 2: Erhitzen 490 Pull - Geschwindigkeit 30 mal 250. 
			Hinweis: Zeiteinheiten entsprechen 0,5 ms pro Einheit; die anderen Geräte sind relativ. Der Wert der Wärme sollte für jedes Filament angepasst werden, nachdem die Rampe-Test durchgeführt wird.</li>
		</ol></li> <li>Mikroskop (35 X Vergrößerung) verwenden, um sicherzustellen, dass der Innendurchmesser der gezogenen Elektrode im Bereich von 7 bis 10 µm ( Abbildung 1A). Speichern Sie die Kapillaren in dicht verschlossenen Behältern, Staub zu verhindern.</li>
	</ol></li> <li>Polieren Feuer <ol><li>Fire Polnisch Kapillaren (80-90 % der maximalen Heizwert für 1-2 s) mit einer Mikro-Schmiede (siehe Tabelle der Materialien). Sicherzustellen, dass der endgültige Innendurchmesser der polierten Elektrode 5 µm ( Abbildung 1A) 27.</li>
	</ol></li> <li>Biegen Hinweis: zwei Biegungen werden vorgenommen, um die Elektrode an der Oberseite des Muskels unter einer hohen Vergrößerung Ziel zu positionieren.
	<ol><li>Verwendung der Apparat in Figur 1 b gezeigt. Korrigieren Sie die Elektrode in der Manipulator und positionieren Sie die Spitze über den Faden, nicht berühren, it.</li>
		<li>Set Erhitzen zu 60-70 % der maximalen Wert und drücken Sie das Pedal der Mikro-Schmiede für 1-2 s (siehe Tabelle der Materialien), das Filament zu heizen. Verwenden Sie eine L-förmigen Nadel vorsichtig herunterziehen der Elektrodenspitze ( Abbildung 1 b vergrößert). Die Biegemarkierung bei ca. 90 o ( Abbildung 1).</li>
		<li>Halten die Elektrode über die Flamme des Brenners von Hand mit Pinzette und machen die zweite Kurve in einem Abstand von 7-10 mm aus der ersten Kurve und in einem Winkel von ca. 120 o ( Abbildung 1).</li>
	</ol></li></ol> <img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56493/56493fig1.jpg" /> Abbildung 1. Letzte Schritte der Herstellung Mikropipette. (A) Elektrode Tipps nach ziehen und Feuer zu polieren. (B. 1) die Einrichtung für Spitze biegen. (B. 2) Box Bereich ist auf der rechten Seite vergrößert dargestellt. Die Elektrode und das Filament sind fixiert, so dass die Elektrodenspitze leicht über den Draht und nicht berührend ist. (C. 1) die Aufnahme-Elektrode. (C. 2) Box Bereich ist auf der rechten Seite vergrößert dargestellt. Die erste Kurve hat einen Winkel von etwa 90 Grad, und der Abstand zwischen der ersten Kurve und die Spitze der Elektrode ist ca. 1 mm. Maßstab bar = 3 mm. <a href="//ecsource.jove.com/files/ftp_upload/56493/56493fig1large.jpg" target="_blank"> Bitte klicken Sie hier für eine größere Version dieser Figur.</a>
 2. zusätzliche vorbereitende Schritte <ol><li>Prepare Heamolymph erinnernden (HL3) Lösung (in mM): 70 NaCl, 5 KCl, 20 MgCl 2 10 Nahco3 3, 5 Trehalose, 115 Saccharose, 5 HEPES und 1 mM CaCl 2; Einstellung des pH-Wertes auf 7,3 - 7.4. die Lösung im Kühlschrank aufbewahren und machen es jede Woche frisch.</li>
	<li>Stimulation Mehrkanalpipette die gleiche Art und Weise wie Aufnahme Glas machen Mehrkanalpipette, außer es keine Notwendigkeit gibt, sie verbiegen. 
	Hinweis: Die Vorbereitung der Stimulation Elektroden ist in 28 und 27 detailliert beschrieben. Den endgültigen Durchmesser nach Feuer polieren sollte im Bereich von 5 bis 7 µm.</li>
	<li>Insert eine Stimulation-Pipette in einer Mikroelektrode Halterung mit einer Spritze verbunden.</li>
	<li>Herstellung Dissektion Platten aus kleinen Petrischalen (35 x 10 mm) mit Silikon-Kautschuk-Epoxid beschichtet (siehe Tabelle der Materialien), wie unter 28. 
	Hinweis: Silikon-Kautschuk sollte vor Gebrauch vollständig ausgehärteten.</li>
	<li>Wählen Sie einen wandernden Larven dritte Instar und sezieren, wie beschrieben in 27 , 28 , 29 , 30. 
	Hinweis: Um Boutons visualisieren, verwenden die Fliege Belastung CD8-GLP (siehe Tabelle der Materialien) <ol><li>mit Zange (siehe Tabelle der Materialien), wählen Sie die 3 rd Instar Larven aus einem Fläschchen.</li>
		<li>Pin die Larven, die Platzierung des ersten Pins posterior und anderen pin anterior (Nähe Mund Haken).</li>
		<li>Lösung hinzufügen HL3.</li>
		<li>Machen Sie einen Schnitt mit Feder Schere (siehe Tabelle der Materialien) ganz aus den oberen Bolzen an der Unterseite eines an der dorsalen Seite der Larven.</li>
		<li>Pin-Larven-Filet, platzieren 2 zusätzliche Pins auf der linken und rechten Seiten der Larven.</li>
		<li>Die Eingeweide und Tracheas mit Pinzette entfernen.</li>
		<li>Die Nerven direkt vor den ventralen Nervenstrang mit Feder Schere geschnitten.</li>
	</ol></li></ol> 3. Elektrische Aufnahmen von EJCs <img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56493/56493fig2.jpg" /> Abbildung 2. Die Recording-Setup. Die Probe NMJ angeheftet, das Silizium beschichteten Petrischale (Pfeil) über die bewegliche Bühne des aufrechten Verbindung Mikroskops ausgestattet mit Epifluoreszenz Fähigkeiten, eine hohe Vergrößerung Objektive und zwei Mikromanipulatoren positioniert ist. Das Mikroskop ist auf eine Anti-Vibrationstisch stationiert. <a href="//ecsource.jove.com/files/ftp_upload/56493/56493fig2large.jpg" target="_blank"> Bitte klicken Sie hier für eine größere Version dieser Figur.</a>
<ol><li>Aufnahme <ol><li>Ort der Petrischale mit der Vorbereitung auf den Mikroskoptisch ( Abbildung 2). Legen Sie die Bezugselektrode in der Badewanne.</li>
		<li>Füllen Sie die Aufnahme-Elektrode mit HL3 Lösung. Unter dem Objektiv 10 X (siehe Tabelle der Materialien), die Elektrode in die Wanne Tauchen und legen Sie es über Muskeln 6 und 7 der Abdominalsegmente 2, 3 oder 4 verwenden der Mikromanipulator () (siehe Tabelle der Materialien) Abbildung 3 A. 1 und a. 2).</li>
		<li>Schalten Sie das Ziel auf 60 X (siehe Tabelle der Materialien). Konzentrieren Sie sich auf den gewünschten Bereich mit Epi-Fluoreszenz oder DIC Optik. Setzen Sie die Spitze der Elektrode auf synaptische Bouton ( Abbildung 3 b. 1-3).</li>
		<li>Drücken Sie die Elektrode sehr sanft auf den Muskel. Übermäßiger Druck kann den NMJ beschädigen oder induzieren eine Erhöhung in spontane synaptische Aktivität. Sicherstellen, dass die Spitze der Elektrode ist nicht verstopft - wenn es it. ersetzen,</li>
		<li>Schalten Sie den Verstärker, A/D-Board und dem Computer.</li>
		<li>Klemme Spannungsmodus am Verstärker wählen.</li>
		<li>Software zur Datenerfassung starten und wählen Sie die &#39; spaltfreie &#39; Modus.</li>
		<li>Das Aussehen der mEJCs auf dem Computerbildschirm zu beobachten.</li>
		<li>Stellen Sie sicher, das ist die Amplitude der mEJCs im Bereich von 0,2 - 0,7 na 
		Hinweis: Kleinere EJCs anzugeben, dass die Aufnahme Elektrode Mängel hat oder es nicht richtig positioniert ist.</li>
	</ol></li></ol> <img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56493/56493fig3.jpg" /> Abbildung 3. Visualisierung der synaptischen Boutons. (A) A Hellfeld Bild eines Hemi-Segments unter 10 x Vergrößerung (a. 1) und den erweiterten Box Bereich zeigt Muskeln 6 und 7 (a. 2, der Pfeil markiert die Aufnahme-Elektrode). Maßstabsleiste = 50 µm. (B) synaptischen Boutons sind in eine Drosophila-Linie mit einem genetisch codierte neuronalen Marker (CD8-GFP) visualisiert mit Epi-Fluoreszenz-Bildgebung (b. 1) oder DIC Optik (b. 2). Bilder sind mit 60 x Objektive und Filter-Cube für die GFP Bildgebung genommen (siehe Tabelle der Materialien). SynaptiC Boutons sind mit Pfeilen gekennzeichnet, und eine Überlagerung von Leuchtstofflampen und DIC Bildern zeigt sich in b. 3. Maßstabsleiste = 10 µm. <a href="//ecsource.jove.com/files/ftp_upload/56493/56493fig3large.jpg" target="_blank"> Bitte klicken Sie hier für eine größere Version dieser Figur.</a>
<ol start="2"> <li>Stimulation <ol><li>mit einem Mikromanipulator unter Sichtkontrolle, legen Sie die Stimulationselektrode in der Nähe der Axon innervieren Abdominalsegmente 2-4.</li>
		<li>Anwenden Unterdruck durch ziehen den Kolben der Spritze, Elektrodenhalter, verbunden, so dass das Axon, im Inneren der Elektrode ( Abbildung 2 gezogen wird).</li>
		<li>Schalten Sie den Stimulator. Drehen Sie den Knopf am Gerät isoliert (siehe Tabelle der Materialien) einen Null Strom festlegen und dann vorsichtig erhöhen, bis EJCs erscheinen (oder der Schwellenwert erreicht ist).</li>
		<li>Führen die Stimulation in einem überschwelligen Regime, mit dem Reizstrom ca. zweimal erhöht, im Vergleich zu den Schwellenwert für die Beobachtung der EJCs. 
		Hinweis: Nach unserer Erfahrung ist solche Reizintensität optimal zur Vermeidung von Aktionspotential Ausfälle und Aktionspotential ausgelöst. Zum Beispiel, wenn EJCs erscheinen bei der Stimulation Strom von 0,2 mA, verwenden Sie einen Strom von 0,4 mA während des Experiments.</li>
	</ol></li> <li>Dichtung Widerstand <ol><li>messen Sie den Siegel-Widerstand der Aufnahme Macropatch Elektrode durch Drehen des Elektrode Widerstand Schalters des Verstärkers zu &#34; Dichtung Test &#34; Position. Beachten Sie, dass der Dichtung Widerstandswert in GΩ in angezeigt wird die &#34; aktueller &#34; Fenster.</li>
		<li>Sicherstellen, dass die Dichtung Widerstand ist im Bereich von 0,5 - 2 M &#8486;. Werte außerhalb dieses Bereichs zeigen, dass die Elektrode nicht richtig poliert oder nicht richtig positioniert ist.</li>
		<li>Dichtung Widerstand während der Aufnahme zu überwachen, damit es während des Experiments konstant bleibt.</li>
	</ol></li></ol> 4. Analyse <ol><li>Analyze Aufnahmen beschäftigt maßgeschneiderte Software für die Analyse (siehe Tabelle der Werkstoffe). 
	Hinweis: Unser Labor verwendet eigenen Software Quantan 31, die für die Erkennung von EJCs und mEJCs fokal aufgenommen ( Abbildung 4) angepasst ist. Diese Software beinhaltet eine Gaußsche Digitalfilter und ermöglicht die Erkennung von Quanten Gipfeln in überlappenden Multi-Quanten Ereignisse ( Abb. 4A). Andere Ansätze sind beschrieben im 17.</li>
</ol> <img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56493/56493fig4.jpg" /> Abbildung 4. Quanten-Analyse. Erkennung von mEJCs (A) und (B) EJCs von Quantan Software. Das Veranstaltungsgelände ist grün markiert, und Spitzen zeichnen sich durch roten Pfeilspitzen. <a href="//ecsource.jove.com/files/ftp_upload/56493/56493fig4large.jpg" target="_blank"> Bitte klicken Sie hier für eine größere Version dieser Figur.</a>

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+methods+for+recording+synaptic+potentials+from+the+NMJ+of+Drosophila+larvae.">Electrophysiological methods for recording synaptic potentials from the NMJ of Drosophila larvae.</a> J Vis Exp. (24), (2009).</li><li>Bykhovskaia, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+quantal+analysis+more+convenient,+fast,+and+accurate:+user-friendly+software+QUANTAN.">Making quantal analysis more convenient, fast, and accurate: user-friendly software QUANTAN.</a> J Neurosci Methods. 168 (2), 500-513 (2008).</li><li>Huntwork, S., Littleton, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+complexin+fusion+clamp+regulates+spontaneous+neurotransmitter+release+and+synaptic+growth.">A complexin fusion clamp regulates spontaneous neurotransmitter release and synaptic growth.</a> Nat Neurosci. 10 (10), 1235-1237 (2007).</li><li>Zhong, Y., Wu, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+synaptic+plasticity+in+Drosophila+memory+mutants+with+a+defective+cyclic+AMP+cascade.">Altered synaptic plasticity in Drosophila memory mutants with a defective cyclic AMP cascade.</a> Science. 251 (4990), 198-201 (1991).</li><li>Delgado, R., Maureira, C., Oliva, C., Kidokoro, Y., Labarca, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+of+vesicle+pools,+rates+of+mobilization,+and+recycling+at+neuromuscular+synapses+of+a+Drosophila+mutant,+shibire.">Size of vesicle pools, rates of mobilization, and recycling at neuromuscular synapses of a Drosophila mutant, shibire.</a> Neuron. 28 (3), 941-953 (2000).</li><li>Melom, J. E., Akbergenova, Y., Gavornik, J. P., Littleton, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+and+evoked+release+are+independently+regulated+at+individual+active+zones.">Spontaneous and evoked release are independently regulated at individual active zones.</a> J Neurosci. 33 (44), 17253-17263 (2013).</li></ol>

Die Autoren haben nichts preisgeben.

Drosophila ist eine vorteilhafte Modellorganismus synaptische Übertragung zu studieren. Mehrere Aufnahme Konfigurationen wurden bei den Larven NMJ, einschließlich intrazelluläre Aufnahmen der synaptischen Potentiale, Aufnahmen der synaptischen Ströme mit zwei Elektroden Spannung Klemme33,34und fokale Macropatch verwendet Aufnahmen der synaptischen Ströme, die hier beschrieben. Diese Technik ermöglicht die präzise Quantifizierung der synaptischen ?...

Drosophila ist ein ausgezeichnetes Modell, die molekularen Mechanismen, die Kontrolle der synaptischen Übertragung zu studieren. Das neuromuskuläre System in Drosophila ist glutamatergen und die Drosophila neuromuskuläre Synapse (NMJ) kann daher verwendet werden, um die konservierten Funktionen des glutamatergen Release zu studieren. Seit Jan und Jan Studie1die dritte Instar Larven weitgehend genutzt, evozierte und spontane synaptische Übertragung durch die Überwachung der exzitatorischen Kreuzung Potentiale (EJPs) oder Strömungen (EJCs) zu studieren. EJPs werden häufig mit einem scharfen Mikro-Glaselektrode intrazellulär aufgezeichnet, und sie reflektieren die Aktivität der gesamten NMJ, einschließlich alle Boutons Synapsen bei der gegebenen Muskelfaser machen.
Im Gegensatz dazu kann die Aktivität von einer begrenzten Anzahl von Standorten der Veröffentlichung fokal durch die Positionierung eines Mikropipette Tipp in der Nähe von neuronalen Terminals oder synaptischen Krampfadern aufgezeichnet werden. Diese Technik wurde ursprünglich von Katz und Miledi2eingesetzt und fokale extrazelluläre Aufnahmen haben erfolgreich an mehreren NMJ Vorbereitungen, einschließlich Frosch3,4,5, Maus6 eingesetzte , 7 , 8, Krustentier9,10,11,12,13,14,15,16und Drosophila17,18,19,20,21,22,23. Dieser Ansatz wurde von Dudel, weiterentwickelt, die Umkodierung Elektroden24,25Macropatch optimiert. Diese Technik abgestimmt Dudel Umsetzung eng locker-Patch-Clamp-Methode26.
Die Drosophila Larven NMJ hat klar definierte synaptischen Boutons und transgene Linien mit genetisch codierte neuronalen fluoreszierende Tags (siehe Tabelle der Materialien) sind leicht zugänglich. Diese Vorteile konnten wir EJCs und mEJCs aus einer ausgewählten synaptische Bouton20,21,22aufnehmen. Hier beschreiben wir diese Technik im Detail.

<table><tbody><tr><td>Sutter P-97</td><td>Sutter instrument</td><td>P-97</td><td>Microelectrode puller</td></tr><tr><td>Narishige MF-830</td><td>Narishige</td><td>MF-830</td><td>Microforge</td></tr><tr><td>WPI MF200</td><td>WPI</td><td>MF200</td><td>Microforge</td></tr><tr><td>Glass capilaries</td><td>WPI</td><td>B150-86-10</td><td>Glass capilaries</td></tr><tr><td>Microtorch 1WG61</td><td>Grainer</td><td>1WG61</td><td>Microtorch</td></tr><tr><td>Sylgard 184 Silicone Elastomer Kit</td><td>Dow Corning</td><td>SYLGARD 184</td><td>Silicone for dissection plates preparation</td></tr><tr><td>Dissection pins</td><td>Amazon</td><td>B00J5PMPJA</td><td>Pins for larvae positioning</td></tr><tr><td>Tweezers</td><td>WPIINC</td><td>500342</td><td>Tweezers for placing pins, removing the guts and tracheas.</td></tr><tr><td>Scissors</td><td>WPIINC</td><td>501778</td><td>Scissors for cutting the cuticula of the larvae and nerves.</td></tr><tr><td>Olympus BX61WI</td><td>Olympus</td><td>BX61WI</td><td>Upright microscope</td></tr><tr><td>Olympus Lumplan FL N 60x</td><td>Olympus</td><td>UPLFLN 60X</td><td>Microscope objective 60X</td></tr><tr><td>Olympus UPlan FL N 10x</td><td>Olympus</td><td>Uplanfl N 10X</td><td>Microscope objective 10X</td></tr><tr><td>Narishige Micromanipulator</td><td>Narishige</td><td>MHW-3</td><td>Three-axis Water Hydraulic Micromanipulator</td></tr><tr><td>npi Electronic GmbH ELC-03XS</td><td>npi Electronic GmbH</td><td>ELC-03XS</td><td>Electrophysiological amplifier</td></tr><tr><td>A.M.P.I Master 8</td><td>A.M.P.I.</td><td>Master 8</td><td>Electrical stimulator</td></tr><tr><td>A.M.P.I Iso-Flex</td><td>A.M.P.I.</td><td>Iso-Flex</td><td>Stimulus isolator</td></tr><tr><td>TMC antivibration table</td><td>TMC</td><td>63-9090</td><td>Antivibration table</td></tr><tr><td>TMC Faraday cage</td><td>TMC</td><td>81-333-90</td><td>Faraday cage</td></tr><tr><td>Digidata 1322A</td><td>Axon Instruments</td><td>Digidata 1322A</td><td>Digidata</td></tr><tr><td>Computer</td><td>Dell</td><td>Dell Dimension 5150</td><td>Computer with Win XP OS</td></tr><tr><td>Electrode holder</td><td>WPI</td><td>MEH3SW</td><td>Electrode holder</td></tr><tr><td>Optical filter</td><td>Omega optical</td><td>XF 115-2</td><td>Filter cube for Green Fluorescent Protein (GFP) detection</td></tr><tr><td>pCLAMP 8</td><td>Axon Instruments</td><td>8.0.0.81</td><td>Software for signal recording</td></tr><tr><td>Quantan</td><td>In-house software</td><td>-</td><td>Software for signal processing</td></tr><tr><td>Canton-S (Wildtype)</td><td>Bloomington Stock Center</td><td>64349</td><td>Control fly line</td></tr><tr><td>cpx SH1</td><td>Generous Gift of J.T. Littleton</td><td>-</td><td>Complexin knock-out fly line with increased spontaneous exocytosis</td></tr><tr><td>CD8-GFP</td><td>Bloomington Stock Center</td><td>5137</td><td>Fly line with neuronal fluorescent (GFP) Tag</td></tr></tbody></table>

focal macropatch recordings of synaptic currents from the drosophila larval neuromuscular junction

Drosophila neuromuskulären Synapse (NMJ) ist ein ausgezeichnetes Modell, glutamatergen synaptische Übertragung zu studieren. Wir beschreiben die Technik der fokalen Macropatch Aufnahmen der synaptischen Ströme von visualisierten Boutons bei der Drosophila Larven NMJ. Diese Technik erfordert maßgeschneiderte Herstellung von Aufnahme Mikropipetten sowie ausgestattet mit einer hohen Vergrößerung, Fernverkehr Wasser eintauchen Ziel, differential Interferenz Kontrast (DIC) Optik und eine fluoreszierende Verbindung Mikroskop Anlage. Die Aufnahme-Elektrode befindet sich auf der Oberseite eine ausgewählte synaptische Bouton visualisiert mit DIC Optik, Epi-Fluoreszenz oder beides. Der Vorteil dieser Technik ist, dass es ermöglicht die Überwachung der synaptischen Aktivität von einer begrenzten Anzahl von Standorten der Veröffentlichung. Die Aufnahme-Elektrode hat einen Durchmesser von mehreren Mikrometern und die Freigabe-Websites außerhalb der Elektrode Felge beeinflussen nicht signifikant die aufgezeichneten Strömungen. Die aufgezeichneten synaptische Ströme haben schnelle Kinetik und können leicht gelöst werden. Diese Vorteile sind besonders wichtig für das Studium der mutierten Fliegenschnüre mit verstärkten spontan oder asynchronen synaptische Aktivität.

Fokale Macropatch Aufnahmen ermöglichen Überwachung synaptische Aktivität von ausgewählten synaptischen Boutons (Abbildung 5). Wenn die Elektrode an der Oberseite eine synaptische Bouton (Abbildung 5A, Seite 1) positioniert ist, die aufgenommenen mEJCs (Abbildung 5, Seite 1) haben eine Amplituden, die deutlich über den Geräuschpegel und scharf steigende Phasen (bei einer Sub-Millisekunden-Berei...

Watch this Scientific Journal Video about Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction at JoVE.com

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Fokale Macropatch Aufnahmen der synaptischen Ströme aus der Drosophila larvalen neuromuskulären Synapse

Synaptische Ströme können fokal aus visualisierten synaptischen Boutons bei der Drosophila dritte Instar Larven neuromuskulären Synapse aufgenommen werden. Diese Technik ermöglicht die Überwachung der Tätigkeit von einem einzigen synaptische Bouton.

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...

focal-macropatch-recordings-synaptic-currents-from-drosophila-larval

Research

JoVE Journal

Neuroscience

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

6.0K Aufrufe.  Wayne State University. Synaptische Ströme können fokal aus visualisierten synaptischen Boutons bei der Drosophila dritte Instar Larven neuromuskulären Synapse aufgenommen werden. Diese Technik ermöglicht die Überwachung der Tätigkeit von einem einzigen synaptische Bouton.

Serie: Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle. This is a direct consequence of the accessibility to both cell types and ability to visually distinguish the single segmental CaP motor-neuron on the basis of morphology and location. This video demonstrates the microscopic methods used to identify a CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type. Identification of the CaP motor-neuron type is confirmed by either dye filling or by the biophysical features such as action potential waveform and cell input resistance. Motor-neuron recordings routinely last for one hour permitting long-term recordings from multiple different target muscle cells. Control over the motor-neuron firing pattern enables measurements of the frequency-dependence of synaptic transmission at the neuromuscular junction. Owing to a large quantal size and the low noise provided by whole cell voltage clamp, all of the unitary events can be resolved in muscle. This feature permits study of basic synaptic properties such as release properties, vesicle recycling, as well as synaptic depression and facilitation. The advantages offered by this in vivo preparation eclipse previous neuromuscular model systems studied wherein the motor-neurons are usually stimulated by extracellular electrodes and the muscles are too large for whole cell patch clamp. The zebrafish preparation is amenable to combining electrophysiological analysis with a wide range of approaches including transgenic lines, morpholino knockdown, pharmacological intervention and in vivo imaging. These approaches, coupled with the growing number of neuromuscular disease models provided by mutant lines of zebrafish, open the door for new understanding of human neuromuscular disorders.

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target skeletal muscle. This video demonstrates the microscopic methods used to identify a segmental CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type.

Gepaart Patch Clamp Messungen von Motor-Neuron-und Target Skelettmuskulatur bei Zebrafisch

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle.  ...

Forschung

Neurowissenschaften

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The Drosophila larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila is well known for the ease of powerful genetic manipulations and the larval nervous system has proven particularly useful in studying not only normal function but also perturbations that accompany some neurological disease (Lloyd and Taylor, 2010). Many key synaptic molecules found in Drosophila are also found in mammals and like most CNS excitatory synapses in mammals, the Drosophila NMJ is glutamatergic and demonstrates activity-dependent remodeling (Kohet al. , 2000). Additionally, Drosophila neurons can be individually identified because their innervation patterns are stereotyped and repetitive making it possible to study identified synaptic terminals, such as those between motor neurons and the body-wall muscle fibers that they innervate (Keshishian and Kim, 2004). The existence of evolutionarily conserved synapse components along with the ease of genetic and physical manipulation make the Drosophila model ideal for investigating the mechanisms underlying synaptic function (Budnik, 1996).
The active zones at synaptic terminals are of particular interest because these are the sites of neurotransmitter release. NC82 is a monoclonal antibody that recognizes the Drosophila protein Bruchpilot (Brp), a CAST1/ERC family member that is an important component of the active zone (Waghet al. , 2006). Brp was shown to directly shape the active zone T-bar and is responsible for effectively clustering Ca2+ channels beneath the T-bar density (Fouquetet al. , 2009). Mutants of Brp have reduced Ca2+ channel density, depressed evoked vesicle release, and altered short-term plasticity (Kittelet al. , 2006). Alterations to active zones have been observed in Drosophila disease models. For example, immunofluorescence using the NC82 antibody showed that the active zone density was decreased in models of amyotrophic lateral sclerosis and Pitt-Hopkins syndrome (Ratnaparkhiet al. , 2008; Zweieret al. , 2009). Thus, evaluation of active zones, or other synaptic proteins, in Drosophila larvae models of disease may provide a valuable initial clue to the presence of a synaptic defect.
Preparing whole-mount dissected Drosophila larvae for immunofluorescence analysis of the NMJ requires some skill, but can be accomplished by most scientists with a little practice. Presented is a method that provides for multiple larvae to be dissected and immunostained in the same dissection dish, limiting environmental differences between each genotype and providing sufficient animals for confidence in reproducibility and statistical analysis.

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The neuromuscular junction (NMJ) of Drosophila melanogaster is an important model system for studying normal synaptic function as well as perturbations to synaptic function found in certain neurological diseases. We present a protocol for dissection of the Drosophila larval motor system and immunostaining for active zone proteins within the NMJ.

Dissection und Imaging von aktiven Zonen in der Drosophila Neuromuskulären Synapse

The Drosophila  larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila  is well known for ...

Electrophysiological Methods for Recording Synaptic Potentials from the NMJ of Drosophila Larvae

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. The larval neuromuscular system is a model synapse for the study of synaptic physiology and neurotransmission, and is a valuable research tool that has defined genetics and is accessible to experimental manipulation. Larvae can be dissected to expose the body wall musculature, central nervous system, and peripheral nerves. The muscles of Drosophila and their innervation pattern are well characterized and muscles are easy to access for intracellular recording. Individual muscles can be identified by their location and orientation within the 8 abdominal segments, each with 30 muscles arranged in a pattern that is repeated in segments A2 - A7. Dissected drosophila larvae are thin and individual muscles and bundles of motor neuron axons can be visualized by transillumination1. Transgenic constructs can be used to label target cells for visual identification or for manipulating gene products in specific tissues. In larvae, excitatory junction potentials (EJP&#8217;s) are generated in response to vesicular release of glutamate from the motoneurons at the synapse. In dissected larvae, the EJP can be recorded in the muscle with an intracellular electrode. Action potentials can be artificially evoked in motor neurons that have been cut posterior to the ventral ganglion, drawn into a glass pipette by gentle suction and stimulated with an electrode. These motor neurons have distinct firing thresholds when stimulated, and when they fire simultaneously, they generate a response in the muscle. Signals transmitted across the NMJ synapse can be recorded in the muscles that the motor neurons innervate. The EJP&#8217;s and minature excitatory junction potentials (mEJP&#8217;s) are seen as changes in membrane potential. Electrophysiological responses are recorded at room temperature in modified minimal hemolymph-like solution2 (HL3) that contains 5 mM Mg2+ and 1.5 mM Ca2+. Changes in the amplitude of evoked EJP&#8217;s can indicate differences in synaptic function and structure. Digitized recordings are analyzed for EJP amplitude, mEJP frequency and amplitude, and quantal content.

Here we describe electrophysiological methods for measuring synaptic transmission at the neuromuscular junction of Drosophila larva. Evoked release is initiated artificially by stimulating the motor neuron axons, and transmission through the NMJ can be measured by the postsynaptic response evoked in the muscle.

Elektrophysiologischen Methoden für die Aufzeichnung Synaptic Potentials aus dem NMJ von Drosophila-Larven

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. ...

Biologie

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

The Drosophila larval neuromuscular preparation has proven to be a useful tool for studying synaptic physiology1,2,3. Currently, the only means available to evoke excitatory junctional potentials (EJPs) in this preparation involves the use of suction electrodes. In both research and teaching labs, students often have difficulty maneuvering and manipulating this type of stimulating electrode. In the present work, we show how to remotely stimulate synaptic potentials at the larval NMJ without the use of suction electrodes. By expressing channelrhodopsin2 (ChR2) 4,5,6 in Drosophila motor neurons using the GAL4-UAS system 7, and making minor changes to a basic electrophysiology rig, we were able to reliably evoke EJPs with pulses of blue light. This technique could be of particular use in neurophysiology teaching labs where student rig practice time and resources are limited.

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

This procedure uses a blue light-activated algal channel and cell-specific genetic expression tools to evoke synaptic potentials with light pulses at the neuromuscular junction (NMJ) in Drosophila larvae. This technique is an inexpensive and easy-to-use alternative to suction electrode stimulation for synaptic physiology studies in research and teaching laboratories.

Channelrhodopsin2 vermittelte Stimulation der synaptischen Potentiale an Drosophila Neuromuskulären Endplatten

The Drosophila larval neuromuscular preparation has proven to be a useful tool for studying synaptic physiology1,2,3. Currently, the only means available ...

Dissecting and Recording from The C. Elegans Neuromuscular Junction

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale. This complex process requires the coordinated activity of many pre- and post-synaptic proteins to ensure appropriate synaptic connectivity, conduction of electrical signals, targeting and priming of secretory vesicles, calcium sensing, vesicle fusion, localization and function of postsynaptic receptors and finally, recycling mechanisms. As neuroscientists it is our goal to elucidate which proteins function in each of these steps and understand their mechanisms of action. Electrophysiological recordings from synapses provide a quantifiable read out of the underlying electrical events that occur during synaptic transmission. By combining this technique with the powerful array of molecular and genetic tools available to manipulate synaptic proteins in C. elegans, we can analyze the resulting functional changes in synaptic transmission. 
The C. elegans NMJs formed between motor neurons and body wall muscles control locomotion, therefore, mutants with uncoordinated locomotory phenotypes (known as unc s) often perturb synaptic transmission at these synapses 1. Since unc mutants are maintained on a rich supply of a bacterial food source, they remain viable as long as they retain some pharyngeal pumping ability to ingest food. This, together with the fact that C. elegans exist as hermaphrodites, allows them to pass on mutant progeny without the need for elaborate mating behaviors. These attributes, coupled with our recent ability to record from the worms NMJs 2,3,7 make this an excellent model organism in which to address precisely how unc mutants impact neurotransmission. 
The dissection method involves immobilizing adult worms using a cyanoacrylic glue in order to make an incision in the worm cuticle exposing the NMJs. Since C. elegans adults are only 1 mm in length the dissection is performed with the use of a dissecting microscope and requires excellent hand-eye coordination. NMJ recordings are made by whole-cell voltage clamping individual body wall muscle cells and neurotransmitter release can be evoked using a variety of stimulation protocols including electrical stimulation, light-activated channel-rhodopsin-mediated depolarization 4 and hyperosmotic saline, all of which will be briefly described.

Application of electrophysiology to accessible synapses provides a quantifiable measure of synaptic activity, useful in analyzing synaptic mutants. This article describes a dissection method used to expose the neuromuscular junctions (NMJ) of Caenorhabditis elegans (C. elegans) and briefly discusses some of the uses to which this preparation can be applied.

Dissecting und Aufnahme von C. elegans neuromuskulären Synapse

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale.   This ...

Preparation of Drosophila Central Neurons for in situ Patch Clamping

Short generation times and facile genetic techniques make the fruit fly Drosophila melanogaster an excellent genetic model in fundamental neuroscience research. Ion channels are the basis of all behavior since they mediate neuronal excitability. The first voltage gated ion channel cloned was the Drosophila voltage gated potassium channel Shaker1,2. Toward understanding the role of ion channels and membrane excitability for nervous system function it is useful to combine powerful genetic tools available in Drosophila with in situ patch clamp recordings. For many years such recordings have been hampered by the small size of the Drosophila CNS. Furthermore, a robust sheath made of glia and collagen constituted obstacles for patch pipette access to central neurons. Removal of this sheath is a necessary precondition for patch clamp recordings from any neuron in the adult Drosophila CNS. In recent years scientists have been able to conduct in situ patch clamp recordings from neurons in the adult brain3,4 and ventral nerve cord of embryonic5,6, larval7,8,9,10, and adult Drosophila11,12,13,14. A stable giga-seal is the main precondition for a good patch and depends on clean contact of the patch pipette with the cell membrane to avoid leak currents. Therefore, for whole cell in situ patch clamp recordings from adult Drosophila neurons must be cleaned thoroughly. In the first step, the ganglionic sheath has to be treated enzymatically and mechanically removed to make the target cells accessible. In the second step, the cell membrane has to be polished so that no layer of glia, collagen or other material may disturb giga-seal formation. This article describes how to prepare an identified central neuron in the Drosophila ventral nerve cord, the flight motoneuron 5 (MN515), for somatic whole cell patch clamp recordings. Identification and visibility of the neuron is achieved by targeted expression of GFP in MN5. We do not aim to explain the patch clamp technique itself.

Preparation of Drosophila Central Neurons for in situ Patch Clamping

In situ patch clamp recordings are used for electrophysiological characterization of neurons in intact circuitry. In the Drosophila genetic model patch clamping is difficult because the CNS is small and surrounded by a robust sheath. This article describes the procedure to remove the sheath and clean neurons for subsequent patch clamp recordings.

Herstellung<em&gt; Drosophila</em&gt; Central Neuronen<em&gt; In situ</em&gt; Patch Clamping

Short generation times and facile genetic techniques make the fruit fly Drosophila melanogaster an excellent genetic model in fundamental neuroscience ...

Electrophysiological Recording in the Drosophila Embryo

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. Whole-cell patch-clamp techniques can then be employed to record from individually-identifiable neurons and somatic muscles. These recording configurations have been used to track the appearance and maturation of ionic currents and action potential propagation in both neurons and muscles. Genetic mutants affecting these electrical properties have been characterized to reveal the molecular composition of ion channels and associated signaling complexes, and to begin exploration of the molecular mechanisms of functional differentiation. A particular focus has been the assembly of synaptic connections, both in the central nervous system and periphery. The glutamatergic neuromuscular junction (NMJ) is most accessible to a combination of optical imaging and electrophysiological recording. A glass suction electrode is used to stimulate the peripheral nerve, with excitatory junction current (EJC) recordings made in the voltage-clamped muscle. This recording configuration has been used to chart the functional differentiation of the synapse, and track the appearance and maturation of presynaptic glutamate release properties. In addition, postsynaptic properties can be assayed independently via iontophoretic or pressure application of glutamate directly to the muscle surface, to measure the appearance and maturation of the glutamate receptor fields. Thus, both pre- and postsynaptic elements can be monitored separately or in combination during embryonic synaptogenesis. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Electrophysiological Recording in the Drosophila Embryo

Electrophysiological recordings from Drosophila embryos allow analyses of developing muscle and neuron electrical properties, as well as characterization of functional synaptogenesis at the glutamatergic neuromuscular junction and central cholinergic and GABAergic synapses.

Elektrophysiologische Aufzeichnung in der Drosophila Embryo

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Mauthner cells (M-cells) are large reticulospinal neurons located in the hindbrain of teleost fish. They are key neurons involved in a characteristic behavior known as the C-start or escape response that occurs when the organism perceives a threat. The M-cell has been extensively studied in adult goldfish where it has been shown to receive a wide range of excitatory, inhibitory and neuromodulatory signals1. We have been examining M-cell activity in embryonic zebrafish in order to study aspects of synaptic development in a vertebrate preparation. In the late 1990s Ali and colleagues developed a preparation for patch clamp recording from M-cells in zebrafish embryos, in which the CNS was largely intact2,3,4. The objective at that time was to record synaptic activity from hindbrain neurons, spinal cord neurons and trunk skeletal muscle while maintaining functional synaptic connections within an intact brain-spinal cord preparation. This preparation is still used in our laboratory today. To examine the mechanisms underlying developmental synaptic plasticity, we record excitatory (AMPA and NMDA-mediated)5,6 and inhibitory (GABA and glycine) synaptic currents from developing M-cells. Importantly, this unique preparation allows us to return to the same cell (M-cell) from preparation to preparation to carefully examine synaptic plasticity and neuro-development in an embryonic organism. The benefits provided by this preparation include 1) intact, functional synaptic connections onto the M-cell, 2) relatively inexpensive preparations, 3) a large supply of readily available embryos 4) the ability to return to the same cell type (i.e. M-cell) in every preparation, so that synaptic development at the level of an individual cell can be examined from fish to fish, and 5) imaging of whole preparations due to the transparent nature of the embryos.

We have developed an intact brain-spinal cord preparation to record and monitor electrical activity via patch clamp recording from the Mauthner neurons and other reticulospinal cells in zebrafish embryos. Thus, we are able to record excitatory and inhibitory synaptic currents, voltage-gated channel activity and action potentials from key neurons in a developing embryo.

Patch-Clamp-Aufnahmen aus embryonalen Zebrafisch-Mauthner-Zellen

Mauthner cells (M-cells) are large  reticulospinal neurons located in the hindbrain of teleost fish. They are key  neurons involved in a characteristic ...

Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae

The Drosophila neuromuscular junction (NMJ) has emerged as a valuable model system in the field of neuroscience. The application of confocal microscopy at the Drosophila NMJ enables researchers to acquire synaptic information, encompassing both quantitative data on synapse abundance and detailed insights into their morphology. However, the diffuse distribution and limited visual range of the TEM present challenges for the ultrastructural analysis. This study introduces an innovative and efficient sample preparation method that surpasses the conventional approach. The procedure begins by placing a metal mesh at the base of a flat-bottomed bottle or test tube, followed by positioning fixed larvae samples onto the mesh. An additional mesh is placed over the samples, ensuring that they are positioned between the two meshes. The fixed samples are thoroughly dehydrated and infiltrated before proceeding with the embedding procedure. Then embedding of the samples in epoxy resin is performed in a flat sheet manner, which allows for the preparation of muscles for positioning and sectioning. Benefiting from these steps, all the muscles of Drosophila larvae can be visualized under light microscopy, therey facilitating subsequent positioning and sectioning. Excess resin is removed after locating the 6th and 7th muscles of body segments A2 and A3. Serial ultra-thin sectioning of the 6th or 7th muscle is performed.

Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae

This report provides a new sample preparation procedure for visualizing neuromuscular junctions in Drosophila Larvae. This method is more effective in preventing the curling of the samples compared to the traditional method and is particularly useful for Drosophila neuromuscular junction ultrastructural analysis.

Optimierung des Probenvorbereitungsprozesses für die Transmissionselektronenmikroskopie von neuromuskulären Verbindungen in Drosophila-Larven

The Drosophila neuromuscular junction (NMJ) has emerged as a valuable model system in the field of neuroscience. The application of confocal microscopy at ...

Drosophila Neuromuscular Junction (NMJ) Quantification: A Method to Assess Synaptic Morphology and Function

Source: Castells-Nobau, A., et al. <a href="https://www.jove.com/video/55395/" target="_blank">Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology</a>. J. Vis. Exp. (2017).
The Drosophila neuromuscular junction (NMJ) is used as a model to study the morphology and function of synapses. This video describes important features that researchers quantify to characterize the NMJ. The example protocol demonstrates a software able to perform the quantification automatically.

Drosophila Quantifizierung der neuromuskulären Verbindung (NMJ): Eine Methode zur Beurteilung der synaptischen Morphologie und Funktion

Source: Castells-Nobau, A., et al. Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology. ...