We thank Joung Jang and Xin Guan for technical assistance. We thank Dr. Rory McQuiston of Virginia Commonwealth University for setting up our first patch clamp rig and advices on experimental procedures. We thank Dr. Samuel Wu for suggestions on voltage clamp recording. The work is supported by NIH grants EY013811, EY022228 and a vision core grant EY002520. C-KC is the Alice R. McPherson Retina Research Foundation Endowed Chair at the Baylor College of Medicine.

Ethische Zustimmung - Verfahren unter Verwendung tierischer Probanden wurden in Übereinstimmung mit den Regeln und Vorschriften der National Institutes of Health Leitlinien für die Forschung Tieren durchgeführt, wie sie in der institutionellen Tierpflege und verwenden Ausschuss von Baylor College of Medicine zugelassen. 1. Externe und interne Lösungen <ol><li> Verwenden Sie Säugetier Ringer-Lösung während der Retina Dissektion und als externe Lösung in den nachfolgenden elektro Aufnahme. Bereiten Sie das Säugetier-Ringer-Lösung von 10x Stammlösung (ohne Calcium) am Tag der Aufnahme, und fügen Sie CaCl 2 tropfenweise nach 15 min von carbogenation (95% O 2 und 5% CO 2). Die endgültige 1x Lösung enthält (in mM): 120 NaCl, 5 KCl, 25 NaHCO 3, 0,8 Na 2 HPO 4, 0,1 NaH 2 PO 4, 2 CaCl 2, 1 MgSO 4 und 10 D-glucose. </li><li> Verwenden Sie zwei interne Lösungen zu SAC Schwingungen charakterisieren. TestDie Nützlichkeit hergestellten Lösungen in längeren whole-cell patch clamp-Aufnahmen auf erwachsenen Maus RGCs und speichert die verifizierten Chargen in 500 &amp; mgr; l Aliquots bei -20 ° C bis zur Verwendung. <ol><li> Für Membranpotential Schwingungs Aufzeichnung, mit einem kaliumhaltigen interne Lösung, die (in mM) enthält: 125 K-Gluconat, 8 NaCl, 4 ATP-Mg 0,5 Li-GTP, 5 EGTA, 10 HEPES und 0,2% Biocytin (w / v). Der pH-Wert auf 7,3 mit KOH. </li><li> Für die Aufnahme von erregenden und hemmenden postsynaptischen Ströme, verwenden Sie eine Cäsium-basierte interne Lösung, die (in mM) enthält: 100 Cs-Methansulfonat, 8 NaCl, 4 ATP-Mg, 0,5 Li-GTP, 5 EGTA, 10 HEPES und 0,2% biocytin ( w / v). Der pH-Wert auf 7,3 mit CsOH. </li></ol></li></ol> 2. Vorbereitung für den Tag der Aufnahme <ol><li> Um eine Nitrocellulosemembran mit offenen Löchern herzustellen, Punsch manuell die Membran mit einer angepassten Puncher aus einem abgestumpften 16 G Spritzennadel und glätten die Membran zwischen zwei saubere Glasplatten. Für eine Groß-Mount Retina, ein zentrales Loch von 0,2 mm Durchmesser machen und umgeben es um 4 größere Löcher, die von 1 bis 1,2 mm im Durchmesser sind. </li><li> Um eine angepasste Verfüllung Glühfaden zum Laden interne Lösung in Elektroden machen, schmelzen, um eine 10 &amp; mgr; l-Pipettenspitze in der Mitte und sanft den geschmolzenen Abschnitt zu verlängern, bis sie abkühlt und erstarrt. Gerade vor dem Gebrauch mit einem sauberen Rasierklinge den Faden zu schneiden, bis er etwas länger als die Patch-Pipetten ist. </li><li> Pull-Patch-Pipetten in einem programmierbaren puller aus Borsilikat Glasrohr mit einem inneren Filaments. Herstellung der Mikro am Tag des Experiments und nutzen sie sofort. HINWEIS: Für die Aufnahme FFH, verwenden wir die folgende Abzieher Einstellung: Wärme: 484; ziehen: 0; Geschwindigkeit: 25; Verzögerungszeit: 1; Druck: 400 und eine Rampenwert von 462. Die OD und ID der Borosilikat-Rohre sind 1,65 mm und 1,0 mm, respectively. Widerstand der Pipetten ist 10-14 MOhm. </li><li> Eine Stunde vor Gewebe Ernte, tauen ein Rohr der internen Lösung von shaking es auf einem Vortex für&gt; 30 min. </li></ol> 3. Retina Dissection <ol><li> Anesthetize das Tier um 4% Isofluran in Sauerstoff, bis das Tier Reaktion auf eine Prise Zehe verliert. Sacrifice das Tier durch Genickbruch. </li><li> Schneiden Sie beide Augäpfel aus den Sehnerven bei 1-2 mm von den Papillen mit geradem zackigen Iris Schere. </li><li> Rollen Sie die Augäpfel auf einem sauberen Papiertuch Blut zu entfernen, und dann tauchen sie in carbogenated Säugetier-Ringer-Lösung. Lochen Sie ein Loch auf dem Limbus mit einer 23 G-Nadel und halbieren die Augäpfel durch entlang des Limbus mit Mikro-Schere schneiden. Entfernen Sie die Hornhaut und Linse mit einer feinen Pinzette. <ol><li> Für retinalen ganze Montage, sanft die gesamte Netzhaut aus dem Pigmentepithel mit einer feinen Pinzette schälen und vier 1,5-2 mm orthogonale Schnitte vom Rand in Richtung des Sehnervenkopfes zu machen. </li></ol></li><li> Tauchen Sie ein gestanztes Nitrocellulosemembran in die Schüssel, und ziehen Sie vorsichtig auf die Netzhaut über sie mitdie GCL Seite nach oben. Platzieren Sie den Sehnervenkopf in das Mittelloch, wenn eine ganze-mount Netzhaut vor. Übertragen Sie die Membran mit der Netzhaut in eine andere saubere Teller. glätten Sie vorsichtig die Netzhaut mit einem feinen Pinsel wie ein Malteserkreuz zu sehen und alle vier Kanten über die Löcher legen. <ol><li> Wenn ein Bruchteil der Netzhaut zu einem Zeitpunkt untersucht werden soll, schneiden sich eyecup aus Schritt hergestellten 3,3 in 3-4 Stücke mit einer Rasierklinge mit der pigmentierten Epithel befestigt bleibt. Schützen Sie die nicht verwendeten Stücke von Licht in carbogenated externe Lösung und innerhalb von 12 Stunden. </li></ol></li><li> Blot die Nitrocellulosemembran mit einem Stück trockenen Filterpapier und entfernen Sie den Glaskörper und innere Grenzmembran mit einer Zange und einem Pinsel. </li><li> Stellen Sie sicher, dass alle retinalen Kanten vollständig an die Membran gebunden sind, bevor die Anordnung in die Aufnahmekammer mit einem verschlossenen Glasdeckglas auf dem Boden zu übertragen. Sichern Sie die Membran mit Vakuumfett auf das Deckglas und rehydrate die Netzhaut mit der externen Lösung. Achten Sie darauf, keine Luftblasen unter der Montage zu stoppen. </li><li> Stellen Sie die Kammer auf die Bühne eines aufrechten Mikroskop. Perfundieren die Kammer mit warm (34-35 ° C) carbogenated externe Lösung mit einer Rate von ~ 3 ml pro min. </li><li> Überprüfen Sie die Netzhaut unter einem 10-fach Objektivlinse zuerst, und dann mit einem 60x Wasser-Immersionslinse GCL und INL Neuronen unter differentiellen Interferenzkontrast (DIC) und / oder Epifluoreszenz zu sehen. Zur Visualisierung FFH tdTomato-exprimierenden, verwenden Sie eine weiße LED-Lichtquelle in Kombination mit einem Anregungsfilter von 554 nm und einem Emissionsfilter von 581 nm. </li></ol> 4. Whole-Cell-Patch-Clamp-Aufzeichnung von Flat-Mount Retina <ol><li> Filtern Sie die interne Lösung durch einen Spritzenfilter in den benutzerdefinierten Verfüllung Filaments. </li><li> Setzen Sie den Faden in einem frisch gezapften Mikropipette und verzichten die interne Lösung in der Nähe der Spitze, bis die Lösung die Silberelektrodendraht für&gt; bedeckt5 mm. Befestigen der Mikropipette auf einen Elektrodenhalter mit einem Saug- Pol, durch den Druck im Inneren der Elektrode kann durch Drücken oder Ziehen des Kolbens aus einem rohr verbunden 10 ml Kunststoffspritze einstellbar. </li><li> Finden Sie die Pipette unter dem Objektiv und bringen es auf ~ 100 &amp; mgr; m über der Netzhaut nach unten. Unter dem Stromfolgemodus (I = 0), verwenden Gleichstrom-Offset des stehenden Gleichspannungssignal auf Null. Messen Sie die Pipette Widerstand unter der Stromklemme (I Klammer) Modus durch feste Amplitude Rechteckströme durch die Pipette eingespritzt , während sie im Bad ist und den Unterschied zu neutralisieren , indem Sie den raccess Knopf drehen. Verwenden Sie den Messwert auf der raccess Knopf Pipette Widerstand der Ohmschen Gesetz zu berechnen. </li><li> Langsam bringen die Elektrode zu ~ 10 &amp; mgr; m über der Netzhaut. Gelten positive Druck auf die Elektrode. Sehen Sie sich das Reflexionsänderung in der Nähe der Pipettenspitze, wie sie die Netzhaut erreicht. Schnell aber sanft die Pipette in die GCL zwingen und die positive pressu reduzierensofort wieder. </li><li> Bewegen Sie die Pipette zu einem markierten Neuron. Vermeiden Sie anderen Neuronen in Kontakt, Blutgefäße und Endfüßen von Müllerzellen. Nehmen positiver Druck, wenn nötig Elektrode ein Verstopfen zu verhindern. </li><li> Positionieren Sie die Pipettenspitze in der Nähe der Mittellinie eines markierten Neuron, bis ein Grübchen sichtbar ist. Lassen Sie den Überdruck und damit die Plasmamembran wieder auf die Pipettenspitze auf die Beine. </li><li> Gelten 20-120 pA negative Ströme an der Pipette die Bildung eines Giga-Ohm-Dichtung zu helfen. Tragen Sie eine sanfte Saugwirkung, wenn notwendig, die Plasmamembran in die Pipette zu ziehen. Warten Sie 5 Minuten nach der Dichtung Bildung, die Zellmembran zu Bruch. Dies ermöglicht die verschüttete interne Lösung von Superfusions gelöscht. Rupture die Membran durch eine sanfte Saugwirkung. </li><li> Nach der Membranbruch und während in der Stromklemme Modus einschalten Brückenabgleich und stellen Sie den raccess Knopf. HINWEIS: Für eine kleine Zelle wie der SAC, nur eine geringe Anpassung notwendig ist, falls vorhanden. Alterntiv, Rekord erregenden und hemmenden postsynaptischen Ströme im voltage clamp Modus (V Clamp) durch die Zelle an Umkehrpotentiale von Chlorid halten (um -75 mV) und Glutamat (um 0 mV) sind. Überprüfen und Pipettenposition anpassen, wenn nötig gelegentliche Netzhaut und / oder Mikromanipulator Drift aufzunehmen. </li><li> Zur Aufzeichnung vor Membranpotential oder Stromänderung, während und nach der pharmakologischen Behandlung, bereiten synaptischen Blocker (zB CNQX, AP5, Picrotoxin, Tubocurarin, etc.) und Kanalmodulatoren (zB Flupirtin, Dopamin, Meclofenaminsäure, etc.) frisch auf der Tag des Experiments aus gefrorenen Beständen. Verdünnen Sie die Medikamente mit carbogenated Ringer-Lösung. Wenden Sie sie in einer Batch-Weise durch Perfusion. </li><li> Nach der Aufnahme entfernen sanft die Pipette aus der soma. Übertragen Sie die Anordnung aus der Kammer zu einer sauberen Schüssel. Lösen Sie erneut an die Netzhaut auf eine andere abgeflachte Nitrocellulosemembran ohne gestanzte holes Retina Flachheit während der Fixierung zu gewährleisten. </li><li> Befestigen Sie die Netzhaut, indem sie es in 4% Paraformaldehyd für 30 min bei RT eingetaucht wird. Entfernen Sie die Netzhaut von der Nitrocellulosemembran und spülen Sie ihn ausgiebig mit 1x PBS. Zeigen die Morphologie der aufgezeichneten Zellen durch O / N Anfärbung mit Farbstoff-konjugiertem Streptavidin , verdünnt in 1x PBS mit 0,4% Triton X-100 bei 4 ° C 14. Montieren Sie die Netzhaut in verblassungshemmenden Medium und beobachten aufgezeichnet Zellen unter Verwendung eines konfokalen Mikroskop. HINWEIS: Verfahren beteiligt Paraformaldehyd, die flüchtig und giftig ist, sollte für die Sicherheit in einem Entwurf Kammer durchgeführt werden. </li></ol>

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The authors have nothing to disclose.

Viele Labors haben von GCL - Neuronen in der aufgezeichneten Flachmontage Vorbereitung 15-18, aber unsere Verfahren von INL Neuronen ermöglichen die Aufnahme. Wir betonen hiermit mehrere Schritte, die für eine erfolgreiche Routine-Aufnahmen von entscheidender Bedeutung sind. Die Frische und die Flachheit der Netzhaut sind wichtig für sie mit einer Aufnahme Pipette eindringen. In dieser Hinsicht ist die feste Anbringung der Netzhaut auf die gestanzten Nitrocellulosemembran ragen...

As an easily accessible part of the central nervous system, the retina has for decades been a useful model in neuroscience studies. Genetic marking of neurons has allowed detailed characterization of synaptic connections in the retina. With many methodologies available to examine function and morphology of retinal neurons, the patch clamp recording technique has been instrumental in our current understanding of vertically transmitted signals in the retina. These signals are originated from photon absorption in photoreceptors and sent to brain visual centers through spiking of retinal ganglion cells (RGCs). Despite a large body of knowledge accumulated thus far, neural diversity in vascularized mammalian retina remains unsolved and obstructs the full appreciation of retinal circuits that subserve normal vision. This is in part because most recordings were performed on retinal slices to trade lateral circuit integrity for access to more proximal retinal neurons1-3. To gain a comprehensive picture on how retina computes visual signals, it is thus desirable to record neurons in flat-mounts wherein lateral connections, large and small, may be better preserved.
When synaptic transmission from photoreceptors to bipolar cells is interrupted due to a defective metabotropic glutamate receptor 6 (mGluR6) signaling pathway in depolarizing bipolar cells4-6 or simply as the result of photoreceptor loss in degenerated retinas7-10, many RGCs exhibit oscillatory activities. These oscillations originate from multiple sources, however the one involving gap junction coupling between AII amacrine cells (AII-ACs) and depolarizing cone bipolar cells (DCBCs) has received the most attention and hence is best understood1,7,11. We have found another source, which persists under pharmacological blockade of the aforementioned AII-AC/DCBC network and drives oscillation of OFF-type SACs in Rho&#916;CTA and Nob mice with deafferentated retinas7,8,12. Here we detail our protocol of preparing retinal flat-mounts for INL neuron recording. This approach uses commercial mouse lines (Jax stock no. 006410 and 007905) to mark cholinergic retinal neurons by fluorescent protein (tdTomato) expression that is identifiable under a fluorescent microscope equipped with contrast enhancing optics. Some experimental results acquired through this approach have been previously reported4,5,7,13.

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		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Fixed-stage fluorescent microscope with DIC</td>
			<td style="width:155px;">Olympus</td>
			<td style="width:219px;">BX51-WI</td>
			<td style="width:419px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Micromanipulators</td>
			<td style="width:155px;">Sutter</td>
			<td style="width:219px;">MP-225</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Patch clamp amplifier</td>
			<td style="width:155px;">A-M System</td>
			<td style="width:219px;">AM2400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">AD converter</td>
			<td style="width:155px;">National Instrument</td>
			<td style="width:219px;">NI-USB-6221</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Heater controller</td>
			<td style="width:155px;">Warner Instrument</td>
			<td style="width:219px;">TC-324B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Inline heater</td>
			<td style="width:155px;">Warner Instrument</td>
			<td style="width:219px;">SC-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Peristaltic pump</td>
			<td style="width:155px;">Rainin</td>
			<td style="width:219px;">Dynamax</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">pipette puller</td>
			<td style="width:155px;">Sutter Instrument</td>
			<td style="width:219px;">P-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Glass tube with filament</td>
			<td style="width:155px;">King Precision Glass</td>
			<td style="width:219px;">Customized</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Stimulator</td>
			<td style="width:155px;">A.M.P.I.</td>
			<td style="width:219px;">Master-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Biocytin</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">B4261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">NaCl</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">S6191</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">KCl</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">P5405</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">NaHCO3</td>
			<td style="width:155px;">Fisher</td>
			<td style="width:219px;">BP328-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Na2HPO4</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">S0876</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">NaH2PO4</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">S5011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">CaCl2</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">C5670</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">MgSO4</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">M1880</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">D-glucose</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">G6152</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">K-gluconate</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">G4500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">ATP-Mg</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">A9187</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Li-GTP</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">G5884</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">EGTA</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">E0396</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">HEPES</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">H4034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">KOH</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">P5958</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Cs-methanesulfonate</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">C1426</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">CsOH</td>
			<td style="width:155px;">Sigma</td>
			<td align="right" style="width:219px;">232041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Syringer filter</td>
			<td style="width:155px;">Nalgene</td>
			<td align="right" style="width:219px;">171</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">1 ml syring</td>
			<td style="width:155px;">Rainin</td>
			<td align="right" style="width:219px;">17013002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">10 ul pipette tip</td>
			<td style="width:155px;">Genesee Scientific</td>
			<td style="width:219px;">24-130RL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Streptavidin-488</td>
			<td style="width:155px;">ThermoFisher</td>
			<td style="width:219px;">S-11223</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">10X PBS</td>
			<td style="width:155px;">Lonza</td>
			<td style="width:219px;">17-517Q</td>
			<td></td>
		</tr>
	</tbody>
</table>

patch clamp recording of starburst amacrine cells in a flat-mount preparation of deafferentated mouse retina

Die Netzhaut von Säugetieren ist ein geschichtetes Gewebe aus mehreren neuronalen Typen zusammen. Um zu verstehen, wie visuelle Signale in seinem komplizierten synaptische Netzwerk verarbeitet werden, werden häufig elektrophysiologischen Ableitungen zu studieren Verbindungen zwischen den einzelnen Neuronen verwendet. Wir haben optimiert, um eine Flachmontage Vorbereitung für die Patch-Clamp-Aufzeichnung von genetisch markierten Neuronen in sowohl GCL (Ganglion Zellschicht) und INL (innere Kernschicht) von Maus-Netzhäuten. Aufnahme INL Neuronen in Flat-Halterungen über Scheiben bevorzugt, weil sowohl vertikale als auch seitliche Anschlüsse in der früheren Konfiguration beibehalten werden, so dass der Netzhaut-Schaltungen mit großen seitlichen Komponenten untersucht werden. Wir haben dieses Verfahren verwendet Reaktionen von Spiegelneuronen partnered in Retinae wie die cholinerge Starburst-Amakrinzellen (SAC) zu vergleichen.

Repräsentative Aufnahmen von On- und OFF-Typ FFH aus einer deafferentated Maus Netzhaut sind in Abbildung 1 dargestellt. Cholinergen Zellen sowohl in der GCL und INL zuverlässig durch tdTomato Fluoreszenz und gezielt für Ganzzell - Patch - Clamp - Aufzeichnung unter DIC (1A) identifiziert werden , offenbaren Schwingung ihrer Membranpotentialen (oben Spuren) und den synaptischen Ströme, die es (unten Spuren, 1B) fahren. Die inhibitori...

Watch this Scientific Journal Video about Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina at JoVE.com

Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina

Dieses Protokoll zeigt, wie Whole-Cell-Patch-Clamp-Aufzeichnung auf die retinale Neuronen aus einem flachen-Mount-Vorbereitung durchzuführen.

Patch-Clamp-Aufzeichnung von Starburst-Amakrinzellen in einer Wohnung Montage Herstellung von Deafferentated Maus Retina

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic ...

patch-clamp-recording-starburst-amacrine-cells-flat-mount-preparation

Research

JoVE Journal

Neuroscience

8.1K Aufrufe.  Baylor College of Medicine.  Dieses Protokoll zeigt, wie Whole-Cell-Patch-Clamp-Aufzeichnung auf die retinale Neuronen aus einem flachen-Mount-Vorbereitung durchzuführen. 

Serie: Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of intracellular events that lead to closure of cyclic-nucleotide-gated channels in the cell membrane. The resulting change in membrane potential leads in turn to reductions in the amount of neurotransmitter release from both rod and cone synaptic terminals. To measure how the light-evoked change in photoreceptor membrane potential leads to downstream activity in the retina, scientists have made electrophysiological recordings from retinal slice preparations for decades1,2. In the past these slices have been cut manually with a razor blade on retinal tissue that is attached to filter paper; in recent years another method of slicing has been developed whereby retinal tissue is embedded in low gelling temperature agar and sliced in cool solution with a vibrating microtome3,4. This preparation produces retinal slices with less surface damage and very robust light-evoked responses. Here we document how this procedure can be done under infrared light to avoid bleaching the visual pigment.

Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.

Patch Clamp Messungen von Mouse Netzhautneuronen in einem Dark-Scheiben angepasst Vorbereitung

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

Forschung

Neurowissenschaften

Culturing and Electrophysiology of Cells on NRCC Patch-clamp Chips

Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard of electrophysiology applied to disease models and pharmaceutical screens alike 1. The method traditionally involves gently contacting a cell with a glass pipette filled by a physiological solution in order to isolate a patch of the membrane under its apex 2. An electrode inserted in the pipette captures ion-channel activity within the membrane patch or, when ruptured, for the whole cell. In the last decade, patch-clamp chips have been proposed as an alternative 3, 4: a suspended film separates the physiological medium from the culture medium, and an aperture microfabricated in the film replaces the apex of the pipette. Patch-clamp chips have been integrated in automated systems and commercialized for high-throughput screening 5. To increase throughput, they include the fluidic delivery of cells from suspension, their positioning on the aperture by suction, and automated routines to detect cell-to-probe seals and enter into whole cell mode. We have reported on the fabrication of a silicon patch-clamp chip with optimized impedance and orifice shape that permits the high-quality recording of action potentials in cultured snail neurons 6; recently, we have also reported progress towards interrogating mammalian neurons 7. Our patch-clamp chips are fabricated at the Canadian Photonics Fabrication Centre 8, a commercial foundry, and are available in large series. We are eager to engage in collaborations with electrophysiologists to validate the use of the NRCC technology in different models. The chips are used according to the general scheme represented in Figure 1: the silicon chip is at the bottom of a Plexiglas culture vial and the back of the aperture is connected to a subterranean channel fitted with tubes at either end of the package. Cells are cultured in the vial and the cell on top of the probe is monitored by a measuring electrode inserted in the channel.The two outside fluidic ports facilitate solution exchange with minimal disturbance to the cell; this is an advantage compared to glass pipettes for intracellular perfusion.
<img alt="Figure 1" src="/files/ftp_upload/3288/3288fig1.jpg" />
Figure 1. Principle of measurement using the NRCC patch-clamp chip
We detail here the protocols to sterilize and prime the chips, load them with medium, plate them with cells, and finally use them for electrophysiological recordings.

We show how planar patch-clamp chips fabricated at the National Research Council of Canada are sterilized, primed, loaded with medium, plated with cells, and used for electrophysiological recordings.

Die Züchtung und Elektrophysiologie von Zellen auf NRCC Patch-Clamp-Chips

Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard ...

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch clamp analyses of the voltage-gated channels in sensory hair cells isolated from a variety of species have been described previously1-4 but this video represents the first application of those techniques to hair cells from zebrafish. Here we demonstrate a method to isolate healthy, intact hair cells from all of the inner ear end-organs: saccule, lagena, utricle and semicircular canals. Further, we demonstrate the diversity in hair cell size and morphology and give an example of the kinds of patch clamp recordings that can be obtained. The advantage of the use of this zebrafish model system over others stems from the availability of zebrafish mutants that affect both hearing and balance. In combination with the use of transgenic lines and other techniques that utilize genetic analysis and manipulation, the cell isolation and electrophysiological methods introduced here should facilitate greater insight into the roles hair cells play in mediating these sensory modalities.

The purpose of this video is to demonstrate procedures for obtaining healthy, intact hair cells from the inner ear organs of adult zebrafish and then using them for patch clamp studies aimed at characterizing the biophysical properties of their voltage-gated channels.

Patch-Clamp-Aufnahmen im inneren Haarsinneszellen Isoliert von Zebrafisch

Patch  clamp analyses of the voltage-gated channels in sensory hair cells isolated  from a variety of species have been described previously1-4 but this  ...

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

Mitochondria are involved in many important cellular functions including metabolism, survival1, development and, calcium signaling2. Two of the most important mitochondrial functions are related to the efficient production of ATP, the energy currency of the cell, by oxidative phosphorylation, and the mediation of signals for programmed cell death3.
The enzyme primarily responsible for the production of ATP is the F1FO-ATP synthase, also called ATP synthase4-5. In recent years, the role of mitochondria in apoptotic and necrotic cell death has received considerable attention. In apoptotic cell death, BCL-2 family proteins such as Bax enter the mitochondrial outer membrane, oligomerize and permeabilize the outer membrane, releasing pro-apoptotic factors into the cytosol6. In classic necrotic cell death, such as that produced by ischemia or excitotoxicity in neurons, a large, poorly regulated increase in matrix calcium contributes to the opening of an inner membrane pore, the mitochondrial permeability transition pore or mPTP. This depolarizes the inner membrane and causes osmotic shifts, contributing to outer membrane rupture, release of pro-apoptotic factors, and metabolic dysfunction. Many proteins including Bcl-xL7 interact with F1FO ATP synthase, modulating its function. Bcl-xL interacts directly with the beta subunit of F1FO ATP synthase, and this interaction decreases a leak conductance within the F1FOATPasecomplex, increasing the net transport of H+ by F1FO during F1FO ATPase activity8 and thereby increasing mitochondrial efficiency. To study the activity and modulation of the ATP synthase, we isolated from rodent brain submitochondrial vesicles (SMVs) containing F1FO ATPase. The SMVs retain the structural and functional integrity of the F1FO ATPase as shown in Alavian et al. Here, we describe a method that we have used successfully for the isolation of SMVs from rat brain and we delineate the patch clamp technique to analyze channel activity (ion leak conductance) of the SMVs.

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

A method to isolate submitochondrial vesicles enriched in F1FO ATP synthase complexes from rat brain is described. These vesicles allow the study of the activity of F1FO ATPase complex and its modulation using the technique of patch clamp recording.

<sub&gt; 1</sub&gt; F<sub&gt; O</sub&gt; ATPase Vesicle Vorbereitung und Technik für Darstellende Patch-Clamp-Recordings von submitochondrialen Vesikelmembranen

Mitochondria are involved in many important cellular  functions including metabolism, survival1, development and, calcium signaling2. Two of the most ...

Whole Cell Patch Clamp for Investigating the Mechanisms of Infrared Neural Stimulation

It has been demonstrated in recent years that pulsed, infrared laser light can be used to elicit electrical responses in neural tissue, independent of any further modification of the target tissue. Infrared neural stimulation has been reported in a variety of peripheral and sensory neural tissue in vivo, with particular interest shown in stimulation of neurons in the auditory nerve. However, while INS has been shown to work in these settings, the mechanism (or mechanisms) by which infrared light causes neural excitation is currently not well understood. The protocol presented here describes a whole cell patch clamp method designed to facilitate the investigation of infrared neural stimulation in cultured primary auditory neurons. By thoroughly characterizing the response of these cells to infrared laser illumination in vitro under controlled conditions, it may be possible to gain an improved understanding of the fundamental physical and biochemical processes underlying infrared neural stimulation.

Infrared nerve stimulation has been proposed as an alternative to electrical stimulation in a range of nerve types, including those associated with the auditory system. This protocol describes a patch clamp method for studying the mechanism of infrared nerve stimulation in a culture of primary auditory neurons.

Whole Cell Patch-Clamp für erforschen die Mechanismen der Infrarot Neural Stimulation

It has been  demonstrated in recent years that pulsed, infrared laser light can be used to  elicit electrical responses in neural tissue, independent of ...

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 ...

Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. Retinal neurons in the network comprise of numerous subtypes. More than 10 subtypes of bipolar cells, ganglion cells, and amacrine cells have been identified by morphological studies. Multiple subtypes of retinal neurons are thought to encode distinct features of visual signaling, such as motion and color, and form multiple neural pathways. However, the functional roles of each neuron in visual signal processing are not fully understood. The patch clamp method is useful to address this fundamental question. Here, a protocol to record light-evoked synaptic responses in mouse retinal neurons using patch clamp recordings in dark-adapted conditions is provided. The mouse eyes are dark-adapted O/N, and retinal slice preparations are dissected in a dark room using infrared illumination and viewers. Infrared light does not activate mouse photoreceptors and thus preserves their light responsiveness. Patch clamp is used to record light-evoked responses in retinal neurons. A fluorescent dye is injected during recordings to characterize neuronal morphological subtypes. This procedure enables us to determine the physiological functions of each neuron in the mouse retina.

We will demonstrate how to prepare retinal slices from the mouse eye and record light responses in retinal neurons. The entire procedure is conducted in dark-adapted conditions.

Recording Licht-evozierte Postsynaptische Antworten in Neuronen im Dunkel-adaptierten, Maus retinalen Scheibe Vorbereitungen Verwendung Patch-Clamp-Techniken

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. ...

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

Perforated-Patch-Clamp-Aufzeichnung der Maus Riechzellen in Intact Neuroepithel: Funktionelle Analyse von Neuronen, die eine bestimmte Geruchsrezeptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

Whole-Cell-Patch-Clamp-Aufnahmen in Hirnschnitten

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in these preparations is perpendicular to the retinal surface, making it ideal for the study of radially oriented neurons like photoreceptors and bipolar cells. However, the large dendritic arbors of horizontal cells, wide-field amacrine cells, and ganglion cells are mostly truncated, leaving markedly reduced synaptic activity in these cells. Whereas ganglion cells and displaced amacrine cells can be studied in a whole-mounted preparation of the retina, horizontal cells and amacrine cells located in the inner nuclear layer are only poorly accessible for electrodes in whole retina tissue.
To achieve maximum accessibility and synaptic integrity, we developed a horizontal slice preparation of the mouse retina, and studied signal transmission at the synapse between photoreceptors and horizontal cells. Horizontal sectioning allows&#160;(1) easy and unambiguous visual identification of horizontal cell bodies for electrode targeting, and (2) preservation of the extended horizontal cell dendritic fields, as a prerequisite for intact and functional cone synaptic input to horizontal cell dendrites.
Horizontal cells from horizontal slices exhibited tonic synaptic activity in the dark, and they responded to brief flashes of light with a reduction of inward current and diminished synaptic activity. Immunocytochemical evidence indicates that almost all cones within the dendritic field of a horizontal cell establish synapses with its peripheral dendrites. The horizontal slice preparation is therefore well suited to study the physiological properties of horizontally extended retinal neurons as well as sensory signal transmission and integration across selected synapses.

We developed a horizontal slice preparation of the adult mammalian retina with the plane of sectioning parallel to the retinal surface. Dendritic fields of nonradially oriented retinal neurons were not truncated, allowing the study of signal processing with patch-clamp and imaging techniques.

Vorbereitung von Horizontal Scheiben der erwachsenen Maus Retina für elektrophysiologische Untersuchungen

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in ...