Diese Arbeit wurde vom NIH Grant EY17606 (APS) unterstützt.

1. Herstellung von Elektroden <ol><li> Bereiten Sie die Aufnahme-und Referenz-Elektroden durch Eintauchen sowohl in 1M NaCl und Fahren 15V zwischen ihnen mit einer 1 Hz Sinuswelle für ~ 20 s. </li><li> Machen Sie die Aufnahme Elektroden mit einer Mikropipette Puller (Sutter Instruments P-97). Für Patch-Pipetten mit einem Borosilikatglas mit einem Außendurchmesser von 1,2 mm und einem Innendurchmesser von 0,69 mm (Sutter Instruments, Kat. Nr. B120-69 bis 10). Messen Sie den Serienwiderstand durch die Spitze der Elektrode. Basierend auf der Größe der Zielzellen wählen Sie eine Pipettenspitze Durchmesser für die Zellkörper ~ 20 um im Durchmesser verwenden ~ 5 MW für Zellkörper ~ 5 um im Durchmesser verwenden ~ 12 MOhm. </li><li> Bereiten Boden / Referenzelektroden. Mit einer Spritze füllen einen kleinen Ausschnitt aus Polyethylen-Schlauch (Intramedic, PE205) mit 1 mM NaCl, dass 3,5% Agar enthält (Sigma; Cat # A7002) in 1M NaCl. Legen Sie dieses Agar Brücke über die Ag / AgCl-Referenzelektrode. </li></ol> 2. Herstellung von Lösungen <ol><li> Externe Badlösung, Ames &#39;Medien mit Bicarbonat (1 L): 1 Flasche Ames&#39; Medium (Sigma; Cat # A1420), 1,9 g NaHCO 3, 7 ml Penicillin-Streptomycin (Sigma, Kat. Nr. P0781) zu verhindern, bakterielle Wachstum. Passen Osmolarität auf ~ 280 mOsm. </li><li> Slicing-Lösung, Ames &#39;Medien mit dem pH-Puffer HEPES (1 L): 1 Flasche Ames&#39; Medium, 0,887 g NaCl, 2,38 g HEPES, 7 ml Penicillin-Streptomycin. Der pH-Wert auf ~ 7,4 mit 1 M NaOH und / oder 1 M HCl. Passen Osmolarität auf ~ 280 mOsm. </li><li> Kalium-Aspartat (K-Asp) Pipette interne Lösung (in mM): 125 K-Aspartat, 10 KCl, 10 HEPES, 5 NMG-HEDTA, 0,5 CaCl 2, 1 ATP-Mg, 0,2 GTP-Mg, pH-Wert wird eingestellt ~ 7,3 mit NMG-OH, und Osmolarität auf ~ 280 mOsm eingestellt. </li><li> Agar Rücklaufsperre für die Stabilisierung niedriger Geliertemperatur Agarose mit der Netzhaut (5 Gew.%), 7,5 g Agar (Sigma; Cat # A7002) in 150 ml dH 2 O. geschmolzen </li></ol> 3. Day of Experiment <ol><li> Thaw ~ 1,5 ml K-Asp Internal Solution und dem Fluorophor Ihrer Wahl aus dem Gefrierschrank. Verdünnen Sie die Fluorophor auf die geeignete Konzentration, die empirisch ermittelt werden sollte. </li><li> Gießen Ames &#39;Medien mit NaHCO 3 (~ 200 ml) in die lichtdichte Vorratsbehälter und legen Sie dann in das Wasserbad (30-32 ° C) und im Gleichgewicht mit 5% CO 2 / 95% O 2-Gas </li><li> Fill-Flasche mit ~ 110 ml Ames &#39;Medien mit HEPES, auf Eis stellen und im Gleichgewicht mit 100% O 2. </li><li> Die restlichen Ames &#39;Medien mit NaHCO 3 in einem beheizten Reservoir auf der Oberseite des Faraday-Käfig, und im Gleichgewicht mit 5% CO 2 / 95% O 2-Gas. Parafilm verwendet werden, um das Gas in den Raum oberhalb der Lösung Falle sein. </li><li> Bereiten Sie die niedrigen Geliertemperatur Agarose (3%) durch Zugabe von 0,75 g (Sigma; Cat # A0701) zu 25 ml Ames &#39;Medien mit HEPES und Wärme der Lösung bei ca. 115 ° C um die Agarose zu schmelzen. Rühren, bis die Lösung klar, ohne Blasen, und speichern Sie in einem Wasserbad (ca. 42 ° C). </li><li> Füllen Sie eine Aufzeichnung Pipette mit dem K-Asp interne Lösung und überprüfen Sie die Spitze Widerstand. </li></ol> 4. Begin Experiment <ol><li> Euthanize der Maus und schnell zu entfernen, die Augen mit einer gebogenen Schere. Infrarot-Schutzbrillen sollten für alle Verfahren, um zu verhindern das Ausbleichen der visuellen Pigment verwendet werden. </li><li> Legen Sie die Augen auf ein Stück Filterpapier, um sie von Rollen, und mit einem dünnen doppelseitigen Klinge zu vermeiden, stellen einen Schlitz in die Hornhaut, während das Auge mit der Pinzette. </li><li> Sobald der Augapfel ist punktiert, übertragen sie Auge in eine Schale mit Ames &#39;Medien gefüllt. </li><li> Mit den Schlitz in die Hornhaut als Einstieg, sind kleine Schere zum Schneiden entfernt die restlichen Teile der Hornhaut, nach dem die Linse und Glaskörper entfernt werden können. Achten Sie darauf, dass die Netzhaut an der Augenmuschel bleibt, und speichern Sie die Augenmuschel in Dunkelheit bei 32 ° C in Ames &#39;media äquilibriert mit 5% CO 2 / 95% O 2. </li></ol> 5. Slicing <ol><li> Hemisect einer der Augenmuscheln und distanzieren der Netzhaut von der retinalen Pigmentepithels. Entfernen Sie die Ränder der Netzhaut, damit das Gewebe flach zu liegen. </li><li> Mit einem kleinen Glas Pasteur Pipette mit einer Latex-Glühlampe auf eine 35 mm Petrischale mit geschmolzenem Agar zu füllen, und ziehen Sie die Zange quer durch den Agar seiner Konsistenz zu testen. </li><li> Wenn Sie anfangen können, finden Sie in der Agar verfestigt: <ol class="lalpha"><li> Übertragen Sie die Netzhaut an die Spitze des Agar. </li><li> Verwenden Sie ein verdrilltes Stück Kimwipe um die überschüssige Lösung rund um die Netzhaut zu absorbieren. </li><li> Legen Sie 2-3 Tropfen Agar direkt auf die Netzhaut und schnell statt einer Kunststoff-Zylinder über die Netzhaut an einer Wand um sie herum zu bilden. Dann tropft zusätzliche geschmolzen Agar an den Seiten der Kunststoff-Zylinder, während die Zentrierung der Netzhaut im Agar. </li><li> Übertragen Sie die Petrischale to ein Eiswasserbad abkühlen lassen. </li></ol></li><li> Nach ~ 45 Sekunden entfernen Sie das Gewebe aus dem Eiswasserbad und mit einem Stößel zu extrudieren die Agar-Block aus der Kunststoff-Zylinder, Schieben von der Seite von der Netzhaut am weitesten. </li><li> Verwenden Sie die einseitigen Rasierklinge herausgeschnitten ein kleiner Block von Agar mit der Netzhaut und Sekundenkleber den Block in Position gegen die Agar Rücklaufsperre auf der Probe-Disc. </li><li> Die Probe-Disc auf die vibrierende Mikrotom Tablett und gießen eiskalten Ames HEPES in das Fach ermöglicht den Block mit Netzhaut vollständig eingetaucht werden. </li><li> Retinal Scheiben mit einer Dicke von ca. 200 mu m kann die maximale Klinge vibriert Zinssenkung und eine vorausschauende Klinge Geschwindigkeit so eingestellt, dass es 4 bis 5 Sekunden dauert, um durch die Netzhaut auf jede Scheibe geschnitten. </li><li> Verwenden Sie einen Nr. 11 Skalpell zu jedem brauchbaren Scheibe ein zu einer Zeit zu entfernen </li><li> Gute Scheiben, die Netzhaut sind in der Aufnahme Kammern gelegt und sich mit Scheibe Anker mit Nylonfäden Kreuzung ihnen gewichtet. Die Nylonfäden halten Sie die Agar-Umgebung der Netzhaut, so dass die Netzhaut ungehindert für Aufnahmen. </li><li> Übertragen Sie die Aufnahme Kammer auf die Bühne des aufrechten Mikroskop, in der Nähe der Vorhang, und schalten Sie den Infrarot-Lichtquelle, um die Scheibe auf eine Infrarot-sensitive Kamera-Ansicht. </li></ol> 6. Aufnahme <ol><li> Sobald eine gute Netzhaut schneiden (mit intakten Photorezeptoren und den entsprechenden Schnittwinkel) identifiziert beginnen Perfusion der Netzhaut mit einer Rate von mehr als 5 ml / min mit beheiztem Ames &#39;Medien, die 35 37 ° C erhitzt wird und äquilibriert mit 5% CO 2 / 95% O 2. </li><li> Einige Beschädigung der Oberfläche kann offensichtlich durch die Slicing-Prozess. In der Regel dünne Schicht von Zellen kann mit Hilfe einer Pipette mit einem gebrochenen Spitze, und sanft Absaugen der Toten Zellkörper. Dies wird in der Regel zeigen lebende Zellen darunter. Identifizieren einer gesunden Zelle Körper, deren Position in den Schichten des Interesses. </li><li> Füllen einer Mikropipette mit dem Kasp interne Lösung und gelten positive (außen-) Druck durch die Spitze der Pipette, und senken Sie die Spitze der Pipette in die Aufnahme Kammer und bis auf die Ebene der Zelle mit einem Mikromanipulator. </li><li> Bewegen Sie die Pipette, so dass es die Zellmembran Grübchen, und wenden Sie sanfte negative (nach innen) Druck auf eine hohe Beständigkeit Dichtung zu machen. Whole-cell-Modus kann durch Anlegen von Unterdruck an der Elektrode in die Zelle zu brechen erreicht werden. </li><li> Förderung der Retina mit LEDs können dann rufen Reaktionen auf Licht. </li><li> Fotografieren Sie die Zelle, aus der die Aufnahme von evozieren die Fluoreszenz des Fluorophors in der Pipette interne Lösung gemacht wurde. </li></ol> 7. Repräsentative Ergebnisse <img src="/files/ftp_upload/2107/2107fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Hier Potenziale einer dunkeladaptierten Retina Neuron zu Lichtblitze von zunehmender Stärke gezeigt Licht hervorgerufen. <img src="/files/ftp_upload/2107/2107fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Fluoreszenz Bild aus einem der Netzhaut schneiden, wo Zellen mit dem Fluorophor, Lucifer Yellow gefüllt wurden. Evozierte Fluoreszenz zeigt die Morphologie der Zellen, aus denen Patch-Aufnahmen gemacht wurden.

<ol>
	<li>Weblin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmission+along+and+between+rods+in+the+tiger+salamander+retina.">Transmission along and between rods in the tiger salamander retina.</a> J Physiol. 280, 449-470 (1978).</li><li>Wu, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+connections+among+retina+neurons+in+living+slices.">Synaptic connections among retina neurons in living slices.</a> J Neurosci Methods. 20, 139-149 (1987).</li><li>Rieke, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+contrast+adaptation+in+salamander+bipolar+cell.">Temporal contrast adaptation in salamander bipolar cell.</a> J Neurosci. 21, 9445-9454 (2001).</li><li>Armstrong-Gold, C., &amp;amp, R. i. e. k. e., F, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bandpass+filtering+at+the+rod+to+second-order+cell+synapse+in+salamander+(Ambystoma+tigrinum)+retina.">Bandpass filtering at the rod to second-order cell synapse in salamander (Ambystoma tigrinum) retina.</a> J Neurosci. 23, 3796-3806 (2003).</li><li>Masland, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fundamental+plan+of+the+retina.">The fundamental plan of the retina.</a> Nat Neurosci. 4, 877-886 (2001).</li><li>Wassle, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+processing+in+the+mammalian+retina.">Parallel processing in the mammalian retina.</a> Nat Rev Neurosci. 5, 747-757 (2004).</li></ol>

Slice-Aufnahme von Wirbeltieren Netzhaut seit Jahrzehnten 1,2 gemacht und waren recht erfolgreich bei der Bereitstellung ein tieferes Verständnis davon, wie die Netzhaut Schaltung kodiert einfallenden Lichts. Die Vorteile des Schneidens mit einem vibrierenden Mikrotom anstatt direkt mit einer Rasierklinge ist, dass die Netzhaut Scheiben weniger Schaden an der Oberfläche entstehen. Mit einer Saugpipette ist es leicht, diese Schäden und Ziel gesunde Zellen direkt unter der Oberfläche, dass ihre Verbindung i...

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<td>Micropipette Puller</td>
<td>&nbsp;</td>
<td>Sutter Instruments</td>
<td>P-97</td>
<td>&nbsp;</td>
<tr>
<td>Borosilicate glass</td>
<td>&nbsp;</td>
<td>Sutter Instruments</td>
<td>B120-69-10</td>
<td>&nbsp;</td>
<tr>
<td>Polyethylene tubing</td>
<td>&nbsp;</td>
<td>Intramedic</td>
<td>PE205</td>
<td>&nbsp;</td>
<tr>
<td>Agar</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A7002</td>
<td>&nbsp;</td>
<tr>
<td>Low gelling temp agarose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A0701</td>
<td>&nbsp;</td>
<tr>
<td>Ames' Medium</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A1420</td>
<td>&nbsp;</td>
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<td>Penicillin-Streptomycin</td>
<td>&nbsp;</td>
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patch clamp recordings from mouse retinal neurons in a dark-adapted slice preparation

Unser visuelles Erlebnis wird eingeleitet, wenn die visuelle Pigment in unserer Netzhaut Photonen des Lichts absorbiert und löst eine Kaskade von intrazellulären Ereignissen, die Schließung von zyklisch-Nukleotid-gesteuerte Kanäle in der Zellmembran führt. Die daraus resultierende Veränderung des Membranpotentials führt wiederum zu Kürzungen in Höhe von Neurotransmittern aus beiden Stäbchen und Zapfen synaptischen Terminals. Um zu messen, wie das Licht hervorgerufene Veränderung der Photorezeptor Membranpotentials führt zu nachgelagerten Aktivitäten in der Netzhaut, haben Wissenschaftler elektrophysiologischen Ableitungen von Netzhaut-Scheibe Vorbereitungen für Jahrzehnte 1,2 gemacht. In der Vergangenheit waren diese Scheiben wurden von Hand mit einer Rasierklinge auf die retinale Gewebe, das auf Filterpapier befestigt wird geschnitten, in den letzten Jahren eine andere Methode des Schneidens wurde entwickelt, wobei Netzhautgewebe in niedrigen Geliertemperatur Agar eingebettet ist und in Scheiben geschnitten in coole Lösung mit einem vibrierenden Mikrotom 3,4. Dieses Präparat produziert Netzhaut Scheiben mit weniger Beschädigung der Oberfläche und sehr robust Licht hervorgerufenen Reaktionen. Hier dokumentieren wir, wie dieses Verfahren unter Infrarot-Licht zu vermeiden, Bleichen der Sehpigment getan werden kann.

Watch this Scientific Journal Video about Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation at JoVE.com

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

Hier beschreiben wir ein Verfahren zur Erzeugung von dunkeladaptierten Scheiben der Maus Netzhaut elektrophysiologischen Ableitungen.

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 ...

patch-clamp-recordings-from-mouse-retinal-neurons-dark-adapted-slice

Research

JoVE Journal

Neuroscience

14.3K Aufrufe.  University of Southern California. Hier beschreiben wir ein Verfahren zur Erzeugung von dunkeladaptierten Scheiben der Maus Netzhaut elektrophysiologischen Ableitungen.

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

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

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 ...

Simultaneous Whole-cell Recordings from Photoreceptors and Second-order Neurons in an Amphibian Retinal Slice Preparation

One of the central tasks in retinal neuroscience is to understand the circuitry of retinal neurons and how those connections are responsible for shaping the signals transmitted to the brain. Photons are detected in the retina by rod and cone photoreceptors, which convert that energy into an electrical signal, transmitting it to other retinal neurons, where it is processed and communicated to central targets in the brain via the optic nerve. Important early insights into retinal circuitry and visual processing came from the histological studies of Cajal1,2 and, later, from electrophysiological recordings of the spiking activity of retinal ganglion cells - the output cells of the retina3,4.
A detailed understanding of visual processing in the retina requires an understanding of the signaling at each step in the pathway from photoreceptor to retinal ganglion cell. However, many retinal cell types are buried deep in the tissue and therefore relatively inaccessible for electrophysiological recording. This limitation can be overcome by working with vertical slices, in which cells residing within each of the retinal layers are clearly visible and accessible for electrophysiological recording.
Here, we describe a method for making vertical sections of retinas from larval tiger salamanders (Ambystoma tigrinum). While this preparation was originally developed for recordings with sharp microelectrodes5,6, we describe a method for dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells in which we manipulate the photoreceptor's membrane potential while simultaneously recording post-synaptic responses in horizontal or bipolar cells. The photoreceptors of the tiger salamander are considerably larger than those of mammalian species, making this an ideal preparation in which to undertake this technically challenging experimental approach. These experiments are described with an eye toward probing the signaling properties of the synaptic ribbon - a specialized synaptic structure found in a only a handful of neurons, including rod and cone photoreceptors, that is well suited for maintaining a high rate of tonic neurotransmitter release7,8 - and how it contributes to the unique signaling properties of this first retinal synapse.

We describe the preparation of thin retinal slices from aquatic tiger salamanders (Ambystoma tigrinum) and explain how we use these slices to study synaptic processing in the retina by obtaining dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells.

Gleichzeitige Ganzzellableitungen von Photorezeptoren und Neuronen zweiter Ordnung in eine Amphibie Retinal Scheibe Vorbereitung

One of the central tasks in retinal neuroscience  is to understand the circuitry of retinal neurons and how those connections are  responsible for shaping ...

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

Isolation von sensorischen Neuronen von<em&gt; Aplysia californica</em&gt; Für Patch-Clamp-Recordings von Glutamaterge Currents

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

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 ...

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

Whole-Cell-Patch-Clamp-Aufnahmen aus Morphologically- und neurochemisch identifizierten Hippocampus Inter

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

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. ...

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 ...

Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding these fundamental functions will shed light on the information transfer in these neurons. Dendritic patch-clamp recordings provide the possibility to directly examine the electrical properties of dendrites and underlying voltage-gated ion channels. However, these fine structures are not easily accessible to patch pipettes because of their small diameter. This report describes a step-by-step procedure to collect stable and reliable recordings from the dendrites of dopaminergic neurons in acute slices. Electrophysiological measurements are combined with post hoc recovery of cell morphology. Successful experiments rely on improved preparation of slices, solutions and pipettes, adequate adjustment of the optics and stability of the pipette in contact with the recorded structure. Standard principles of somatic patch-clamp recording are applied to dendrites but with a gentler approach of the pipette. These versatile techniques can be implemented to address various questions concerning the excitable properties of dendrites.

Subcellular patch-clamp recordings offer the possibility to investigate the functional properties of dendrites. However these fine structures are not easily accessible due to their small diameter. The protocol described here aims to facilitate the collection of stable and reliable recordings from the dendrites of dopamine neurons in slices in vitro.

Subzellulärer Patch-Clamp-Aufnahmen aus der somatodendritische Domain nigraler Dopamine Neurons

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding ...