1. Mitochondrien-Isolation (Angepasst von Braun MR et al. 9) <ol><li> Sacrifice die Ratte unter Verwendung von Methoden der Institutional Animal Care und Use Committee (IACUC) zugelassen. </li><li> Schneiden Sie den Kopf des Tieres durch Enthauptung, schneiden Sie die Haut und setzen den Schädel. </li><li> Öffnen Sie den Schädel sanft durch Schneiden mit einer Schere oder Zange. Entfernen Sie das Gehirn. </li><li> Mince fein das Gehirn ohne Kleinhirn in Isolation Buffer (siehe Tabelle 1) und überträgt es auf eine 5-ml-Glas / Teflon-Homogenisator (siehe Geräteliste). </li><li> Homogenisieren Gewebe sanft 10 mal (keine Blasen), ca. 5 min. </li><li> Zentrifuge Probe bei 1.500 × g für 10 min bei 4 ° C in einer Tischzentrifuge. </li><li> Speichern Sie den Überstand (Mitochondrien und Synaptosome) und entsorgen Sie die Pellet (Kernmaterial und Zelltrümmer). Zentrifuge bei 16.000 × g für 10 min bei 4 ° C in einer Tischzentrifuge. </li><li> VerwerfenÜberstand und resuspendieren Pellet in 500 ul Isolation Buffer. Stören Synaptosome mit einem Zellaufschluss Schiffes (siehe Geräteliste). Anwenden eines Druck von 1200 psi für 10 min, gefolgt von schnellen Dekompression. </li><li> Schicht der Mischung auf Ficoll-Gradienten (siehe Tabelle 2), in SW-50.1 Rotor und Zentrifuge bei 126.500 × g für 20 min bei 4 ° C in einer Ultrazentrifuge (siehe Geräteliste). Das Pellet wird das gereinigte Mitochondrien, ist die Ebene zwischen den unterschiedlichen Dichten von Ficoll ungestörten Synaptosomen. </li><li> Waschen Pellet durch Zentrifugation in Isolation Puffer bei 16.000 × g für 10 min bei 4 ° C in einer Tischzentrifuge. </li></ol> 2. Submitochondrialen Vesikel (SMV) Isolation (Angepasst von Chan et al. 10) <ol><li> Re-suspend in 200 ul Isolation Buffer mit einem gleichen Volumen von 1% Digitonin kombiniert Mitochondrien und damit auf Eis für 15 min sitzen. </li><li> Fügen Sie mehr Isolation Buffer und Zentrifuge bei 16.000 × g für 10 min bei 4 ° C in einer Tischzentrifuge. Tun Sie dies zweimal. </li><li> Re-suspend Pellet in 200 ul Isolation Buffer und fügen 2 ul 10% Lubrol PX (C12E9). Mische und lasse auf Eis für 15 min sitzen. </li><li> Layer-Mischung auf Isolation Buffer, in SW-50.1 Rotor und Zentrifuge bei 182.000 × g bei 4 ° C für 1 Stunde. </li><li> Waschen Endpellet durch Zentrifugieren in einer Tischzentrifuge in Isolation Puffer bei 16.000 × g für 10 min bei 4 ° C. </li></ol> 3. Elektrophysiologische Recording <ol><li> Eine typische Elektrophysiologie rig einen Verstärker, einen PC mit einer Digidata 1440A Analog-zu-Digital-Wandler-Schnittstelle in Verbindung mit pClamp10.0 Software Manipulatoren, ein Mikroskop, eine Schwingungsisolation Tabelle Faraday-Käfig ausgestattet. </li><li> Borosilikatglas Kapillaren werden in einem Flaming / Brown Feinpipettenziehvorrichtung Modell P-87 eingesetzt. Eine Pipette-Abzieher Programm wird optimierterzeugen Pipetten mit Widerständen zwischen 80 bis 100 MOhm. </li><li> SMVS in einer physiologischen intrazellulären Lösung (ATP wird zu einem geeigneten Zeitpunkt während der Aufzeichnung hinzugefügt) (Tabelle 3) angeordnet ist. Patch-Clamp-Pipetten mit der gleichen Lösung (ohne ATP) gefüllt. Aufnahmen werden durch Bildung eines Giga-Ohm-Dichtung auf SMVS bei Raumtemperatur durchgeführt. Vesikel werden visualisiert durch Phasenkontrast-Mikroskopie mit einer Nikon oder Zeiss inversen Mikroskop. </li><li> Das Membranpotential bei Spannungen im Bereich von -100 mV bis +100 mV für die Dauer von 10 Sekunden aufrechterhalten. Aufnahmen werden bei 5 kHz über die Verstärkerschaltung filtriert. Niveau von streunenden elektrische oder unspezifische Rauschen von weniger als 1 pA sind wünschenswert für eine erfolgreiche Aufnahme. </li><li> Die Daten werden mit 10,0 Clampfit Software beispielsweise in Frequenz und Amplitude der Einkanal Ereignisse zu bestimmen analysiert. Spannung Abhängigkeit wird durch Auftragen einer Strom-Spannungs-Beziehung bestimmt. </li></ol>

<ol>
	<li>Cheng, W. C., Berman, S. B., Ivanovska, I., Jonas, E. A., Lee, S. J., Chen, Y., Kaczmarek, L. K., Pineda, F., Hardwick, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+factors+with+dual+roles+in+death+and+survival.">Mitochondrial factors with dual roles in death and survival.</a> Oncogene. 25, 4697-4705 (2006).</li><li>Duchen, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+and+calcium+in+health+and+disease.">Mitochondria and calcium in health and disease.</a> Cell Calcium. 44, 1-5 (2008).</li><li>Lemasters, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+mitochondrial+membrane+permeability+in+pathogenesis,+autophagy+and+control+of+metabolism.">Modulation of mitochondrial membrane permeability in pathogenesis, autophagy and control of metabolism.</a> J. Gastroenterol. Hepatol. 22, S31-S37 (2007).</li><li>Cox, G. B., Jans, D. A., Fimmel, A. L., Gibson, F., Hatch, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothesis.+The+mechanism+of+ATP+synthase.+Conformational+change+by+rotation+of+the+beta-subunit.">Hypothesis. The mechanism of ATP synthase. Conformational change by rotation of the beta-subunit.</a> Biochim. Biophys. Acta. 768, 201-208 (1984).</li><li>Cox, G. B., Fimmel, A. L., Gibson, F., Hatch, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+ATP+synthase:+a+reassessment+of+the+functions+of+the+b+and+a+subunits.">The mechanism of ATP synthase: a reassessment of the functions of the b and a subunits.</a> Biochim. Biophys. Acta. 849, 62-69 (1986).</li><li>Cory, S., Huang, D. C., Adams, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Bcl-2+family:+roles+in+cell+survival+and+oncogenesis.">The Bcl-2 family: roles in cell survival and oncogenesis.</a> Oncogene. 22, 8590-8607 (2003).</li><li>Vander Heiden, M. G., Thompson, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bcl-2+proteins:+regulators+of+apoptosis+or+of+mitochondrial+homeostasis.">Bcl-2 proteins: regulators of apoptosis or of mitochondrial homeostasis.</a> Nat. Cell Biol. 1, 209-216 (1999).</li><li>Alavian, K. N., Li, H., Collis, L., Bonanni, L., Zeng, L., Sacchetti, S., Lazrove, E., Nabili, P., Flaherty, B., Graham, M., Chen, Y., Messerli, S. M., Mariggio, M. A., Rahner, C., McNay, E., Shore, G. C., Smith, P. J. S., Hardwick, J. M., Jonas, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bcl-xL+regulates+metabolic+efficiency+of+neurons+through+interaction+with+the+mitochondrial+F1FO+ATP+synthase.">Bcl-xL regulates metabolic efficiency of neurons through interaction with the mitochondrial F1FO ATP synthase.</a> Nat. Cell Biol. 13 (10), 1224-1233 (2011).</li><li>Brown, M. R., Sullivan, P. G., Dorenbos, K. A., Modafferi, E. A., Geddes, J. W., Steward, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitrogen+disruption+of+synaptoneurosomes:+an+alternative+method+to+isolate+brain+mitochondria.">Nitrogen disruption of synaptoneurosomes: an alternative method to isolate brain mitochondria.</a> Journal of Neuroscience Methods. 137, 299-303 (2004).</li><li>Chan, T. L., Greenawalt, J. W., Pedersen, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+and+ultrastructural+properties+of+a+mitochondrial+inner+membrane+fraction+deficient+in+outer+membrane+and+matrix+activities.">Biochemical and ultrastructural properties of a mitochondrial inner membrane fraction deficient in outer membrane and matrix activities.</a> J. Cell Biol. 45 (2), 291-305 (1970).</li><li>Young, H. K. o., Delannoy, M., Hullihen, J., Chiu, W., Pedersen, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+ATP+Synthasomes.">Mitochondrial ATP Synthasomes.</a> J. Biol. Chem. 278 (14), 12305-12309 (2003).</li></ol>

Keine Interessenskonflikte erklärt.

Die hierin beschriebenen Verfahren ermöglichen die Gewinnung von reinem Mitochondrien am Ende von Schritt 1 und submitochondrialen Vesikel (SMVS) nach Schritt 2 aus ganzen Gehirn ohne Unterschied der Zelle durch dieses Verfahren phenotypes.SMVspurified im wesentlichen frei von Verunreinigung durch andere subzelluläre Organellen, wie in Abbildung 1 und unserer bisherigen Arbeit (Alavian KN et al. 8) und behalten ihre strukturelle und funktionelle Integrität vor dem Einfrieren. Nach...

<table border="1">
 <tbody>
 <tr>
 <td>Name </td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Potter-Elvehjem Tissue Grinder withPTFEPestle</td>
 <td>Krackeler Scientific, Inc.</td>
 <td>1-7725T-5 </td>
 </tr>
 <tr>
 <td>Eppendorf Centrifuge 5424 </td>
 <td>Eppendorf </td>
 <td>5424 000.410</td>
 </tr>
 <tr>
 <td>4639 Cell Disruption Vessel</td>
 <td>Parr Instrument Company</td>
 <td>4639</td>
 </tr>
 <tr>
 <td>Ficoll</td>
 <td>Sigma-Aldrich</td>
 <td>F5415</td>
 </tr>
 <tr>
 <td>Polycarbonate centrifuge tubes</td>
 <td>Beckman Coulter</td>
 <td>P20314</td>
 </tr>
 <tr>
 <td>SW-50.1 rotor </td>
 <td>Beckman Coulter</td>
 <td></td>
 </tr>
 <tr>
 <td>L8-70M Ultracentrifuge</td>
 <td>Beckman Coulter</td>
 <td></td>
 </tr>
 <tr>
 <td>Digitonin</td>
 <td>Sigma-Aldrich</td>
 <td>D5628 </td>
 </tr>
 <tr>
 <td>Lubrol PX (C12E9) </td>
 <td>Calbiochem</td>
 <td>205534</td>
 </tr>
 <tr>
 <td>Axopatch 200B </td>
 <td>Axon Instruments </td>
 <td></td>
 </tr>
 <tr>
 <td>Digidata 1440A </td>
 <td>Molecular Device</td>
 <td></td>
 </tr>
 <tr>
 <td>pClamp10.0</td>
 <td>Molecular Device</td>
 <td></td>
 </tr>
 <tr>
 <td>Manipulator</td>
 <td>Sutter Instrument</td>
 <td></td>
 </tr>
 <tr>
 <td>Borosilicate glass capillary</td>
 <td>World Precision Instruments</td>
 <td>1308325</td>
 </tr>
 <tr>
 <td>Flaming/Brown Micropipette Puller Model P-87</td>
 <td>Sutter Instrument</td>
 <td></td>
 </tr>
 </tbody>
</table>

f1fo atpase vesicle preparation and technique for performing patch clamp recordings of submitochondrial vesicle membranes

Mitochondrien sind in vielen wichtigen zellulären Funktionen wie Stoffwechsel, Überleben 1, Entwicklung und Calcium-Signalgebung 2 beteiligt. Zwei der wichtigsten mitochondrialen Funktionen sind auf die effiziente Produktion von ATP, die Energie Währung der Zelle, die durch oxidative Phosphorylierung und die Vermittlung von Signalen für den programmierten Zelltod 3 bezogen. Das Enzym in erster Linie verantwortlich für die Produktion von ATP ist die F1FO-ATP-Synthase, auch als ATP-Synthase 4-5. In den letzten Jahren hat sich die Rolle der Mitochondrien in apoptotischen und nekrotischen Zelltod erhebliche Aufmerksamkeit erhalten. In apoptotischen Zelltod, geben Bcl-2-Proteine ​​wie Bax äußeren Mitochondrienmembran, Oligomerisierung und Permeabilisierung der äußeren Membran, die Freigabe pro-apoptotische Faktoren in das Cytosol 6. In der klassischen nekrotischen Zelltod, wie die durch Ischämie oder Exzitotoxizität in Neuronen eine größ produzierte, schlecht reguliert Erhöhung Matrix Calcium trägt zur Öffnung der inneren Membran Poren, die mitochondriale Permeabilität Übergang Pore oder mPTP. Diese depolarisiert die innere Membran und verursacht osmotischen Verschiebungen, einen Beitrag zur äußeren Membran Ruptur, die Freisetzung von pro-apoptotischen Faktoren und Stoffwechselstörungen. Viele Proteine ​​einschließlich Bcl-xL 7 interagieren F1FO ATP-Synthase, stetig seine Funktion. Bcl-xL interagiert direkt mit der beta-Untereinheit der ATP-Synthase F1FO, und diese Interaktion vermindert ein Leck Leitwert innerhalb der F1FOATPasecomplex, die Erhöhung der Netto-Transport von H + durch F1FO während F1FO ATPase Aktivität 8 und wodurch mitochondrialen Effizienz. Um die Aktivität und Modulation der ATP-Synthase studieren isolierten wir aus Nagergehirn submitochondrialen Vesikel (SMVS) enthält F1FO ATPase. Die SMVS behalten die strukturelle und funktionelle Integrität des F1FO ATPase nach Alavian et al. Hier beschreiben wir eine Methodedass wir erfolgreich für die Isolierung von SMVS aus Rattenhirn verwendet und wir Abgrenzung der Patch-Clamp-Technik, um Kanal-Aktivität zu analysieren (ion Leck Leitwert) der SMVS.

Der erste Schritt der Protokoll ermöglicht für die Isolierung von Mitochondrien gereinigt, wie durch Western-Blot in Fig. 1 gezeigt. In Abbildung 2 ist ein Beispiel für einen brain-derived submitochondrialen Vesikel Patch Aufnahme gezeigt. Mit der inside-out Patch Konfiguration zeigen wir Kanalaktivität moduliert durch ATP. Die Steuerung (CTL) die Aufnahme (links) zeigt Multi-Kanal-Aktivität Leitfähigkeit mit einem Spitzenwert Leitfähigkeit von 600 pS im Durchschnitt. das wurde u...

Watch this Scientific Journal Video about F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes at JoVE.com

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

Eine Methode, um submitochondrialen Vesikel in F1FO ATP-Synthase-Komplexe aus Rattenhirn angereichert isolieren beschrieben. Diese Vesikel erlauben das Studium der Aktivität von F1FO ATPase-Komplex und dessen Modulation unter Verwendung der Technik der Patch-Clamp-Aufnahme.

<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 ...

f1fo-atpase-vesicle-preparation-technique-for-performing-patch-clamp

Research

JoVE Journal

Neuroscience

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

10.2K Aufrufe.  Yale University. Eine Methode, um submitochondrialen Vesikel in F1FO ATP-Synthase-Komplexe aus Rattenhirn angereichert isolieren beschrieben. Diese Vesikel erlauben das Studium der Aktivität von F1FO ATPase-Komplex und dessen Modulation unter Verwendung der Technik der Patch-Clamp-Aufnahme.

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

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

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.  ...

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  ...

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 ...

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 ...

Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate electrical processes in target cells. In this context, ion selectivity of a channelrhodopsin is of particular importance. This article describes the investigation of chloride selectivity for a recently identified anion-conducting channelrhodopsin of Proteomonas sulcata via electrophysiological patch-clamp recordings on HEK293 cells. The experimental procedure for measuring light-gated photocurrents demands a fast switchable &#8211; ideally monochromatic &#8211; light source coupled into the microscope of an otherwise conventional patch-clamp setup. Preparative procedures prior to the experiment are outlined involving preparation of buffered solutions, considerations on liquid junction potentials, seeding and transfection of cells, and pulling of patch pipettes. The actual recording of current-voltage relations to determine the reversal potentials for different chloride concentrations takes place 24 h to 48 h after transfection. Finally, electrophysiological data are analyzed with respect to theoretical considerations of chloride conduction.

This article describes how the ion selectivity of channelrhodopsin is determined with electrophysiological whole-cell patch-clamp recordings using HEK293 cells. Here, the experimental procedure for investigating chloride selectivity of an anion-selective channelrhodopsin is demonstrated. However, the procedure is transferable to other channelrhodopsins of distinct selectivity.

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