Wir danken dem Leben finanzieren, University of Oklahoma Undergraduate Research Opportunities Program, und dem Department of Zoology für die Finanzierung dieser Arbeit. Wir danken auch Dr. Mariëlle Hoefnagels für redaktionelle Hilfe.

1. Pre-Recording, Live Animal Vorbereitung <ol><li> Entfernen Sie den Skorpion aus dem Hause Krug und legen Sie sie in einem vorgekühlten Glasbehälter; nächstes legen Sie die jar mit dem Skorpion in einen kalten Umgebung (-5 ° C) für mindestens zwei Minuten. Diese Zeit kann je nach Alter unterschiedlich, Größe, Art, etc. der spezifischen Skorpion untersuchten. Aber im Allgemeinen ist das Einfrieren Umwelt-Phase abgeschlossen, wenn das Tier regungslos. </li><li> Nach zwei Minuten wird das immobilisierte Skorpion aus dem kalten Glas-Container, und legen Sie sie Bauchseite bis auf einen Objektträger. Sichern Sie den Stachel, Schwanz, Beine und Pedipalpen mit formbaren Ton (auf Ölbasis, Van Aken Plastalina). </li><li> Machen Sie eine Plattform, auf der der Skorpion ist Pektine kannst. Stellen Sie die Plattform der Länge und Breite je nach Größe des pecten. In der Regel sind unsere Plattformen von Mikroskop Deckglas gebaut und sind ca. 10 (L) x 18 (W) mm. </li><li> Beachten doppelseitigen Klebeband auf die pecten Plattform, Trimmen des Bandes (falls notwendig) zu montieren. </li><li> Um eine pectinal Kammer, legte drei der Plattform vier Seiten in pre-geschmolzenes Wachs (wir verwenden 100% Bienenwachs-Chips) auf eine Tiefe von 3-4 mm. Wir schmelzen das Wachs, indem sie die Chips in einer Glas-Petrischale, auf einer heißen Platte entfernt. Wenn das Wachs an den Rändern der pectinal Kammer auf Raumtemperatur abgekühlt ist, sollte diese Wände aus Wachs ca. 1 mm hohen erstellen. </li><li> Auf das Tier, suchen Sie den Punkt pectinal Einfügen, und legen Sie die un-gewachst Rand der Plattform (Bandseite-up) direkt hinter sie. Danach Stabilisierung der Plattform (so dass es später nicht mehr bewegen) durch leichten Druck seiner rechten und linken peripheren Kanten gegen den Lehm verwendet, um die Beine des Tieres zu beschränken. </li><li> Verwenden Sie spitzen Pinzette vorsichtig die pecten oder Pektine in der Kammer. Genauer gesagt, mit einer Pinzette, halten Sie die pecten entlang der pectinal Wirbelsäule und sanft bringen pecten nicht unterhalb der Glasabdeckung und senken Sie es auf den Klebstoff-Plattform Boden. Stellen Sie eine pecten-to-Tape-Bindung durch eine sorgfältige Anwendung von Druck auf die pectinal Wirbelsäule. </li><li> Jetzt bewerben geschmolzenes Wachs (wir verwenden eine vorgewärmte Metallspatel), um die un-gewachst Rand der Plattform. Der Zweck ist zwei-fach, die pecten zu sichern und vollständig umschließen die Kammer für zukünftige Mineralöl Einführung. Vorsicht beim Auftragen des Wachses über das Tier pecten, wie es möglich ist, die pecten mit Wachs zu heiß ist beschädigt. </li><li> Die pecten Kammer-Konstruktion abgeschlossen ist. Als nächstes gründen eine indifferente Elektrode Zusammenhang mit dem Tier Hämolymphe durch Einfügen eines Silberdraht zwischen zwei Heck-Segmente. </li></ol> 2. Gleichzeitige Aufnahme und extrazelluläre chemische Stimulation <ol><li> Wir bereiten das Tier 24 h vor der Durchführung extrazellulären, tip-Aufnahmen, die unter anderen Gründen (zB Tier-Anpassung an die feste Position) ermöglicht Zeit, um individuell zu Stimulans Pipetten einer bestimmten (Sensillum-abhängig) Tip-Durchmesser. Wir verwenden eine Mikropipette Abzieher für die Erreichung dieser Vorgaben. Im Idealfall ist der Durchmesser der Pipetten ≈ 2 pm größer als die sensillar Porendurchmesser. Wir passen unsere Mikropipette Spezifikationen entsprechend für jedes Tier. </li><li> 1 h vor der Aufnahme, fügen wir 5 ul Mineralöl auf die pectinal Kammer, das Eintauchen des pecten unter Öl. Dann positionieren wir das Tier unter einem Hochleistungs-Mikroskop, das lange gearbeitet hat Objektive und Auflicht. </li><li> Um chemisch anzuregen peg Sensillen stellen wir wässrigen Geschmacksstoffe (zB Zitronensäure und Salz) durch das Öl Medium. Erstens verwenden wir eine Mikropipette Füllstoff zur Stimulans in die Stimulans Pipette injiziert. Hinweis: Alle Stimulans Lösungen sollten ein Elektrolyt für die elektrische Leitfähigkeit enthalten. </li><li> Als nächstes wird nach der Positionierung des Stimulans Pipette auf einem Mikromanipulator (in der Aufnahme rig), legen Sie die Aufnahme-Elektrode in die stumpfe, offene Ende des Stimulans Pipette. Es ist wichtig, den Kontakt zwischen der metallischen Elektrode und Elektrolyt-stimulierende Lösung zu gewährleisten. </li><li> Nach der Vorbereitung ist geerdet (über die indifferente Elektrode), einfach wieder senkt die Pipette durch das Öl mittel-und auf eine sensillar Pore. Die Dauer der chemischen Stimulation pro pectinal Sensillum kann von so viel wie 30 Minuten, um so wenig wie 1 Sekunde variieren. Im Allgemeinen können wir Probe mehrere Stifte (&gt; 30) pro Pipette. Für den Fall, dass die stimulierende Pipette verstopft mit winzigen Schmutz wird, ersetzen Sie einfach die verstopfte Pipette für eine neue, unbenutzte Pipette. </li></ol> 3. Vorbereitung eines Tier für mehrere Tage der Experimente <ol><li> Wenn das Tier über einen längeren Zeitraum verwendet werden, empfehlen wir die Wiederverwendung der pectinal Kammer und Austausch der Tropfen Mineralöl für jede Aufnahme-Session. Nach einem bestimmten Tag der Aufnahme-Session beendet ist, entfernen Sie das Tier von der Recording-, und führen Sie ein Mineralöl zu waschen. </li><li> Specifically, fügen Sie einen Tropfen (5 ul) von Mineralöl in die Kammer, und alles Öl. Dies minimiert die Gegenwart von restlichem Stimulans, die aus der Pipette kann zugespielt haben während der Aufzeichnung. </li><li> Vor der nächsten Sitzung einen Tropfen Öl wie zuvor beschrieben. </li></ol> 4. Repräsentative Ergebnisse: Je nach dem Kontrollgerät verwendet werden (zB die besondere Verstärker, Digitalisierungs-Hardware, etc.), ist ein repräsentatives Ergebnis einer extrazellulären Aufnahme mit einem S: N-Verhältnis von mindestens 3:1. Erfolgreiche Bau der Mineralöl-Kammer ist notwendig für die Isolierung chemische Stimulation einzelner Sensillen, da sie ein Medium für die Bereitstellung von polaren (Wasser-) chemischen Geschmacksstoffe 8 bietet. Wenn es zu wenig Öl oder gar keine, wird der stimulierende Lösung aus den aufgezeichneten Sensillum zu seinen benachbarten Sensillen, die zu einem länger anhaltenden, unkontrollierten Anfall von chemischen Stimulation 8 Ursache zu verbreiten.

<ol>
	<li>Hodgson, E. S., Lettvin, J. Y., Roeder, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiology+of+a+Primary+Chemoreceptor+unit.">Physiology of a Primary Chemoreceptor unit.</a> Science. 12, 417-418 (1955).</li><li>Newland, P. L., Christensen, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taste+processing+in+the+insect+nervous+system.">Taste processing in the insect nervous system.</a> Methods in Insect Sensory Neuroscience. , 289-318 (2005).</li><li>Foelix, R. F., M&#252;ller-Vorholt, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fine+structure+of+scorpion+sensory+organs+II.+Pecten+sensilla.">The fine structure of scorpion sensory organs II. Pecten sensilla.</a> Bull. Br. Arachnol. Soc. 6, 68-74 (1983).</li><li>Carthy, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fine+structure+and+function+of+the+sensory+pegs+on+the+scorpion+pecten.">Fine structure and function of the sensory pegs on the scorpion pecten.</a> Experientia. 22, 89-91 (1966).</li><li>Gaffin, D. D., Brownell, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+properties+of+chemosensory+peg+sensilla+on+the+pectines+of+scorpions.">Response properties of chemosensory peg sensilla on the pectines of scorpions.</a> J. Comp. Physiol. A. 181, 291-300 (1997).</li><li>Wolf, 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+pectine+organs+of+the+scorpion,+Vaejovis+spinigerus:+Structure+and+(glomerular)+central+projections.">The pectine organs of the scorpion, Vaejovis spinigerus: Structure and (glomerular) central projections.</a> Arthropod Struct. Dev. 37, 67-80 (2008).</li><li>Gaffin, D. D., Brownell, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+evidence+of+synaptic+interactions+within+chemosensory+sensilla+of+scorpion+pectines.">Electrophysiological evidence of synaptic interactions within chemosensory sensilla of scorpion pectines.</a> J. Comp. Physiol. A. 181, 301-307 (1997).</li><li>Knowlton, E. D., Gaffin, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+approach+to+examining+scorpion+peg+sensilla:+the+mineral+oil+flood+technique.">A new approach to examining scorpion peg sensilla: the mineral oil flood technique.</a> J. Arachnol. 37, 379-382 (2009).</li><li>Knowlton, E. D., Gaffin, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+tip-recording+method+to+test+scorpion+pecten+chemoresponses+to+water-soluble+stimulants.">A new tip-recording method to test scorpion pecten chemoresponses to water-soluble stimulants.</a> J. Neurosci Methods. , .</li><li>Haupt, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antennal+sucrose+perception+in+the+honey+bee+(Apis+mellifera+L.):+behaviour+and+electrophysiology.">Antennal sucrose perception in the honey bee (Apis mellifera L.): behaviour and electrophysiology.</a> J. Comp. Physiol. A. 190, 735-745 (2004).</li><li>Bland, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensilla+on+the+antennae,+mouthparts,+and+body+of+the+larva+of+the+alfalfa+weevil,+hypera+postica+(gyllenhal)+(Coleoptera+:+Curculionidae).">Sensilla on the antennae, mouthparts, and body of the larva of the alfalfa weevil, hypera postica (gyllenhal) (Coleoptera : Curculionidae).</a> Int. J. Insect Morphol. Embryol. 12, 261-272 (1983).</li><li>Cate, H. S., Derby, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hooded+Sensilla+Homologues:+Structural+Variations+of+a+Widely+Distributed+Bimodal+Chemomechanosensillum.">Hooded Sensilla Homologues: Structural Variations of a Widely Distributed Bimodal Chemomechanosensillum.</a> J. Comp. Neurol. 444, 345-357 (2002).</li></ol>

Wir haben bereits diese Methoden in beiden Zeitschriften Knowlton und Gaffin (2009) and Knowlton und Gaffin (in press) veröffentlicht.

Das Protokoll über beschrieben, wie eine Wüste Grünland Skorpion (Paruroctonus utahensis) für elektrophysiologische Untersuchung vorzubereiten. Insbesondere zeigen wir, wie eine Kammer für die kontrollierte extrazelluläre tip-Aufnahmen von pectinal chemosensorischen Neuronen unter Öl zu bauen. Da Öl und Wasser nicht mischen, ist es möglich, chemische Stimulation (mit Wasser-basierte Stimulanzien) zu einzelnen Sensillen zu isolieren. Es sollte betont werden, dass P. utahensis, ist ein relativ kleines Tier (≈ 2,5-5 cm) und die Anwendung dieses Protokolls, um größere Tiere können viele size Anpassungen, wie zB die Größe des Tieres Plattform-, Kammer-und Ölmenge angewandt erfordern. Wir empfehlen Pilotstudien, die minimale Menge an Öl, die für kontrollierte Abgabe von Wasser-basierte chemische Stimulanzien testen. Darüber hinaus fanden wir keinen Einfluss von Öl auf der Grundlinie neuronale Aktivität innerhalb pectinal Sensillen 8. Dies sollte für andere Modellsysteme bestätigt auch sein, vor der Beurteilung chemoresponsiveness. Diese Methode kann in Verbindung mit einer anderen Methode zur chemoresponse Wechselwirkungen zwischen den Sensillen in der sensorischen Feldtest eingesetzt werden. Zum Beispiel ist es möglich, tip-Record (unter Öl) von einem Sensillum wie wir Basis-Datensatz (über eine Wolfram-Elektrode) aus einem benachbarten Sensillum 9. Eine solche Aufnahme-Konfiguration verwendet werden, um festzustellen, ob auch chemisch nicht stimulierend Sensillum beeinflusst die neuronale Aktivität der Basis-recorded Sensillum werden. Bis heute hat niemand diese für scorpion pectinal Sensillen getestet, und es bleibt eine offene Frage für andere chemosensorischen Systemen. Zusammenfassend lässt sich sagen, dass wir die Spitze-Recording-Methode unter Öl Fortschritte Rahmen der elektrophysiologischen Untersuchungen der neuronalen Basis von Arthropoden gustation. In diesem Manuskript, bieten wir ein Protokoll für die Vorbereitung Tiere für diese Methode, und wir hoffen, es bietet eine solide Grundlage für weitere Untersuchungen der peripheren sensorischen Nervensystem.

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<td>Lift-N-Press tab</td>
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<td>Ted Pella, Inc.</td>
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electrophysiology of scorpion peg sensilla

Wir beschreiben eine Änderung einer bestehenden tip-Aufnahmetechnik 1,2 für elektrophysiologisch untersucht kurzen, zapfenförmigen sensorischen Sensillen 3,4. Auf der Mitte der ventralen Oberfläche aller Skorpione sind zwei Anhängsel namens Pektine, die dichten Bereichen haben mechano-und chemosensorischen peg Sensillen 5,6. Eine Methode zur Beurteilung chemoresponsiveness dieser Sensillen verwendet eine Wolfram-Elektrode für extrazellulär Aufnahme neuronaler Aktivität innerhalb eines Sensillum als flüchtige Riechstoff der sensorischen Bereich 5,7 eingeführt wird. Die Grenzen dieser Methode sind langsame Datensammlung und unkontrollierte Stimulans Einführung und Entnahme aus der Zapfen Feld. Um diese Einschränkungen zu überwinden, entwickelten wir einen neuen Tipp-Aufnahmetechnik, dass unpolare Mineralöl als Medium, durch die Wasser-Basis Geschmacksstoffe zu einzelnen peg Sensillen 8,9 zu liefern. Wir haben erfolgreich diese Methode angewendet, um sensillar chemoresponses Zitronensäure, Ethanol und Salz zu erhalten. Hier beschreiben wir die experimentelle Protokoll für eine solche Studie 9. Wir denken, diese neue Methode kann nützlich sein für die Untersuchung der Reaktion Eigenschaften von anderen Arthropoden chemosensorischen-Systeme einschließlich derer von Insekten 10, 11 und Krebstiere 12.

Watch this Scientific Journal Video about Electrophysiology of Scorpion Peg Sensilla at JoVE.com

Electrophysiology of Scorpion Peg Sensilla

Dieser Artikel beschreibt eine elektrophysiologische Methode zur Isolierung chemische Stimulation einzelner Sensillen via extrazelluläre, tip-Aufnahmen unter Mineralöl.

Elektrophysiologie des Scorpion Peg Sensillen

We describe a modification of an existing tip-recording technique1,2 for electrophysiologically investigating short, peg-like sensory sensilla3,4. On the ...

electrophysiology-of-scorpion-peg-sensilla

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Neuroscience

8.9K Aufrufe.  University of Oklahoma. Dieser Artikel beschreibt eine elektrophysiologische Methode zur Isolierung chemische Stimulation einzelner Sensillen via extrazelluläre, tip-Aufnahmen unter Mineralöl.

Serie: Electrophysiology of Scorpion Peg Sensilla

Simultaneous fMRI and Electrophysiology in the Rodent Brain

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in which functional MRI data and in vivo intracortical recording can be performed simultaneously. The combination of MRI and electrical recording is technically challenging because the electrodes used for recording distort the MRI images and the MRI acquisition induces noise in the electrical recording. To minimize the mutual interference of the two modalities, glass microelectrodes were used rather than metal and a noise removal algorithm was implemented for the electrophysiology data. In our studies, two microelectrodes were separately implanted in bilateral primary somatosensory cortices (SI) of the rat and fixed in place. One coronal slice covering the electrode tips was selected for functional MRI. Electrode shafts and fixation positions were not included in the image slice to avoid imaging artifacts. The removed scalp was replaced with toothpaste to reduce susceptibility mismatch and prevent Gibbs ringing artifacts in the images. The artifact structure induced in the electrical recordings by the rapidly-switching magnetic fields during image acquisition was characterized by averaging all cycles of scans for each run. The noise structure during imaging was then subtracted from original recordings. The denoised time courses were then used for further analysis in combination with the fMRI data. As an example, the simultaneous acquisition was used to determine the relationship between spontaneous fMRI BOLD signals and band-limited intracortical electrical activity. Simultaneous fMRI and electrophysiological recording in the rodent will provide a platform for many exciting applications in neuroscience in addition to elucidating the relationship between the fMRI BOLD signal and neuronal activity.

We have developed a method for simultaneous functional magnetic resonance imaging and electrophysiological recording in the rodent brain, providing a platform for the investigation of the relationship between neural activity and the blood oxygenation level dependent (BOLD) MRI signal.

Gleichzeitige fMRI und Elektrophysiologie in der Nager Gehirn

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in ...

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Neurowissenschaften

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

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

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

Spinal Cord Elektrophysiologie II: Extrazelluläre Saugelektrode Fabrication

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

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 ...

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the researcher to measure gustatory responses directly and quantitatively, reflecting the sensory input that the insect nervous system receives from taste stimuli in its environment. This protocol outlines all key steps in performing this technique. The critical steps in assembling an electrophysiology rig, such as selection of necessary equipment and a suitable environment for recording, are delineated. We also describe how to prepare for recording by making appropriate reference and recording electrodes, and tastant solutions. We describe in detail the method used for preparing the insect by insertion of a glass reference electrode into the fly in order to immobilize the proboscis. We show traces of the electrical impulses fired by taste neurons in response to a sugar and a bitter compound. Aspects of the protocol are technically challenging and we include an extensive description of some common technical challenges that may be encountered, such as lack of signal or excessive noise in the system, and potential solutions. The technique has limitations, such as the inability to deliver temporally complex stimuli, observe background firing immediately prior to stimulus delivery, or use water-insoluble taste compounds conveniently. Despite these limitations, this technique (including minor variations referenced in the protocol) is a standard, broadly accepted procedure for recording Drosophila neuronal responses to taste compounds.

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

This protocol describes extracellular recording of the action potential responses fired by labellar taste neurons in Drosophila.

Elektrophysiologische Aufnehmen von<em&gt; Drosophila</em&gt; Labellar Geschmack Sensillen

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the ...

Real-time Electrophysiology: Using Closed-loop Protocols to Probe Neuronal Dynamics and Beyond

Experimental neuroscience is witnessing an increased interest in the development and application of novel and often complex, closed-loop protocols, where the stimulus applied depends in real-time on the response of the system. Recent applications range from the implementation of virtual reality systems for studying motor responses both in mice1 and in zebrafish2, to control of seizures following cortical stroke using optogenetics3. A key advantage of closed-loop techniques resides in the capability of probing higher dimensional properties that are not directly accessible or that depend on multiple variables, such as neuronal excitability4 and reliability, while at the same time maximizing the experimental throughput. In this contribution and in the context of cellular electrophysiology, we describe how to apply a variety of closed-loop protocols to the study of the response properties of pyramidal cortical neurons, recorded intracellularly with the patch clamp technique in acute brain slices from the somatosensory cortex of juvenile rats. As no commercially available or open source software provides all the features required for efficiently performing the experiments described here, a new software toolbox called LCG5 was developed, whose modular structure maximizes reuse of computer code and facilitates the implementation of novel experimental paradigms. Stimulation waveforms are specified using a compact meta-description and full experimental protocols are described in text-based configuration files. Additionally, LCG has a command-line interface that is suited for repetition of trials and automation of experimental protocols.

Closed-loop protocols are becoming increasingly widespread in modern day electrophysiology. We present a simple, versatile and inexpensive way to perform complex electrophysiological protocols in cortical pyramidal neurons in vitro, using a desktop computer and a digital acquisition board.

Echtzeit-Elektrophysiologie: Mit Closed-loop Protokolle Probe neuronaler Dynamik und darüber hinaus

Experimental neuroscience is witnessing an increased interest in the development and application of novel and often complex, closed-loop protocols, where ...

Spiral Ganglion Neuron Explant Culture and Electrophysiology on Multi Electrode Arrays

Spiral ganglion neurons (SGNs) participate in the physiological process of hearing by relaying signals from sensory hair cells to the cochlear nucleus in the brain stem. Loss of hair cells is a major cause of sensory hearing loss. Prosthetic devices such as cochlear implants function by bypassing lost hair cells and directly stimulating SGNs electrically, allowing for restoration of hearing in deaf patients. The performance of these devices depends on the functionality of SGNs, the implantation procedure and on the distance between the electrodes and the auditory neurons. 
We hypothesized, that reducing the distance between the SGNs and the electrode array of the implant would allow for improved stimulation and frequency resolution, with the best results in a gapless position. Currently we lack in vitro culture systems to study, modify and optimize the interaction between auditory neurons and electrode arrays and characterize their electrophysiological response. To address these issues, we developed an in vitro bioassay using SGN cultures on a planar multi electrode array (MEA). With this method we were able to perform extracellular recording of the basal and electrically induced activity of a population of spiral ganglion neurons. We were also able to optimize stimulation protocols and analyze the response to electrical stimuli as a function of the electrode distance. This platform could also be used to optimize electrode features such as surface coatings.

We present a protocol to culture primary murine spiral ganglion neuron explants on multi electrode arrays to study neuronal response profiles and optimize stimulation parameters. Such studies aim to improve the neuron-electrode interface of cochlear implants to benefit hearing in patients as well as the energy consumption of the device.

Spiralganglion Neuron Explantation Kultur und Elektrophysiologie auf Multi-Elektroden-Arrays

Spiral ganglion neurons (SGNs) participate in the physiological process of hearing by relaying signals from sensory hair cells to the cochlear nucleus in ...

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the experimental time, and increases the number of animals that are utilized per study. This limitation specifically impacts protocols such as calcium imaging that require prolonged pre-incubation with bath-applied dyes. Exponential bacterial growth within 3 - 4 h after slicing is tightly correlated with a decrease in tissue health. This study describes a method for&#160;limiting the proliferation of bacteria in acute preparations to maintain viable neuronal tissue for prolonged periods of time (&#62;24 h) without the need for antibiotics, sterile procedures, or tissue culture media containing growth factors. By cycling the extracellular fluid through UV irradiation and keeping the tissue in a custom holding chamber at 15 - 16 &#176;C, the tissue shows no difference in electrophysiological properties, or calcium signaling through intracellular calcium dyes at &#62;24 h postdissection. These methods will not only extend experimental time for those using acute neuronal tissue, but will reduce the number of animals required to complete experimental goals, and will set a gold standard for acute neuronal tissue incubation.

Once removed from the body, neuronal tissue is greatly affected by environmental conditions, leading to eventual degradation of the tissue after 6 - 8 h. Using a unique incubation method, which closely monitors and regulates the extracellular environment of the tissue, tissue viability can be significantly extended for &#62;24 h.

Längerer Inkubation von Akute neuronales Gewebe für Elektrophysiologie und Calcium-Bildgebung

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility

Insects rely on their sense of smell to guide a wide range of behaviors that are critical for their survival, such as food-seeking, predator avoidance, oviposition, and mating. Myriad chemicals of varying volatilities have been identified as natural odorants that activate insect Olfactory Receptor Neurons (ORNs). However, studying the olfactory responses to low-volatility odorants has been hampered by an inability to effectively present such stimuli using conventional odor-delivery methods. Here, we describe a procedure that permits the effective presentation of low-volatility odorants for in vivo Single-Sensillum Recording (SSR). By minimizing the distance between the odor source and the target tissue, this method allows for the application of biologically salient but hitherto inaccessible odorants, including palmitoleic acid, a stimulatory pheromone with a demonstrated effect on ORNs involved in courtship and mating behavior1. Our procedure thus affords a new avenue to assay a host of low-volatility odorants for the study of insect olfaction and pheromone communication.

Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility

The overall goal of this protocol is to demonstrate how to present odorants of low volatility for single-sensillum recording from Drosophila olfactory receptor neurons that respond to long-chain cuticular pheromones.

Elektrophysiologische Aufzeichnung von<em&gt; Drosophila</em&gt; Trichoid Sensilla in Reaktion auf Odoranten der niedrigen Volatilität

Insects rely on their sense of smell to guide a wide range of behaviors that are critical for their survival, such as food-seeking, predator avoidance, ...

Single Sensillum Recordings for Locust Palp Sensilla Basiconica

The palps of locust mouthparts are considered to be conventional gustatory organs that play an important role in a locust&#39;s food selection, especially for the detection of non-volatile chemical cues through sensilla chaetica (previously named terminal sensilla or crested sensilla). There is now increasing evidence that these palps also have an olfactory function. An odorant receptor (LmigOR2) and an odorant-binding protein (LmigOBP1) have been localized in the neurons and accessory cells, respectively, in the sensilla basiconica of the palps. Single sensillum recording (SSR) is used for recording the responses of odorant receptor neurons, which is an effective method for screening active ligands on specific odorant receptors. SSR is used in functional studies of odorant receptors in palp sensilla. The structure of the sensilla basiconica located on the dome of the palps differs somewhat from the structure of those on the antennae. Therefore, when performing an SSR elicited by odorants, some specific advice may be helpful for obtaining optimum results. In this paper, a detailed and highly effective protocol for an SSR from insect palp sensilla basiconica is introduced.

This paper describes a detailed and highly effective protocol for single sensillum recordings from the sensilla basiconica on the palps of insect mouthparts.

Einzelne Sensillum Aufnahmen für Locust spürbare Sensillen Basiconica

The palps of locust mouthparts are considered to be conventional gustatory organs that play an important role in a locust&#39;s food selection, especially ...

Evaluation of Synaptic Multiplicity Using Whole-cell Patch-clamp Electrophysiology

In the central nervous system, a pair of neurons often form&#160;multiple synaptic contacts and/or functional neurotransmitter release sites (synaptic multiplicity). Synaptic multiplicity is plastic and changes throughout development and in different physiological conditions, being an important determinant for the efficacy of synaptic transmission. Here, we outline experiments for estimating the degree of multiplicity of synapses terminating onto a given postsynaptic neuron using whole-cell patch clamp electrophysiology in acute brain slices. Specifically, voltage-clamp recording is used to compare the difference between the amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) and miniature excitatory postsynaptic currents (mEPSCs). The theory behind this method is that afferent inputs that exhibit multiplicity will show large, action potential-dependent sEPSCs due to the synchronous release that occurs at each synaptic contact. In contrast, action potential-independent release (which is asynchronous) will generate smaller amplitude mEPSCs. This article outlines a set of experiments and analyses to characterize the existence of synaptic multiplicity and discusses the requirements and limitations of the technique. This technique can be applied to investigate how different behavioral, pharmacological or environmental interventions in vivo affect the organization of synaptic contacts in different brain areas.

Here, we present a protocol for evaluating the functional synaptic multiplicity using whole-cell patch clamp electrophysiology in acute brain slices.

Bewertung der synaptischen Multiplizität mit Whole-Cell Patch-Clamp-Elektrophysiologie

In the central nervous system, a pair of neurons often form&#160;multiple synaptic contacts and/or functional neurotransmitter release sites (synaptic ...