Nous remercions le Fonds de la vie, Université de l&#39;Oklahoma Undergraduate Research Opportunities Program, et le département de zoologie du financement de ce travail. Nous remercions également le Dr Hoefnagels Mariëlle de l&#39;aide éditoriale.

1. Pré-enregistrement, la préparation des animaux vivants <ol><li> Retirez le scorpion de son pot d&#39;origine et le placer dans un récipient en verre pré-réfrigérés; Ensuite, placez le pot contenant le scorpion dans un environnement de congélation (-5 ° C) pendant un minimum de deux minutes. Ce temps peut varier en fonction de l&#39;âge, la taille, espèces, etc spécifiques de l&#39;scorpion sous étude. Mais généralement, la phase de l&#39;environnement de congélation est terminée une fois que l&#39;animal est immobile. </li><li> Après deux minutes, retirez le immobilisé scorpions du récipient en verre à froid, et placez-le face ventrale sur une lame de microscope. Fixez le dard, la queue, les pattes et pédipalpes avec de l&#39;argile malléable (à base d&#39;huile, Van Aken Plastalina). </li><li> Faire une plate-forme sur laquelle mettre pectines du scorpion. Ajuster la longueur de la plate-forme et la largeur en fonction de la taille du pecten. Typiquement, nos plates-formes sont construites à partir de couvercle en verre de microscope et sont environ 10 (L) x 18 (W) mm. </li><li> Adhérer ruban adhésif double-face à la plateforme pecten, parage de la bande (si nécessaire) pour s&#39;adapter. </li><li> Pour faire une chambre pectinal, mettez trois des quatre côtés de la plateforme en pré-fondu de cire (nous utilisons des puces de cire d&#39;abeille 100%) à une profondeur de 3-4 mm. Nous fondre la cire en plaçant les jetons dans un plat en verre de Pétri, situé sur une plaque chauffante. Une fois la cire sur les bords de la chambre de pectinal est refroidi à température ambiante, ce qui devrait créer des murs de la cire d&#39;environ 1 mm de haut. </li><li> Sur l&#39;animal, repérer le point d&#39;insertion pectinal, et placez le bord non ciré de la plateforme (bandes latérales-up) juste derrière elle. Par la suite, de stabiliser la plate-forme (afin qu&#39;elle ne bouge pas plus tard) en appuyant doucement sur sa droite et à gauche contre les bords périphériques de l&#39;argile utilisée pour empêcher les jambes de l&#39;animal. </li><li> Utilisez une pince à pointe fine pour placer soigneusement le pecten ou pectines intérieur de la chambre. Plus précisément, en utilisant une pince, tenez le pecten long de la colonne pectinal, et ramenez doucement le pecten de dessous le couvercle en verre et abaissez-le sur le plancher de la nacelle adhésif. Établir un lien pecten-à-bande en appliquant soigneusement la pression de la colonne vertébrale pectinal. </li><li> Maintenant, appliquez la cire fondue (nous utilisons une spatule métallique préalablement chauffé) au bord de l&#39;ONU-ciré de la plateforme. Le but est double, pour sécuriser le pecten et à entourer complètement la chambre pour l&#39;introduction d&#39;huile minérale à venir. Faites preuve de prudence lors de l&#39;application de la cire sur pecten de l&#39;animal, car il est possible d&#39;endommager le pecten avec de la cire trop chaude. </li><li> La construction de chambre pecten est terminée. Ensuite, établir une connexion avec électrode indifférente hémolymphe de l&#39;animal par l&#39;insertion d&#39;un fil d&#39;argent entre deux segments de la queue. </li></ol> 2. Simultanée d&#39;enregistrement extracellulaire et chimiques de stimulation <ol><li> Nous préparons l&#39;animal 24 h avant de mener extracellulaire, pointe-enregistrements, qui, entre autres raisons (l&#39;ajustement des animaux à savoir la position fixe), permet le temps de fabriquer sur les pipettes d&#39;un stimulant spécifique (sensillum-dépendant) pointe diamètre. Nous utilisons un extracteur micropipette pour réaliser de telles spécifications. Idéalement, le diamètre de la pipette est ≈ 2 um plus grand que le diamètre des pores sensillar. Nous ajustons nos spécifications micropipette conséquence pour chaque animal. </li><li> 1 h avant l&#39;enregistrement, nous ajoutons 5 ul d&#39;huile minérale dans la chambre de pectinal, submergeant le pecten dans l&#39;huile. Ensuite, nous avons la position de l&#39;animal sous un microscope très puissant, qui a longtemps travaille objectifs de distance et épi-illumination. </li><li> Pour stimuler chimiquement peg sensilles, nous introduisons sapides aqueuse (c&#39;est à dire l&#39;acide citrique et de sel) par le moyen du pétrole. Tout d&#39;abord, nous utilisons une charge micropipette pour injecter le stimulant dans la pipette stimulant. Remarque: toutes les solutions doivent contenir des stimulants de l&#39;électrolyte pour la conductance électrique. </li><li> Ensuite, après le positionnement de la pipette stimulant sur un micromanipulateur (dans le banc d&#39;enregistrement), insérez l&#39;électrode d&#39;enregistrement dans le émoussé, extrémité ouverte de la pipette stimulant. Il est important pour assurer le contact entre l&#39;électrode métallique et une solution électrolytique stimulants. </li><li> Une fois la préparation est électriquement à la terre (via l&#39;électrode indifférente), tout simplement inférieure de la pipette à travers le milieu du pétrole et sur un pore sensillar. La durée de la stimulation chimique par sensillum pectinal peut varier d&#39;autant que trente minutes pour aussi peu que d&#39;une seconde. Généralement, nous pouvons l&#39;échantillon chevilles multiples (&gt; 30) par pipette. Dans le cas où la pipette stimulants devient obstrué par des débris minute, il suffit d&#39;échanger la pipette bouchée d&#39;une nouvelle pipette inutilisés. </li></ol> 3. Préparation d&#39;un animal pendant plusieurs jours d&#39;expérimentation <ol><li> Si l&#39;animal est à utiliser sur une période prolongée, nous recommandons de réutiliser la chambre pectinal et le remplacement du goutte d&#39;huile minérale pour chaque session d&#39;enregistrement. Après la session d&#39;enregistrement d&#39;un jour donné est complet, enlever l&#39;animal de l&#39;enregistrement mis en place et effectuer un lavage de l&#39;huile minérale. </li><li> Specifically, ajouter une autre goutte (5 pi) de l&#39;huile minérale dans la chambre, et enlever toute l&#39;huile. Cela permet de minimiser la présence de stimulants résiduelle, qui peut avoir filtré de la pipette lors de la session d&#39;enregistrement. </li><li> Avant la prochaine session, ajouter une goutte d&#39;huile comme décrit précédemment. </li></ol> 4. Les résultats représentatifs: Selon le matériel d&#39;enregistrement utilisé (par exemple l&#39;amplificateur particulier, la numérisation du matériel, etc), un résultat représentatif est un enregistrement extracellulaire avec un grand S: N ratio d&#39;au moins 3:1. La construction réussie de la chambre de l&#39;huile minérale est nécessaire pour isoler la stimulation chimique de sensilles individu, car il fournit un moyen d&#39;offrir sapides chimiques polaires (à base d&#39;eau) 8. S&#39;il ya trop peu d&#39;huile ou pas du tout, la solution stimulants se propage à partir du sensillum enregistrées à son sensilles voisins, ce qui est susceptible de provoquer une période prolongée, Bout incontrôlée de stimulation chimique 8.

<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>

Nous avons déjà publié ces méthodes dans deux revues, Knowlton et Gaffin (2009) et Knowlton et Gaffin (sous presse).

Le protocole ci-dessus décrit comment préparer un désert prairies scorpion (Paruroctonus utahensis) pour l&#39;étude électrophysiologique. Plus précisément, nous montrons comment construire une chambre pour contrôler extracellulaire pointe des enregistrements de neurones pectinal chimiosensoriels dans l&#39;huile. Parce que l&#39;huile et l&#39;eau ne se mélangent pas, il est possible d&#39;isoler la stimulation chimique (à base d&#39;eau stimulants) pour sensilles unique. Il convient de souligner que P. utahensis, est un animal relativement petit (≈ 2,5-5 cm de) et en appliquant ce protocole pour les grands animaux peut nécessiter des ajustements de taille beaucoup, tels que la taille de la plate-forme de l&#39;animal, de chambre, et la quantité d&#39;huile appliquée. Nous recommandons la réalisation d&#39;études pilotes pour tester la quantité minimale d&#39;huile nécessaire pour la livraison contrôlée de stimulants chimiques à base d&#39;eau. Par ailleurs, nous n&#39;avons trouvé aucun effet de l&#39;huile sur l&#39;activité neuronale au sein de base pectinal sensilles 8. Cela devrait être confirmé pour les systèmes d&#39;autres modèles ainsi, avant d&#39;évaluer chemoresponsiveness. Cette méthode peut être utilisée en conjonction avec une autre méthode pour tester les effets d&#39;interaction entre chemoresponse sensilles dans le domaine sensoriel. Par exemple, il est possible de la pointe-record (dans l&#39;huile) d&#39;un sensillum que nous basons-enregistrer (via une électrode de tungstène) à partir d&#39;un sensillum voisins 9. Une telle configuration de l&#39;enregistrement peut être utilisé pour évaluer si oui ou non chimiquement stimulant une sensillum affecte l&#39;activité neuronale de l&#39;sensillum base enregistré. À ce jour, personne n&#39;a testé cela pour scorpion pectinal sensilles, et il reste une question ouverte pour d&#39;autres systèmes chimiosensoriels ainsi. Pour résumer, nous pensons que la méthode de la pointe-enregistrement sous les avances du pétrole de la portée des enquêtes électrophysiologiques dans les bases neurales de la gustation arthropodes. Dans ce manuscrit, nous fournissons un protocole de préparation des animaux pour cette méthode, et nous espérons qu&#39;il constituera une base solide pour une étude plus approfondie des périphériques sensoriels du système nerveux.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Lift-N-Press tab</td>
<td>&nbsp;</td>
<td>Ted Pella, Inc.</td>
<td>16082</td>
<td>Doubled-sided adhesive</td>
</tr>
<tr>
<td>MicroFil</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Inc.</td>
<td>MF34G</td>
<td>Micropipette filler</td>
</tr>
<tr>
<td>Standard Glass Capillaries</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Inc.</td>
<td>1B100F-6</td>
<td>Glass pipettes</td>
</tr>
<tr>
<td>Microelectrode Holder</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Inc. </td>
<td>MEH3SBW10</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

electrophysiology of scorpion peg sensilla

Nous décrivons une modification d&#39;un 1,2 technique de pointe d&#39;enregistrement existants pour enquêter électrophysiologiquement court, comme le PEG-sensorielle sensilles 3,4. Sur la surface la mi-ventrale de tous les scorpions sont deux appendices appelés pectines, qui ont des champs denses de mécano-et chimiosensoriels peg sensilles 5,6. Une méthode pour évaluer chemoresponsiveness de ces sensilles utilise une électrode de tungstène pour l&#39;enregistrement extracellulaire activité neuronale au sein d&#39;un sensillum comme une substance odorante volatile est introduit dans le champ sensoriel 5,7. Les limites de cette méthode comprennent ralentir la collecte de données et l&#39;introduction incontrôlée aux stimulants, et l&#39;enlèvement d&#39;, le champ cheville. Pour surmonter ces limitations, nous avons développé une nouvelle pointe d&#39;enregistrement technique qui utilise de l&#39;huile minérale non polaires comme un moyen par lequel nous livrer à base d&#39;eau sapides au PEG individuels sensilles 8,9. Nous avons appliqué avec succès cette méthode pour obtenir chemoresponses sensillar à l&#39;acide citrique, l&#39;éthanol, et le sel. Nous décrivons ici le protocole expérimental pour une telle étude 9. Nous pensons que cette nouvelle méthode peut être utile pour étudier les propriétés de réponse des autres arthropodes systèmes chimiosensoriels, y compris ceux des insectes 10, 11 et 12 de crustacés.

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

Electrophysiology of Scorpion Peg Sensilla

Cet article décrit une méthode électrophysiologique pour isoler la stimulation chimique de sensilles individuels via extracellulaire, pointe-enregistrements sous huile minérale.

Électrophysiologie de Scorpion Peg Sensilles

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

Research

JoVE Journal

Neuroscience

8.9K vues.  University of Oklahoma. Cet article décrit une méthode électrophysiologique pour isoler la stimulation chimique de sensilles individuels via extracellulaire, pointe-enregistrements sous huile minérale.

Vidéo: 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.

IRMf simultanées et l&#39;électrophysiologie dans le cerveau de rongeurs

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

Recherche

Neurosciences

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

La moelle épinière électrophysiologie II: fabrication des électrodes extracellulaires d&#39;aspiration

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.

La culture et l&#39;électrophysiologie des cellules sur la SCGDV de 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 ...

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We developed a novel nanoinjection/electrophysiology assay that allows the detection of bioactive modulatory effects of compounds on the function of a neuronal circuit that mediates the escape response in Drosophila melanogaster3,4. Our in vivo assay, which uses the Drosophila Giant Fiber System (GFS, Figure 1) allows screening of different types of compounds, such as small molecules or peptides, and requires only minimal quantities to elicit an effect. In addition, the Drosophila GFS offers a large variety of potential molecular targets on neurons or muscles. The Giant Fibers (GFs) synapse electrically (Gap Junctions) as well as chemically (cholinergic) onto a Peripheral Synapsing Interneuron (PSI) and the Tergo Trochanteral Muscle neuron (TTMn)5. The PSI to DLMn (Dorsal Longitudinal Muscle neuron) connection is dependent on D&alpha;7 nicotinic acetylcholine receptors (nAChRs)6. Finally, the neuromuscular junctions (NMJ) of the TTMn and the DLMn with the jump (TTM) and flight muscles (DLM) are glutamatergic7-12. Here, we demonstrate how to inject nanoliter quantities of a compound, while obtaining electrophysiological intracellular recordings from the Giant Fiber System13 and how to monitor the effects of the compound on the function of this circuit. We show specificity of the assay with methyllycaconitine citrate (MLA), a nAChR antagonist, which disrupts the PSI to DLMn connection but not the GF to TTMn connection or the function of the NMJ at the jump or flight muscles.
Before beginning this video it is critical that you carefully watch and become familiar with the JoVE video titled "Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster " from Augustin et al7, as the video presented here is intended as an expansion to this existing technique. Here we use the electrophysiological recordings method and focus in detail only on the addition of the paired nanoinjections and monitoring technique.

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

A rapid in vivo assay to test for neuromodulatory compounds using the Giant Fiber System (GFS) of Drosophila melanogaster is described. Nanoinjections in the head of the animal along with electrophysiological recordings of the GFS can reveal bioactivity of compounds on neurons or muscles.

Nanoinjection paires et test d&#39;électrophysiologie à l&#39;écran de la bioactivité des composés en utilisant le Drosophila melanogaster Système de fibre géant

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We ...

An Isolated Semi-intact Preparation of the Mouse Vestibular Sensory Epithelium for Electrophysiology and High-resolution Two-photon Microscopy

Understanding vestibular hair cells function under normal conditions, or how trauma, disease, and aging disrupt this function is a vital step in the development of preventative approaches and/or novel therapeutic strategies. However, the majority of studies looking at abnormal vestibular function have not been at the cellular level but focused primarily on behavioral assays of vestibular dysfunction such as gait analyses and vestibulo-ocular reflex performance. While this work has yielded valuable data about what happens when things go wrong, little information is gleaned regarding the underlying causes of dysfunction. Of the studies that focus on the cellular and subcellular processes that underlie vestibular function, most have relied on acutely isolated hair cells, devoid of their synaptic connections and supporting cell environment. Therefore, a major technical challenge has been access to the exquisitely sensitive vestibular hair cells in a preparation that is least disrupted, physiologically. Here we demonstrate a semi-intact preparation of the mouse vestibular sensory epithelium that retains the local micro-environment including hair cell/primary afferent complexes.

Analysis of vestibular hair cell function is complicated by their location deep within the hardest part of the skull, the petrous temporal bone. Most functional hair cell studies have used acutely isolated hair cells. Here we describe a semi-intact preparation of mouse vestibular epithelium for electrophysiological and two-photon microscopy studies.

Une préparation semi-intact d&#39;isolement de la souris vestibulaire épithélium sensoriel pour électrophysiologie et haute résolution microscopie à deux photons

Understanding vestibular hair cells function  under normal conditions, or how trauma, disease, and aging disrupt this  function is a vital step in the ...

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.

Enregistrement électrophysiologique De<em&gt; Drosophila</em&gt; Labellar Goût Sensilles

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.

Électrophysiologie en temps réel: en utilisant des protocoles en boucle fermée à la sonde neuronaux Dynamics et au-delà

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

Electrophysiology on Isolated Brainstem-spinal Cord Preparations from Newborn Rodents Allows Neural Respiratory Network Output Recording

While it is well known that the central respiratory drive is located in the brainstem, several aspects of its basic function, development, and response to stimuli remain to be fully understood. To overcome the difficulty of accessing the brainstem in the whole animal, isolation of the brainstem and part of the spinal cord is performed. This preparation is maintained in artificial cerebro-spinal fluid where gases, concentrations, and temperature are controlled and monitored. The output signal from the respiratory network is recorded by a suction electrode placed on the fourth ventral root. In this manner, stimuli can be directly applied onto the brainstem, and the effect can be recorded directly. The signal recorded is linked to the inspiratory signal sent to the diaphragm via the phrenic nerve, and can be described as bursts (around 8 bursts per minute). Analysis of these bursts (frequency, amplitude, length, and area under the curve) allows precise characterization of the stimulus effect on the respiratory network. The main limitation of this method is the viability of the preparation beyond the early post-natal stages. Thus, this method greatly focuses on the study of the whole network without the peripheral inputs in the newborn rat.

The central respiratory drive is located in the brainstem. Spontaneous respiratory motor output from an isolated brainstem-spinal cord is recorded by placing an electrode on the fourth ventral root. This experimental approach is valuable for pharmacological investigations or the assessment of respiratory challenges and genetic manipulations on rhythmic motor behavior.

Électrophysiologie sur isolés Cord Préparatifs du tronc cérébral-spinal de la naissance Rongeurs Permet Neural respiratoire Réseau d&#39;enregistrement de sortie

While it is well known that the central respiratory drive is located in the brainstem, several aspects of its basic function, development, and response to ...

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.

Spiral Ganglion Neuron Explantation Culture et électrophysiologie sur multi-électrodes 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.

L&#39;incubation prolongée de aiguë Neuronal tissus pour électrophysiologie et Calcium-imagerie

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