L&#39;auteur tient à remercier Peter Castro et Matthew Dinday pour leurs premiers efforts pour mettre en place le poisson zèbre dans le laboratoire. Ce travail a été financé par le National Institutes of Health EUREKA subvention (# R01NS079214-01).

1. Production d&#39;oeufs et de Collection <ol><li> Le poisson zèbre élevage suit des procédures standard décrites précédemment 8. En bref, le poisson-zèbre adultes sont mis en place dans l&#39;élevage de réservoirs avec des intercalaires en place. Lorsque les lumières dans la salle viennent le lendemain matin, les diviseurs sont retirés de l&#39;élevage et les poissons des réservoirs sont autorisées environ 20 à 60 min de la période de reproduction sans être dérangé. </li><li> Les oeufs de bassins d&#39;élevage sont collectées dans une passoire et rincer à l&#39;eau d&#39;œuf. Les œufs sont ensuite transférés dans une boîte de Pétri avec de l&#39;eau œuf. Les œufs non fécondés et les débris sont enlevés à l&#39;aide d&#39;une pipette de transfert. </li><li> Lieu boîte de Petri contenant les œufs recueillis dans un incubateur (28-32 ° C). Après éclosion des œufs, environ deux jours après la fécondation (dpf), retirez chorion et tout autre débris avec une pipette de transfert. </li><li> Le jour de votre choix après la fécondation (3 à 8 dpf) retirer boîte de Pétri contenant des larves nageant librement et placer sur paillasse à température ambiante. </li></ol><ol><li> L&#39;utilisation d&#39;un extracteur micropipette, tirez de 1,2 mm de diamètre extérieur en verre de borosilicate capillaire dans deux aiguilles et les stocker dans un plat de Pétri de 150 mm ou une boîte vide pipette en posant sur des rampes Silly Putty. Aiguilles peut être tiré à l&#39;avance. Différents OD capillaires en verre peuvent être utilisés en fonction du type de headstage amplificateur disponible. </li><li> Backloading la microélectrode avec une solution d&#39;enregistrement extracellulaire (2 M de NaCl) à l&#39;aide d&#39;une seringue de 1 ml avec un filtre Nalgene seringue (4-mm) et Microfil filament (Warner Instruments de précision) ci-joint. Secouer ou tapoter le bol vers la pointe de l&#39;aiguille jusqu&#39;à ce qu&#39;il n&#39;y pas ou peu de bulles restantes. </li></ol> 3. Immobilisation en gélose <ol><li> Préparer une solution fraîche de 1,2% d&#39;agar bas point de fusion dans l&#39;eau d&#39;œuf. Solution d&#39;agar place dans un ensemble bain-marie à 37 ° C ~ </li><li> Préparer une lamelle de chambre d&#39;enregistrement et mettez au congélateur (5-10 minutes). La chambre d&#39;enregistrement doit MATCh celui utilisé sur la plate-forme d&#39;électrophysiologie. Nous utilisons un profil bas chambre ouverte imagerie diamant bain de Warner Instruments (modèle RC-26GLP). </li><li> L&#39;utilisation d&#39;un Pasteur ou une pipette de transfert, placez un poisson zèbre larvaire dans une petite goutte d&#39;eau oeuf dans une boîte de Pétri plat propre. À cette gouttelette, ajouter une goutte de solution contenant un anesthésique (0,02% tricaïne) et de l&#39;agent paralysant (1 mg / ml α-bungarotoxine). </li><li> Surveiller le poisson zèbre pour la perte de mouvement (5 à 10 mn). </li><li> Enlever les lamelles / chambre d&#39;enregistrement du congélateur. Placez chambre sur la scène d&#39;un stéréomicroscope. À l&#39;aide d&#39;une pipette de transfert, mélanger quelques millilitres de 1,2% d&#39;agarose à bas point de fusion avec des gouttelettes et de la solution de transfert (avec poisson) à la chambre de lamelle / enregistrement. </li><li> En utilisant une pointe de pipette ou l&#39;aiguille fine mousse, les larves de position sous la loupe binoculaire de telle sorte que la face dorsale du poisson est exposé à la surface du gel d&#39;agarose. Laisser durcir agarose (5-10 minutes), puis en utilisant une spatule plate transfert du b agarverrouiller à une chambre d&#39;enregistrement mis en place sur une plate-forme d&#39;électrophysiologie. </li></ol> 3. Field Recording extracellulaire <ol><li> Ajouter 2-5 ml de supports d&#39;enregistrement de poisson zèbre à la chambre d&#39;enregistrement. Si des modifications sont nécessaires drogues, chambre perfuse avec une solution d&#39;enregistrement à une vitesse d&#39;environ 1 ml / min. Pas d&#39;oxygénation de la solution d&#39;enregistrement est nécessaire. </li><li> Insérer une microélectrode (environ 1 um diamètre de pointe, 2-7 MQ) dans la headstage amplificateur monté sur un micromanipulateur à trois dimensions. Vérifier que le micromanipulateur est dans une position appropriée pour permettre une amplitude de mouvement et d&#39;ajustement. Apportez la pointe de microélectrodes dans le plan de vision du microscope, haut au-dessus de la scène, et en utilisant un réglage grossier inférieure à un point juste au-dessus et légèrement en avant de la tête du poisson-zèbre. </li><li> Avec l&#39;amplificateur en «courant-clamp&quot; mode zéro de l&#39;électrode. </li><li> Utiliser un réglage grossier inférieure de la pointe de l&#39;électrode de sorte qu&#39;il touche à peine le surface du poisson zèbre, un peu en avant du cerveau antérieur. </li><li> En utilisant des étapes de réglage fin avancer la pointe de l&#39;électrode jusqu&#39;à ce qu&#39;elle perfore la peau du poisson zèbre. Laisser reposer, puis avancez l&#39;électrode de quelques microns dans le cerveau antérieur. </li><li> Enregistrer l&#39;activité électrique dans le courant de serrage mode à l&#39;aide du logiciel Axoscope. Les données sont acquises à un taux d&#39;échantillonnage de 10 kHz. </li></ol>

<ol>
	<li>Clark, K. J., 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=Stressing+zebrafish+for+behavioral+genetics.">Stressing zebrafish for behavioral genetics.</a> Reviews in Neuroscience. 22 (1), 49 (2011).</li><li>Rinkwitz, S., 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=Zebrafish:+an+integrative+system+for+neurogenomics+and+neurosciences.">Zebrafish: an integrative system for neurogenomics and neurosciences.</a> Progress in Neurobiology. 93 (2), 231 (2011).</li><li>Penberthy, W. T., 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=The+zebrafish+as+a+model+for+human+disease.">The zebrafish as a model for human disease.</a> Frontiers in Bioscience. 7, d1439 (2002).</li><li>Letamendia, A., 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=Development+and+validation+of+an+automated+high-throughput+system+for+zebrafish+in+vivo+screenings.">Development and validation of an automated high-throughput system for zebrafish in vivo screenings.</a> PLoS One. 7, e36690 (2012).</li><li>Nasevicius, A., Ekker, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+targeted+gene+'knockdown'+in+zebrafish.">Effective targeted gene 'knockdown' in zebrafish.</a> Nature Genetics. 26 (2), 216 (2000).</li><li>Haffter, P., 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=Mutations+affecting+development+of+the+zebrafish+inner+ear+and+lateral+line.">Mutations affecting development of the zebrafish inner ear and lateral line.</a> Development. 123, 1 (1996).</li><li>Suster, M. L., 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=Transgenesis+in+zebrafish+with+the+tol2+transposon+system.">Transgenesis in zebrafish with the tol2 transposon system.</a> Methods Molecular Biology. 561, 41 (2009).</li><li>Rosen, J. N., Sweeney, M. F., Mably, 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=Microinjection+of+Zebrafish+Embryos+to+Analyze+Gene+Function.">Microinjection of Zebrafish Embryos to Analyze Gene Function.</a> J. Vis. Exp. (25), e1115 (2009).</li><li>Baraban, S. C., 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=Pentylenetetrazole+induced+changes+in+zebrafish+behavior,+neural+activity+and+c-fos+expression.">Pentylenetetrazole induced changes in zebrafish behavior, neural activity and c-fos expression.</a> Neuroscience. 131 (3), 759 (2005).</li><li>Baraban, S. C., 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=A+large-scale+mutagenesis+screen+to+identify+seizure-resistant+zebrafish.">A large-scale mutagenesis screen to identify seizure-resistant zebrafish.</a> Epilepsia. 48 (6), 1151 (2007).</li><li>Williams, P., 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=The+use+of+radiotelemetry+to+evaluate+electrographic+seizures+in+rats+with+kainate-induced+epilepsy.">The use of radiotelemetry to evaluate electrographic seizures in rats with kainate-induced epilepsy.</a> Journal of Neuroscience Methods. 155 (1), 39 (2006).</li><li>Marsh, E. D., 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=Interictal+EEG+spikes+identify+the+region+of+electrographic+seizure+onset+in+some,+but+not+all,+pediatric+epilepsy+patients.">Interictal EEG spikes identify the region of electrographic seizure onset in some, but not all, pediatric epilepsy patients.</a> Epilepsia. 51 (4), 592 (2010).</li><li>Zhu, C., 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=Evaluation+and+application+of+modularly+assembled+zinc-finger+nucleases+in+zebrafish.">Evaluation and application of modularly assembled zinc-finger nucleases in zebrafish.</a> Development. 138 (20), 4555 (2011).</li></ol>

Aucun conflit d&#39;intérêt déclaré.

La méthode d&#39;enregistrement extracellulaire présentée ici permet une analyse très sensible et rapide de l&#39;activité cérébrale. Ces enregistrements sont analogues à électroencéphalographique (EEG) de surveillance couramment utilisé pour évaluer la présence de décharges électriques anormales (par exemple, la saisie) dans des modèles rongeurs d&#39;épilepsie 11 et les patients de 12. Enregistrements extracellulaires peuvent être combinés avec des manipulations pharma...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires (optionnel) </td></tr><tr><td> Agarose bas point de fusion </td><td> Fisher Scientific- </td><td> BP1360-100 </td><td> Dissoudre dans les médias embryon à 1,2% </td></tr><tr><td> Supports d&#39;enregistrement </td><td> Fisher Scientific- </td><td> BP3581, P330-3, BP410-1, BP214-500, D16-1, C77-500 </td><td> 1 mM NaCl, 2,9 mM de KCl, 10 mM d&#39;HEPES, 1,2 mM MgCl 2, 10 mM de dextrose, 2,1 mM de CaCl 2 pH à environ 7,3 avec NaOH 1 N </td></tr><tr><td> Tricaïne </td><td> Labs Argent </td><td> MS-222 </td><td> 0,02% </td></tr><tr><td> α-bungarotoxine </td><td> Bioscience Tocris </td><td> 2133 </td><td> 1 mg / ml </td></tr><tr><td> Tube capillaire en verre </td><td> Warner Instruments </td><td> G120TF-3 </td><td> Tirer à une résistance de 2 -7 MQ </td></tr><tr><td> Amplificateur de patch-clamp </td><td> Warner Instruments </td><td> PC-505B </td><td> Nous utilisons un amplificateur de Warner en mode courant clamp; Gain fixé à 2 mV / pA et filtre de Bessel fixé à 2K. Des modèles comparables peuvent être utilisés selon les instructions du fabricant. </td></tr><tr><td> Filtre / amplificateur </td><td> Cygnus Technology </td><td> FLA-01 </td><td> Nous utilisons une Cygnus pré-amplificateur de gain fixé à 10-20; Fréquence de coupure fixée à 1-2K; filtre Notch IN. Des modèles comparables peuvent être utilisés selon les instructions du fabricant. </td></tr><tr><td> Axon A / N et des logiciels Axoscope </td><td> Molecular Devices </td><td> Axon Digidata 1320A; Axoscope 8,2 </td><td> Les données sont collectées dans le mode d&#39;acquisition utilisant Axoscope sans jeu; échantillonnage à 10 kHz. Des modèles comparables et programmes peuvent être utilisés selon les instructions du fabricant. </td></tr><tr><td> L&#39;eau d&#39;œuf </td><td&gt; Instant Ocean </td><td></td><td> 3 g Instant Ocean sel de mer, 2 bleu de méthylène ml 0,1% dans 10 ml d&#39;eau déminéralisée </td></tr></tbody></table>

forebrain electrophysiological recording in larval zebrafish

L&#39;épilepsie touche près de 3 millions de personnes aux Etats-Unis et jusqu&#39;à 50 millions de personnes à travers le monde. Définie comme la survenue de crises non provoquées spontanées, l&#39;épilepsie peut être acquise à la suite d&#39;une insulte au cerveau ou une mutation génétique. Les efforts de crises modèle chez l&#39;animal ont principalement utilisé acquise insultes (médicaments anticonvulsivants, la stimulation ou une lésion cérébrale) et les manipulations génétiques (knockdown antisens, la recombinaison homologue ou transgénèse) chez les rongeurs. Le poisson zèbre constituent un système modèle vertébré 1-3 qui pourrait fournir une alternative intéressante aux rongeurs à base de recherche sur l&#39;épilepsie. Le poisson zèbre sont largement utilisés dans l&#39;étude de la génétique des vertébrés ou de développement, présentent un degré élevé de similitude génétique des mammifères et d&#39;exprimer homologues pour environ 85% des mutations connues de l&#39;homme épilepsie d&#39;un seul gène. En raison de leur petite taille (4-6 mm de longueur), les larves de poisson zèbre peut être maintenu dans des volumes de fluide aussi bas que 100 pi au cours du développement précoce et arrayed en plaques multi-puits. Les réactifs peuvent être ajoutés directement à la solution dans laquelle les embryons se développent, ce qui simplifie l&#39;administration du médicament et permettant une réaction rapide criblage in vivo de composés d&#39;essai 4. Oligonucléotides synthétiques (morpholinos), mutagénèse, de zinc et de nucléases doigt approches transgéniques peut être utilisé pour générer rapidement démontable ou mutation du gène chez le poisson zèbre 5-7. Ces propriétés payer les études de poisson zèbre sans précédent avantage de pouvoir sur l&#39;analyse statistique des rongeurs dans l&#39;étude des troubles neurologiques comme l&#39;épilepsie. Parce que le «gold standard» pour la recherche en épilepsie est de surveiller et d&#39;analyser les rejets anormaux électriques qui prennent naissance dans une structure du cerveau central (c.-à-crises d&#39;épilepsie), une méthode efficace pour enregistrer l&#39;activité cérébrale chez les larves du poisson zèbre est décrite ici. Cette méthode est une adaptation de techniques classiques d&#39;enregistrement extracellulaire et permet un fonctionnement stable à long terme de surveillance de l&#39;activité cérébrale chez les larves de poisson zèbre intacte. Senregistrements amples sont présentés pour les crises aiguës induites par l&#39;application de médicaments convulsivants bain et saisies spontanées enregistrées dans un poisson génétiquement modifié.

Des exemples de saisie de type électrographique décharge enregistrée dans le cerveau antérieur de l&#39;agar-noyé poisson zèbre est représenté sur la figure 1. Grande amplitude de décharge éclatement multi-pic dans ces échantillons a été évoqué par l&#39;application de bain d&#39;un médicament anticonvulsivant, 40 mM de pilocarpine (en A; 6 dpf) ou 1 mM picrotoxine (en B, 8 dpf). Dans ces enregistrements, immobilisé et l&#39;agar-intégrés poisson zèbre sont surveillés en permanence ...

Watch this Scientific Journal Video about Forebrain Electrophysiological Recording in Larval Zebrafish at JoVE.com

Forebrain Electrophysiological Recording in Larval Zebrafish

Une méthode simple pour enregistrer les potentiels de champ extracellulaires dans le cerveau antérieur larvaire du poisson zèbre est décrite. La méthode fournit une robuste<em&gt; In vivo</em&gt; Lecture de saisie-comme l&#39;activité. Cette technique peut être utilisée avec poisson zèbre génétiquement modifié portant des gènes liés à l&#39;épilepsie ou de convulsions provoquées par l&#39;administration de médicaments anticonvulsivants.

Prosencéphale enregistrement électrophysiologique des larves dans le poisson-zèbre

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked ...

forebrain-electrophysiological-recording-in-larval-zebrafish

Research

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Neuroscience

17.0K vues.  University of California, San Francisco. Une méthode simple pour enregistrer les potentiels de champ extracellulaires dans le cerveau antérieur larvaire du poisson zèbre est décrite. La méthode fournit une robuste In vivo Lecture de saisie-comme l'activité. Cette technique peut être utilisée avec poisson zèbre génétiquement modifié portant des gènes liés à l'épilepsie ou de convulsions provoquées par l'administration de médicaments anticonvulsivants.

Vidéo: Forebrain Electrophysiological Recording in Larval Zebrafish

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

Imagerie time-lapse en direct de relation clonale cellules progénitrices neurales dans le cerveau antérieur développement Zebrafish

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

Recherche

Neurosciences

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram recordings. This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments, allowing recording of neural activity. Immobilization of the adult requires a mechanism to deliver dissolved oxygen to the gills in lieu of buccal and opercular movement. With our technique, animals are immobilized and perfused with habitat water to fulfill this requirement. A craniotomy is performed under tricaine methanesulfonate (MS-222; tricaine) anesthesia to provide access to the brain. The primary electrode is then positioned within the craniotomy window to record extracellular brain activity. Through the use of a multitube perfusion system, a variety of pharmacological compounds can be administered to the adult fish and any alterations in the neural activity can be observed. The methodology not only allows for observations to be made regarding changes in neurological activity, but it also allows for comparisons to be made between larval and adult zebrafish. This gives researchers the ability to identify the alterations in neurological activity due to the introduction of various compounds at different life stages.

This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

Enregistrement électrophysiologique dans le cerveau d&#39;Intact poisson zèbre adulte

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram ...

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 ...

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

Spinal Cord Transection dans le poisson zèbre larvaire

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing system was developed that allows for the injection of a drug while recording electrophysiological signals from the affected neuronal population. This method provides a simple and economical alternative to commercial solutions. A glass pipette was modified by combining it with a hypodermic needle and a silver filament. The injectrode is attached to commercial microsyringe pump for drug delivery. This results in a technique that provides real-time pharmacodynamics feedback through multi-unit extracellular signals originating from the site of drug delivery. As a proof of concept, we recorded neuronal activity from the superior colliculus elicited by flashes of light in rats, concomitantly with delivery of drugs through the injectrode. The injectrode recording capacity permits the functional characterization of the injection site favoring precise control over the localization of drug delivery. Application of this method also extends far beyond what is demonstrated here, as the choice of chemical substance loaded into the injectrode is vast, including tracing markers for anatomic experiments.

Here, we craft a glass pipette with dual functions: inhibition of deep brain structures by microinjections of drugs and real-time monitoring of their effects through simultaneous electrophysiological recordings.

Simultanée électrophysiologique Enregistrement et micro-injections d&#39;agents inhibiteurs dans le cerveau des rongeurs

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing ...

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.

Enregistrement électrophysiologique de<em&gt; Drosophila</em&gt; Sensilla Trichoid en Réponse aux Odorants de Faible Volatilité

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, ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

En imagerie calcique Vivo des cellules ciliées ligne latérale chez le poisson zèbre larvaire

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...

Investigating Long-term Synaptic Plasticity in Interlamellar Hippocampus CA1 by Electrophysiological Field Recording

The study of synaptic plasticity in the hippocampus has focused on the use of the CA3-CA1 lamellar network. Less attention has been given to the longitudinal interlamellar CA1-CA1 network. Recently however, an associational connection between CA1-CA1 pyramidal neurons has been shown. Therefore, there is the need to investigate whether the longitudinal interlamellar CA1-CA1 network of the hippocampus supports synaptic plasticity.
We designed a protocol to investigate the presence or absence of long-term synaptic plasticity in the interlamellar hippocampal CA1 network using electrophysiological field recordings both in vivo and in vitro. For in vivo extracellular field recordings, the recording and stimulation electrodes were placed in a septal-temporal axis of the dorsal hippocampus at a longitudinal angle, to evoke field excitatory postsynaptic potentials. For in vitro extracellular field recordings, hippocampal longitudinal slices were cut parallel to the septal-temporal plane. Recording and stimulation electrodes were placed in the stratum oriens (S.O) and the stratum radiatum (S.R) of the hippocampus along the longitudinal axis. This enabled us to investigate the directional and layer specificity of evoked excitatory postsynaptic potentials. Already established protocols were used to induce long-term potentiation (LTP) and long-term depression (LTD) both in vivo and in vitro. Our results demonstrated that the longitudinal interlamellar CA1 network supports N-methyl-D-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) with no directional or layer specificity. The interlamellar network, however, in contrast to the transverse lamellar network, did not present with any significant long-term depression (LTD).

We used recording and stimulation electrodes in longitudinal hippocampal brain slices and longitudinally positioned recording and stimulation electrodes in the dorsal hippocampus in vivo to evoke extracellular postsynaptic potentials and demonstrate long-term synaptic plasticity along the longitudinal interlamellar CA1.

Investigating Long-term Synaptic Plasticity in Interlamellar Hippocampus CA1 par Electrophysiological Field Recording

The study of synaptic plasticity in the hippocampus has focused on the use of the CA3-CA1 lamellar network. Less attention has been given to the ...

Wireless Electrophysiological Recording of Neurons by Movable Tetrodes in Freely Swimming Fish

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain activity from freely moving fish would advance research on the neural basis of fish behavior considerably. Moreover, precise control of the recording location in the brain is critical to studying coordinated neural activity across regions in fish brain. Here, we present a technique that records wirelessly from the brain of freely swimming fish while controlling for the depth of the recording location. The system is based on a neural logger associated with a novel water-compatible implant that can adjust the recording location by microdrive-controlled tetrodes. The capabilities of the system are illustrated through recordings from the telencephalon of goldfish.

A novel wireless technique for recording extracellular neural signals from the brain of freely swimming goldfish is presented. The recording device is composed of two tetrodes, a microdrive, a neural data logger, and a waterproof case. All parts are custom-made except for the data logger and its connector.

Enregistrement électrophysiologique sans fil des neurones par les tetrodes mobiles dans les poissons nageant librement

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain ...