Financé par le NIH (NS-18205).

1. Préparation des poissons pour les enregistrements appariés <ol><li> Avant de commencer la procédure, préparer cinq solutions clés, qui comprennent: Solution de bain, la solution de bain avec tricaïne (MS222), solution du bain avec le formamide, Neuron interne Solution et Solution musculaire interne. </li><li> Ensuite, recueillir une seule larve de poisson zèbre âgés de 72 à 96 heures et le placer dans la solution de bain avec tricaïne. En une minute d&#39;exposition à l&#39;anesthésie, le poisson ne répond pas à toucher. </li><li> Placer le poisson sur le dessus d&#39;un plat de plastique et, en utilisant un microscope stéréoscopique, décapiter avec une lame de rasoir à double tranchant. </li><li> Transférer le poisson à une chambre d&#39;enregistrement de verre revêtu d&#39;Sylgard et rempli avec la solution de bain. Fixer à chaque extrémité en insérant broches de tungstène électrolytiquement aiguisé par la notochorde. </li><li> Créer un lambeau de peau à proximité de la fin rostrale du poisson avec une épingle qui offrira une adhérence suffisante pour une paire de pinces fines. Retirez la peau recouvrant en le saisissant à proximité de la broche rostrale avec une paire de pinces fines. Peler lentement dans la direction caudale pour enlever la peau. </li><li> Traiter le poisson avec 3 ml de solution du bain avec le formamide pendant 3-5 minutes pour bloquer les contractions musculaires qui pourraient interférer avec les enregistrements. Laver 5 fois avec la solution de bain normal. Retirer une partie de la couche superficielle des muscles pendant 5 à 6 segments par un léger casse avec le côté d&#39;une épingle de tungstène. </li><li> Transférer le poisson à un microscope droit situé dans une cage de Faraday pour protéger du bruit électrique. Le microscope est équipé d&#39;une scène fixe, longue distance de travail objective 40x et zoom de 0,5 à 4x. Le microscope est monté sur une platine motorisée XY de traduction afin de déplacer la préparation entre le nerf et le muscle sous haute puissance. L&#39;utilisation de l&#39;optique interférence différentielle de contraste permet de visualiser les neurones. </li><li> Moins de 0.5 x zoom, utilisez un large alésage 15 microns pipette pour retirer les muscles sus-et exposer la moelle épinière. Une pression négative est appliquée à travers une seringue de 3 cc jetables et les cellules musculaires sont enlevés un par un. Cette procédure est répétée pour 5 à 6 segments dorsaux du muscle squelettique. La même largeur alésage pipette est utilisée aussi enlever les deux couches supérieures du muscle ventral pour obtenir cibles muscle rapide du squelette. </li><li> Ceci achève la préparation pour les enregistrements appariés et le zoom microscope est porté à environ 1.6x. </li></ol> 2. Enregistrements paires de CaP motoneurone et les cellules musculaires cibles <ol><li> Les segments nettoyées sont scannés pour localiser les neurones de la PAC. Chaque hémi-segment de la moelle épinière contient un grand diamètre de 10 microns, Cap motoneurone. Cette forme de larme Soma est situé superficiellement sous la mère spinale à proximité de l&#39;extrémité la plus caudale de la frontière segmentaire. Un neurone PAC est choisi pour l&#39;enregistrement et du muscle cible ventrale dans le segment adjacent caudale est scanné pour vérifier l&#39;intégrité. </li><li> Deux électrodes de patch sont positionnés pour l&#39;enregistrement, un pour le neurone et un second pour le muscle. L&#39;électrode supérieure a un cône peu profonde pour la pénétration de la dure-mère spinale et l&#39;ouverture de pointe d&#39;environ 2 microns. L&#39;électrode inférieure a une pente plus conique pour l&#39;enregistrement de la résistance pince basse tension du muscle squelettique </li><li> Position de l&#39;électrode neurone à un angle de 30 degrés environ pour pénétrer le spinale, environ un demi-segment rostrale au neurone CaP sélectionnés. Advance l&#39;électrode à l&#39;aide d&#39;un manipulateur d&#39;entraînement axial tout en appliquant une légère pression positive continue d&#39;environ 60 millimètres de mercure. Comme l&#39;électrode à travers les pauses-mère, le neurone CAP peuvent être déplacées par la perfusion de l&#39;électrode, nécessitant un réajustement de la mise au point. </li><li> Quand l&#39;électrode touche la PAC neurone Soma pression positive est libéré et se traduit généralement par la formation immédiate d&#39;un sceau gigaohm. Toutefois, dans certains cas où un joint ne se forme, il devient nécessaire d&#39;appliquer une pression douce négative. Après la formation du joint, le potentiel de l&#39;électrode est ajustée à 80 mV et l&#39;application d&#39;une pression négative des ruptures de la membrane cellulaire. Le neurone PAC est caractérisée par une résistance d&#39;entrée de 140 à 180 méga-ohms et un potentiel d&#39;action que les dépassements 40 millivolts. Cela a été testé en injectant progressivement croissante étapes de dépolarisation sous pince de courant jusqu&#39;à ce que le potentiel d&#39;action est déclenchée. </li><li> Ensuite, le microscope est relocalisé au muscle ventral à l&#39;aide du traducteur XY motorisée de moins de puissance élevée. Une cellule de muscle rapide est choisi parmi le champ cible. </li><li> L&#39;électrode de muscle est avancé vers la cellule musculaire cible sous pression positive, qui, lorsqu&#39;il est libéré, forme un joint gigaohm. Le potentiel d&#39;électrode est ajustée à -50 millivolts pour inactiver les canaux sodiques et sous aspirer doucement la membrane cellulaire est rompue. Une baisse de la résistance de 100 à 200 méga-ohms et l&#39;apparition soudaine de la grande entrée de signaux transitoires capacitifs. </li><li> Pour testerpour l&#39;enregistrement couplé, le motoneurone est dépolarisée par des injections progressivement plus de courant jusqu&#39;à ce qu&#39;un potentiel d&#39;action est déclenchée. A ce moment un courant plaque motrice devrait être obtenue dans le muscle. Une stimulation continue de la motoneurones au 1 résultats Hertz dans les courants de plaque motrice sans échec. Comme la fréquence de stimulation est augmentée à 100 Hertz plaque motrice des courants fluctuent en amplitude et en subissent l&#39;épuisement fonctionnels dans les 10 secondes de stimulation. </li></ol> 3. Les résultats représentatifs Généralement, l&#39;enregistrement neurone est stable jusqu&#39;à une heure, mais les cellules musculaires se détériorent comme en témoignent comme une augmentation de la résistance série. Lorsque cela se produit l&#39;expérience est terminée soit ou d&#39;une autre cellule musculaire est serré correctif. Tous les enregistrements doivent être terminées dans une heure. <img src="/files/ftp_upload/2351/2351fig1.jpg" alt="Figure 1" /> Figure 1. Enregistrements du potentiel d&#39;action des motoneurones (AP, en haut) et le plateau vertébral musculaire associée actuelle (EPC, en bas). Le potentiel d&#39;action pour un neurone PAC devrait dépassement 40 mV et l&#39;EPC devrait augmenter à moins de 300 msec et la décomposition le long d&#39;un cours à temps exponentiel avec une constante de temps de moins de 1 ms. <table border="1"><tbody><tr><td> Nom </td><td> Recette </td></tr><tr><td> Solution de bain (en mM) </td><td> 134 NaCl, KCl 2,9, CaCl 2 2,1, 1,2 MgCl 2, 10 de glucose, 10 Na-HEPES, pH 7,8 </td></tr><tr><td> Solution de bain avec tricaïne </td><td> Solution bain contenant 0,02% tricaïne </td></tr><tr><td> Solution de bain avec le formamide </td><td> Solution bain contenant 2M formamide </td></tr><tr><td> Neuron interne de la solution (en mM) </td><td> 115 K-gluconate, 15 KCl, MgCl 2 2, 10 K-HEPES, 5 K-EGTA, 4 Mg-ATP, pH 7,2 </td></tr><tr><td> Solution musculaire interne (en mM) </td><td> 120 KCl, 10 K-HEPES, 5 BAPTA, pH 7,4 </td></tr></tbody></table> Tableau 1. Solutions.

<ol>
	<li>Fetcho, J. R., O'Malley, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neuronal+networks+in+behaving+animals.">Imaging neuronal networks in behaving animals.</a> Current Opinion in Neurobiology. 7, 832-838 (1997).</li><li>Fetcho, J. R., S, H. i. g. a. s. h. i. j. i. m. a. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+and+genetic+approaches+toward+understanding+neuronal+circuits+in+zebrafish.">Optical and genetic approaches toward understanding neuronal circuits in zebrafish.</a> Integr Comp Biol. 44, 57-70 (2004).</li><li>Iba&#241;ez-Tallon, I., Wen, H., Miwa, J., Xing, J., Aslantas-Tekinay, A., Ono, F., Brehm, P., Heintz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tethering+naturally+occurring+peptide+toxins+for+cell+autonomous+modulation+of+ion+channels+and+receptors+in+vivo.">Tethering naturally occurring peptide toxins for cell autonomous modulation of ion channels and receptors in vivo.</a> Neuron. 43, 305-311 (2004).</li><li>Li, W., Ono, F., Brehm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+measurements+of+presynaptic+release+in+mutant+zebrafish+lacking+postsynaptic+receptors.">Optical measurements of presynaptic release in mutant zebrafish lacking postsynaptic receptors.</a> J. Neuroscience. , 23-10467 (2003).</li><li>Ono, F., Mandel, G., Brehm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetylcholine+receptors+direct+rapsyn+clusters+to+the+neuromuscular+synapse+in+zebrafish.">Acetylcholine receptors direct rapsyn clusters to the neuromuscular synapse in zebrafish.</a> J. Neuroscience. 24, 475-5481 (2004).</li><li>Ono, F., Shcherbatko, A., Higashijima, S., Fetcho, J., Mandel, G., Brehm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paralytic+zebrafish+lacking+ACh+receptors+fail+to+localize+rapsyn+clusters+to+the+synapse.">Paralytic zebrafish lacking ACh receptors fail to localize rapsyn clusters to the synapse.</a> J. Neuroscience. 21, 5439-5448 (2001).</li><li>Ono, F., Shcherbatko, A., Higashijima, S., Mandel, G., Brehm, P. <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+motility+mutant+twitch+once+reveals+new+roles+for+rapsyn+in+synaptic+function.">The zebrafish motility mutant twitch once reveals new roles for rapsyn in synaptic function.</a> J. Neuroscience. 22, 6491-6498 (2002).</li><li>Wang, M., Wen, H., Brehm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+neuromuscular+synapses+in+the+zebrafish+choline-acetyltransferase+mutant+bajan.">Function of neuromuscular synapses in the zebrafish choline-acetyltransferase mutant bajan.</a> J Neurophysiol. , 100-104 (2008).</li><li>Wen, H., Brehm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paired+motor-neuron+muscle+recordings+in+zebrafish+test+the+receptor+blockade+model+for+shaping+synaptic+current.">Paired motor-neuron muscle recordings in zebrafish test the receptor blockade model for shaping synaptic current.</a> J. Neuroscience. 25, 8104-8111 (2005).</li></ol>

Nous avons utilisé le poisson-zèbre enregistrements appariés (Wen et Brehm, 2005) principalement à étudier les processus de libération des vésicules et de recyclage lors de la stimulation à haute fréquence. Ce processus est perturbé en mutants motilité où la transmission synaptique normale est compromise en raison de mutations ponctuelles dans les principaux composants synaptiques. Par exemple, les mutations qui perturbent les agrégats des récepteurs post-synaptiques (Ono et al., 2001, 2002, 2004) et la synthèse de l&#39;émetteur présynaptique (Wang et al., 2008) résulte de la non-coordination de natation. Enregistrements appariés de fournir les informations qui peuvent identifier la source de l&#39;anomalie fonctionnelle, reliant ainsi le comportement, la génétique et la physiologie. Il devrait maintenant être possible de disséquer les processus qui sous-tendent la transmission synaptique par des moyens d&#39;expression transitoire de la PAC à moteur neurones en utilisant des promoteurs judicieux. De cette façon dominante négative ou gènes mutés peuvent être spécifiquement exprimée dans les neurones et les deux conséquences comportementales et électrophysiologiques déterminée. L&#39;avantage supplémentaire offert par la configuration cellule entière permet de dialyse du soma PAC avec des agents qui peuvent potentiellement modifier le recyclage des vésicules, comme les toxines clostridiales et les tampons de calcium. En raison de la courte distance entre le soma et le muscle, la diffusion devrait se produire ainsi dans le délai d&#39;enregistrements. Le potentiel de cette préparation a seulement commencé à être exploitées. La transparence du poisson à cet endroit âge et superficielle des synapses facilite également l&#39;utilisation d&#39;outils optiques (Fetcho et O&#39;Malley, 1997; Fetcho et Higashijima, 2004). Nous avons eu beaucoup de succès dans la mesure de l&#39;endo et d&#39;exocytose dans les poissons qui vivent avec des teintures FM et indicateurs génétiques comme Synaptophluorin (Li et al., 2003). En outre, les toxines fluorescents conjugués peuvent être utilisés pour étiqueter les protéines synaptiques pour l&#39;imagerie de poissons vivants (Ibanez-Tallon et al., 2004). Compte tenu de la simplicité de cette préparation, il est très prometteur pour des études futures.

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<td>Pneumatic transducer tester</td>
<td>&nbsp;</td>
<td>Fluke Biomedical Instruments</td>
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<td>0.002 x 3 inch tungsten rod</td>
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<td>A-M Systems Inc</td>
<td>715000</td>
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<td>Sylgard 184 Elastomer</td>
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<td>The thickness of application will affect the DIC optics</td>
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paired patch clamp recordings from motor-neuron and target skeletal muscle in zebrafish

Les larves de poisson zèbre constituent le système de premier vertébré modèle pour permettre l&#39;enregistrement simultané de patch-clamp à partir d&#39;un motoneurone spinal et de cibler des muscles. Ceci est une conséquence directe de l&#39;accessibilité pour les deux types de cellules et la capacité de distinguer visuellement la PAC seule segmentaire motoneurone sur la base de la morphologie et la localisation. Cette vidéo illustre les méthodes microscopiques utilisées pour identifier un CAP motoneurone et les cellules musculaires cibles ainsi que les méthodologies pour l&#39;enregistrement de chaque type de cellule. Identification de la PAC de type motoneurone est confirmée par le remplissage de teinture soit ou par les caractéristiques biophysiques comme forme d&#39;onde du potentiel d&#39;action et de résistance d&#39;entrée cellulaire. Motoneurone enregistrements régulièrement dernière pendant une heure permettant à long terme des enregistrements de plusieurs différentes cellules du muscle cible. Le contrôle sur le modèle de mise à feu du neurone moteur permet des mesures de la fréquence-dépendance de la transmission synaptique à la jonction neuromusculaire. Grâce à une grande taille quantique et le faible niveau de bruit fournies par la cellule de tension pince ensemble, tous les événements unitaires peuvent être résolus dans le muscle. Cette fonctionnalité permet d&#39;étudier les propriétés de base synaptiques tels que les propriétés de libération, le recyclage des vésicules, ainsi que la dépression synaptique et la facilitation. Les avantages offerts par cette in vivo la préparation précédente éclipse les systèmes modèles étudiés neuromusculaire où les neurones moteurs-sont généralement stimulé par des électrodes extracellulaires et les muscles sont trop gros pour pince cellules correctif ensemble. La préparation de poisson zèbre se prête à une analyse électrophysiologique combinant avec un large éventail d&#39;approches, notamment des lignées transgéniques, knockdown morpholino, pharmacologiques et d&#39;imagerie in vivo. Ces approches, couplé avec le nombre croissant de modèles de maladies neuromusculaires fournies par lignées mutantes du poisson zèbre, ouvrir la porte à une nouvelle compréhension de l&#39;humain des troubles neuromusculaires.

Watch this Scientific Journal Video about Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish at JoVE.com

Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish

Les larves de poisson zèbre constituent le système de premier vertébré modèle pour permettre l&#39;enregistrement simultané de patch-clamp à partir d&#39;un motoneurone spinal et de cibler des muscles squelettiques. Cette vidéo illustre les méthodes microscopiques utilisées pour identifier un CAP segmentaire motoneurone et les cellules musculaires cibles ainsi que les méthodologies pour l&#39;enregistrement de chaque type de cellule.

Jumelés Recordings Patch Clamp du motoneurone et de muscle ciblé squelettiques chez le poisson zèbre

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle.  ...

paired-patch-clamp-recordings-from-motor-neuron-target-skeletal

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Neuroscience

17.0K vues.  Oregon Health and Sciences University. Les larves de poisson zèbre constituent le système de premier vertébré modèle pour permettre l'enregistrement simultané de patch-clamp à partir d'un motoneurone spinal et de cibler des muscles squelettiques. Cette vidéo illustre les méthodes microscopiques utilisées pour identifier un CAP segmentaire motoneurone et les cellules musculaires cibles ainsi que les méthodologies pour l'enregistrement de chaque type de cellule.

Vidéo: Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish

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

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of intracellular events that lead to closure of cyclic-nucleotide-gated channels in the cell membrane. The resulting change in membrane potential leads in turn to reductions in the amount of neurotransmitter release from both rod and cone synaptic terminals. To measure how the light-evoked change in photoreceptor membrane potential leads to downstream activity in the retina, scientists have made electrophysiological recordings from retinal slice preparations for decades1,2. In the past these slices have been cut manually with a razor blade on retinal tissue that is attached to filter paper; in recent years another method of slicing has been developed whereby retinal tissue is embedded in low gelling temperature agar and sliced in cool solution with a vibrating microtome3,4. This preparation produces retinal slices with less surface damage and very robust light-evoked responses. Here we document how this procedure can be done under infrared light to avoid bleaching the visual pigment.

Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.

Enregistrements Patch Clamp à partir de neurones rétiniens souris dans une préparation adaptés à l&#39;obscurité Slice

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

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Neurosciences

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch clamp analyses of the voltage-gated channels in sensory hair cells isolated from a variety of species have been described previously1-4 but this video represents the first application of those techniques to hair cells from zebrafish. Here we demonstrate a method to isolate healthy, intact hair cells from all of the inner ear end-organs: saccule, lagena, utricle and semicircular canals. Further, we demonstrate the diversity in hair cell size and morphology and give an example of the kinds of patch clamp recordings that can be obtained. The advantage of the use of this zebrafish model system over others stems from the availability of zebrafish mutants that affect both hearing and balance. In combination with the use of transgenic lines and other techniques that utilize genetic analysis and manipulation, the cell isolation and electrophysiological methods introduced here should facilitate greater insight into the roles hair cells play in mediating these sensory modalities.

The purpose of this video is to demonstrate procedures for obtaining healthy, intact hair cells from the inner ear organs of adult zebrafish and then using them for patch clamp studies aimed at characterizing the biophysical properties of their voltage-gated channels.

Les enregistrements de patch-clamp en cellules ciliées de l&#39;oreille Isolé du poisson zèbre

Patch  clamp analyses of the voltage-gated channels in sensory hair cells isolated  from a variety of species have been described previously1-4 but this  ...

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

Mitochondria are involved in many important cellular functions including metabolism, survival1, development and, calcium signaling2. Two of the most important mitochondrial functions are related to the efficient production of ATP, the energy currency of the cell, by oxidative phosphorylation, and the mediation of signals for programmed cell death3.
The enzyme primarily responsible for the production of ATP is the F1FO-ATP synthase, also called ATP synthase4-5. In recent years, the role of mitochondria in apoptotic and necrotic cell death has received considerable attention. In apoptotic cell death, BCL-2 family proteins such as Bax enter the mitochondrial outer membrane, oligomerize and permeabilize the outer membrane, releasing pro-apoptotic factors into the cytosol6. In classic necrotic cell death, such as that produced by ischemia or excitotoxicity in neurons, a large, poorly regulated increase in matrix calcium contributes to the opening of an inner membrane pore, the mitochondrial permeability transition pore or mPTP. This depolarizes the inner membrane and causes osmotic shifts, contributing to outer membrane rupture, release of pro-apoptotic factors, and metabolic dysfunction. Many proteins including Bcl-xL7 interact with F1FO ATP synthase, modulating its function. Bcl-xL interacts directly with the beta subunit of F1FO ATP synthase, and this interaction decreases a leak conductance within the F1FOATPasecomplex, increasing the net transport of H+ by F1FO during F1FO ATPase activity8 and thereby increasing mitochondrial efficiency. To study the activity and modulation of the ATP synthase, we isolated from rodent brain submitochondrial vesicles (SMVs) containing F1FO ATPase. The SMVs retain the structural and functional integrity of the F1FO ATPase as shown in Alavian et al. Here, we describe a method that we have used successfully for the isolation of SMVs from rat brain and we delineate the patch clamp technique to analyze channel activity (ion leak conductance) of the SMVs.

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

A method to isolate submitochondrial vesicles enriched in F1FO ATP synthase complexes from rat brain is described. These vesicles allow the study of the activity of F1FO ATPase complex and its modulation using the technique of patch clamp recording.

<sub&gt; 1</sub&gt; F<sub&gt; O</sub&gt; ATPase Vesicle Préparation et Technique for the Performing Patch enregistrements de serrage des membranes des vésicules submitochondriales

Mitochondria are involved in many important cellular  functions including metabolism, survival1, development and, calcium signaling2. Two of the most ...

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

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

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

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

Isolation des neurones sensoriels de<em&gt; Aplysia californica</em&gt; Patch pour les enregistrements de serrage des courants glutamatergiques

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

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Mauthner cells (M-cells) are large reticulospinal neurons located in the hindbrain of teleost fish. They are key neurons involved in a characteristic behavior known as the C-start or escape response that occurs when the organism perceives a threat. The M-cell has been extensively studied in adult goldfish where it has been shown to receive a wide range of excitatory, inhibitory and neuromodulatory signals1. We have been examining M-cell activity in embryonic zebrafish in order to study aspects of synaptic development in a vertebrate preparation. In the late 1990s Ali and colleagues developed a preparation for patch clamp recording from M-cells in zebrafish embryos, in which the CNS was largely intact2,3,4. The objective at that time was to record synaptic activity from hindbrain neurons, spinal cord neurons and trunk skeletal muscle while maintaining functional synaptic connections within an intact brain-spinal cord preparation. This preparation is still used in our laboratory today. To examine the mechanisms underlying developmental synaptic plasticity, we record excitatory (AMPA and NMDA-mediated)5,6 and inhibitory (GABA and glycine) synaptic currents from developing M-cells. Importantly, this unique preparation allows us to return to the same cell (M-cell) from preparation to preparation to carefully examine synaptic plasticity and neuro-development in an embryonic organism. The benefits provided by this preparation include 1) intact, functional synaptic connections onto the M-cell, 2) relatively inexpensive preparations, 3) a large supply of readily available embryos 4) the ability to return to the same cell type (i.e. M-cell) in every preparation, so that synaptic development at the level of an individual cell can be examined from fish to fish, and 5) imaging of whole preparations due to the transparent nature of the embryos.

We have developed an intact brain-spinal cord preparation to record and monitor electrical activity via patch clamp recording from the Mauthner neurons and other reticulospinal cells in zebrafish embryos. Thus, we are able to record excitatory and inhibitory synaptic currents, voltage-gated channel activity and action potentials from key neurons in a developing embryo.

Patch-clamp enregistrements de cellules embryonnaires de poisson zèbre Mauthner

Mauthner cells (M-cells) are large  reticulospinal neurons located in the hindbrain of teleost fish. They are key  neurons involved in a characteristic ...

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

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

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

De cellules entières de patch-clamp enregistrements de Morphologically- et interneurones hippocampiques neurochemically identifiés

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

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

Appariés enregistrements de cellules entières dans organotypiques coupes d&#39;hippocampe

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with simultaneous intracellular biocytin filling allows a correlated analysis of their structural and functional properties. With this method it is possible to identify and characterize both pre- and postsynaptic neurons by their morphology and electrophysiological response pattern. Paired recordings allow studying the connectivity patterns between these neurons as well as the properties of both chemical and electrical synaptic transmission. Here, we give a step-by-step description of the procedures required to obtain reliable paired recordings together with an optimal recovery of the neuron morphology. We will describe how pairs of neurons connected via chemical synapses or gap junctions are identified in brain slice preparations. We will outline how neurons are reconstructed to obtain their 3D morphology of the dendritic and axonal domain and how synaptic contacts are identified and localized. We will also discuss the caveats and limitations of the paired recording technique, in particular those associated with dendritic and axonal truncations during the preparation of brain slices because these strongly affect connectivity estimates. However, because of the versatility of the paired recording approach it will remain a valuable tool in characterizing different aspects of synaptic transmission at identified neuronal microcircuits in the brain.

Patch-clamp recordings and simultaneous intracellular biocytin filling of synaptically coupled neurons in acute brain slices allow a correlated analysis of their structural and functional properties. The aim of this protocol is to describe the essential technical steps of electrophysiological recording from neuronal microcircuits and their subsequent morphological analysis.

Électrophysiologiques et Caractérisation morphologique des neurones Les circuits dans aiguë tranches cerveau en utilisant jumelés patch-clamp enregistrements

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with ...

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

Recordings cellules entières Patch-clamp en tranches cérébrales

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

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

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

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

Subcellulaires Recordings Patch-clamp du Domaine somato de nigrales Dopamine Neurones

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