Ce travail a été soutenu par des subventions du NIH EY17606 (APS).

1. Électrodes Préparation <ol><li> Préparer les électrodes d&#39;enregistrement et de référence en les trempant dans deux 1M de NaCl et de conduite 15V entre eux utilisant une onde sinusoïdale de 1 Hz pour ~ 20 s. </li><li> Faire les électrodes d&#39;enregistrement à l&#39;aide d&#39;un tire-micropipette (Sutter Instruments P-97). Pour les pipettes de patch-utiliser un verre borosilicate avec un diamètre extérieur de 1,2 mm et un diamètre intérieur de 0,69 mm (Sutter Instruments, Cat # B120-69-10). Mesurer la résistance série à travers la pointe de l&#39;électrode. Basé sur la taille des cellules cibles choisir un diamètre de pointe de la pipette, car les corps cellulaires ~ 20 um d&#39;utilisation diamètre ~ 5 MW pour les corps cellulaires ~ 5 um d&#39;utilisation de diamètre ~ 12 MQ. </li><li> Préparez-chaussée / électrodes de référence. En utilisant une seringue de remplir un tube en polyéthylène petite section (Intramedic, PE205) avec 1 mM de NaCl qui comprend Agar 3,5% (Sigma, Cat # A7002) dans NaCl 1M. Placez ce pont agar sur l&#39;électrode de référence Ag / AgCl. </li></ol> 2. La préparation de solutions <ol><li> Solution du bain externe, «les médias contenant du bicarbonate (1 L): 1 bouteille de Ames Ames Moyenne (Sigma, Cat # A1420), 1,9 g de NaHCO 3, 7 ml de pénicilline-streptomycine (Sigma, Cat # P0781) pour empêcher des bactéries la croissance. Ajustez l&#39;osmolarité à ~ 280 mOsm. </li><li> Tranchage solution, «support contenant le tampon HEPES pH (1 L): 1 bouteille de Ames Ames moyen, 0,887 g de NaCl, 2,38 g d&#39;HEPES, 7 ml de pénicilline-streptomycine. Ajuster le pH à 7,4 en utilisant ~ 1M NaOH et / ou HCl 1M. Ajustez l&#39;osmolarité à ~ 280 mOsm. </li><li> Potassium-aspartate (K-Asp), pipeter solution interne (en mM): 125 K-aspartate, 10 KCl, 10 HEPES, 5 NMG-HEDTA, 0,5 CaCl2, une ATP-Mg, 0,2 GTP-Mg; pH est ajusté à ~ 7.3 avec NMG-OH, et l&#39;osmolarité est ajusté à ~ 280 mOsm. </li><li> Filet Agar pour la stabilisation de basse température de gélification agarose contenant de la rétine (5% en poids), 7,5 g d&#39;Agar (Sigma, Cat # A7002) fondu dans 150 ml de dH 2 O. </li></ol> 3. Journée de l&#39;expérience <ol><li> Dégel ~ 1,5 ml de K-Asp solution interne et le fluorophore de votre choix du congélateur. Diluer le fluorophore à la concentration appropriée, qui doit être déterminée empiriquement. </li><li> Verser les médias Ames contenant NaHCO 3 (~ 200 ml) dans le conteneur de stockage étanche à la lumière puis placez-le dans le bain d&#39;eau (30-32 ° C) et s&#39;équilibrer avec 5% de CO 2 / 95% d&#39;O 2 du gaz </li><li> Remplissez la bouteille avec ~ 110 mL de milieu d&#39;Ames contenant HEPES, placer sur la glace et équilibrer avec 100% d&#39;O 2. </li><li> Placez le support restant Ames contenant NaHCO 3 dans un réservoir chauffé au-dessus de la cage de Faraday, et s&#39;équilibrer avec 5% de CO 2 / 95% d&#39;O 2 du gaz. Parafilm peut être utilisé pour piéger le gaz dans l&#39;espace ci-dessus la solution. </li><li> Préparer la faible température de gélification agarose (3%) en ajoutant 0,75 g (Sigma, Cat # A0701) à 25 ml de milieu d&#39;Ames »avec HEPES et chauffer la solution à ~ 115 ° C pour faire fondre l&#39;agarose. Remuer jusqu&#39;à ce que la solution claire, sans bulles, et stocker dans un bain d&#39;eau (~ 42 ° C). </li><li> Remplir une pipette d&#39;enregistrement avec la solution K-Asp interne et vérifier la résistance de pointe. </li></ol> 4. Commencez l&#39;expérience <ol><li> Euthanasier la souris et supprimer rapidement les yeux avec des ciseaux courbes. Des lunettes à infrarouge doit être utilisé pour toutes les procédures visant à prévenir le blanchiment du pigment visuel. </li><li> Placez les yeux sur un morceau de papier filtre pour les empêcher de rouler, et en utilisant un mince lame à double face, faire une fente dans la cornée de l&#39;œil tout en maintenant avec la pince. </li><li> Dès le globe oculaire est percé, le transfert de leur oeil dans un plat rempli de média Ames. </li><li> Utilisation de la fente de la cornée comme un point d&#39;entrée, petits ciseaux sont utilisés pour couper les portions restantes de la cornée, après quoi le cristallin et du vitré peut être enlevé. Veillez à ce que la rétine reste attaché à l&#39;œilleton, et de stocker l&#39;œilleton dans l&#39;obscurité à 32 ° C dans les médias équilibrée avec 5% de CO 2 / 95% O 2 Ames. </li></ol> 5. Tranchage <ol><li> Hemisect l&#39;un des œilletons et dissocier la rétine de l&#39;épithélium pigmentaire rétinien. Retirer les bords de la rétine afin de permettre le tissu à plat. </li><li> Utilisez une petite pipette Pasteur en verre avec une ampoule de latex pour remplir un plat de Pétri de 35 mm avec de l&#39;agar fondu, et faites glisser la pince travers grâce à l&#39;agar-agar pour tester sa cohérence. </li><li> Lorsque vous pouvez commencer à voir la gélose se solidifier: <ol class="lalpha"><li> Transfert de la rétine au sommet de l&#39;agar-agar. </li><li> Utilisez un morceau tordu de Kimwipe pour absorber l&#39;excès de solution autour de la rétine. </li><li> Placer 2-3 gouttes d&#39;Agar directement sur la rétine et de placer rapidement un cylindre en plastique sur la rétine pour former un mur autour d&#39;elle. Puis au goutte à goutte supplémentaires gélose fondue sur les côtés du cylindre en plastique tout en centrant la rétine dans l&#39;agar. </li><li> Transférer la boîte de Petri to un bain d&#39;eau glacée pour se rafraîchir. </li></ol></li><li> Après ~ 45 secondes enlever le tissu du bain d&#39;eau glacée et l&#39;utilisation d&#39;un piston pour extruder la gélose de bloc hors du cylindre en plastique, poussant du côté le plus éloigné de la rétine. </li><li> Utilisez la lame de rasoir unilatérale de couper un petit bloc de gélose contenant de la rétine et la superglue le bloc en place contre la butée gélose sur la platine. </li><li> Transfert du disque spécimen sur le plateau vibrant microtome et versez glacée HEPES Ames dans le bac permettant le bloc avec la rétine pour être pleinement immergé. </li><li> Tranches de la rétine avec une épaisseur de ~ 200 um peut être coupé au taux maximum de la lame vibrante et une vitesse de lame vers l&#39;avant ensemble tel que cela prend 4 à 5 secondes pour couper à travers la rétine sur chaque tranche. </li><li> Utilisez une lame de bistouri n ° 11 pour supprimer chaque une tranche utilisable à la fois </li><li> Bonne tranches contenant la rétine sont placés dans des chambres d&#39;enregistrement et sont lestés avec des ancres tranche avec fils de nylon en les croisant. Le fils de nylon maintenez enfoncée la gélose entourant la rétine, laissant la rétine dégagée pour les enregistrements. </li><li> Transférer la chambre d&#39;enregistrement de la platine du microscope debout, fermer le rideau et tourner sur la source de lumière infrarouge pour voir la tranche sur une caméra infrarouge sensible. </li></ol> 6. Enregistrement <ol><li> Une fois une bonne tranche de rétine (avec des photorécepteurs intact et l&#39;angle de coupe appropriée) est identifié commencent perfusant la rétine à un taux supérieur à 5 mL / min avec les médias chauffés Ames qui est chauffé à 35 37 ° C et équilibrée avec 5% de CO 2 / 95% d&#39;O 2. </li><li> Quelques dégâts de surface peut être apparente en raison du processus de tranchage. Cette couche généralement mince de cellules peuvent être retirés à l&#39;aide d&#39;une pipette avec une pointe cassée, et doucement sucer enlever les corps des cellules mortes. Ce sera généralement révèlent des cellules vivantes en dessous. Identifier un corps cellulaire saine, dont la position se situe dans les strates d&#39;intérêt. </li><li> Remplir une micropipette avec la solution Kasp internes et d&#39;appliquer positif (vers l&#39;extérieur) de pression à travers la pointe de la pipette, et inférieure de la pointe de la pipette dans la chambre d&#39;enregistrement et jusqu&#39;au niveau de la cellule à l&#39;aide d&#39;un micromanipulateur. </li><li> Placez l&#39;extrémité de la pipette de telle sorte qu&#39;il fossettes de la membrane cellulaire, et appliquer doucement négatives (flux entrants) de pression pour faire un joint à haute résistance. Cellules entières mode peut être obtenu en appliquant une pression négative à l&#39;électrode de pénétrer dans la cellule. </li><li> Stimuler le tissu rétinien utilisant des LED peut alors évoquer des réponses à la lumière. </li><li> Photographie de la cellule à partir de laquelle l&#39;enregistrement a été réalisé en évoquant la fluorescence du fluorophore dans la pipette solution interne. </li></ol> 7. Les résultats représentatifs <img src="/files/ftp_upload/2107/2107fig1.jpg" alt="Figure 1" /> Figure 1. Ici, la lumière des potentiels évoqués d&#39;une adaptés à l&#39;obscurité neurone rétinien d&#39;éclairs de lumière de force croissante sont présentés. <img src="/files/ftp_upload/2107/2107fig2.jpg" alt="Figure 2" /> Figure 2. Image de fluorescence d&#39;une tranche où les cellules rétiniennes ont été remplis avec du fluorophore, Lucifer Yellow. Fluorescence évoqués montre la morphologie des cellules à partir desquelles des enregistrements de patch ont été faites.

<ol>
	<li>Weblin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmission+along+and+between+rods+in+the+tiger+salamander+retina.">Transmission along and between rods in the tiger salamander retina.</a> J Physiol. 280, 449-470 (1978).</li><li>Wu, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+connections+among+retina+neurons+in+living+slices.">Synaptic connections among retina neurons in living slices.</a> J Neurosci Methods. 20, 139-149 (1987).</li><li>Rieke, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+contrast+adaptation+in+salamander+bipolar+cell.">Temporal contrast adaptation in salamander bipolar cell.</a> J Neurosci. 21, 9445-9454 (2001).</li><li>Armstrong-Gold, C., &amp;amp, R. i. e. k. e., F, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bandpass+filtering+at+the+rod+to+second-order+cell+synapse+in+salamander+(Ambystoma+tigrinum)+retina.">Bandpass filtering at the rod to second-order cell synapse in salamander (Ambystoma tigrinum) retina.</a> J Neurosci. 23, 3796-3806 (2003).</li><li>Masland, R. 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+fundamental+plan+of+the+retina.">The fundamental plan of the retina.</a> Nat Neurosci. 4, 877-886 (2001).</li><li>Wassle, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+processing+in+the+mammalian+retina.">Parallel processing in the mammalian retina.</a> Nat Rev Neurosci. 5, 747-757 (2004).</li></ol>

L&#39;enregistrement tranche de rétines de vertébrés ont été faites depuis des décennies 1,2, et ont assez bien réussi à fournir une compréhension plus profonde de la façon dont les circuits rétiniens encode la lumière incidente. Les avantages de la coupe avec un microtome vibrant plutôt que directement avec une lame de rasoir, c&#39;est que les tranches de la rétine encourent moins de dégâts à la surface. Avec une pipette d&#39;aspiration, il est facile d&#39;enlever ces dommages et les cell...

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<td>Micropipette Puller</td>
<td>&nbsp;</td>
<td>Sutter Instruments</td>
<td>P-97</td>
<td>&nbsp;</td>
<tr>
<td>Borosilicate glass</td>
<td>&nbsp;</td>
<td>Sutter Instruments</td>
<td>B120-69-10</td>
<td>&nbsp;</td>
<tr>
<td>Polyethylene tubing</td>
<td>&nbsp;</td>
<td>Intramedic</td>
<td>PE205</td>
<td>&nbsp;</td>
<tr>
<td>Agar</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A7002</td>
<td>&nbsp;</td>
<tr>
<td>Low gelling temp agarose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A0701</td>
<td>&nbsp;</td>
<tr>
<td>Ames' Medium</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A1420</td>
<td>&nbsp;</td>
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<td>Penicillin-Streptomycin</td>
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patch clamp recordings from mouse retinal neurons in a dark-adapted slice preparation

Notre expérience visuelle est déclenchée lorsque le pigment visuel dans nos photorécepteurs rétiniens absorbe les photons d&#39;énergie de la lumière et déclenche une cascade d&#39;événements intracellulaires qui conduisent à la fermeture de nucléotide cyclique-dépendants des canaux dans la membrane cellulaire. Le changement résultant dans le potentiel de membrane entraîne à son tour une réduction du montant de la libération des neurotransmetteurs à la fois de la tige et les terminaux cône synaptique. Pour mesurer la façon dont le changement de lumière évoquée dans le potentiel de membrane des photorécepteurs conduit à une activité en aval dans la rétine, les scientifiques ont fait des enregistrements électrophysiologiques de préparations tranche de la rétine pour 1,2 décennies. Dans le passé, ces tranches ont été coupées manuellement avec une lame de rasoir sur le tissu rétinien qui est attaché à un papier filtre; ces dernières années une autre méthode de découpage a été développé selon lequel le tissu rétinien est intégré dans l&#39;agar gélifiant à basse température et tranchées dans une solution fraîche avec un vibrant microtome 3,4. Cette préparation produit des tranches de la rétine avec moins de dommages de surface et des réponses très robuste lumière évoquée. Ici nous montrons comment cette procédure peut être faite sous une lumière infrarouge pour éviter le blanchiment du pigment visuel.

Watch this Scientific Journal Video about Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation at JoVE.com

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

Nous décrivons ici une procédure pour générer adaptés à l&#39;obscurité des tranches de la rétine de souris pour les enregistrements électrophysiologiques.

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 ...

patch-clamp-recordings-from-mouse-retinal-neurons-dark-adapted-slice

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Neuroscience

14.3K vues.  University of Southern California. Nous décrivons ici une procédure pour générer adaptés à l'obscurité des tranches de la rétine de souris pour les enregistrements électrophysiologiques.

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

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

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle. This is a direct consequence of the accessibility to both cell types and ability to visually distinguish the single segmental CaP motor-neuron on the basis of morphology and location. This video demonstrates the microscopic methods used to identify a CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type. Identification of the CaP motor-neuron type is confirmed by either dye filling or by the biophysical features such as action potential waveform and cell input resistance. Motor-neuron recordings routinely last for one hour permitting long-term recordings from multiple different target muscle cells. Control over the motor-neuron firing pattern enables measurements of the frequency-dependence of synaptic transmission at the neuromuscular junction. Owing to a large quantal size and the low noise provided by whole cell voltage clamp, all of the unitary events can be resolved in muscle. This feature permits study of basic synaptic properties such as release properties, vesicle recycling, as well as synaptic depression and facilitation. The advantages offered by this in vivo preparation eclipse previous neuromuscular model systems studied wherein the motor-neurons are usually stimulated by extracellular electrodes and the muscles are too large for whole cell patch clamp. The zebrafish preparation is amenable to combining electrophysiological analysis with a wide range of approaches including transgenic lines, morpholino knockdown, pharmacological intervention and in vivo imaging. These approaches, coupled with the growing number of neuromuscular disease models provided by mutant lines of zebrafish, open the door for new understanding of human neuromuscular disorders.

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target skeletal muscle. This video demonstrates the microscopic methods used to identify a segmental CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type.

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.  ...

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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 ...

Simultaneous Whole-cell Recordings from Photoreceptors and Second-order Neurons in an Amphibian Retinal Slice Preparation

One of the central tasks in retinal neuroscience is to understand the circuitry of retinal neurons and how those connections are responsible for shaping the signals transmitted to the brain. Photons are detected in the retina by rod and cone photoreceptors, which convert that energy into an electrical signal, transmitting it to other retinal neurons, where it is processed and communicated to central targets in the brain via the optic nerve. Important early insights into retinal circuitry and visual processing came from the histological studies of Cajal1,2 and, later, from electrophysiological recordings of the spiking activity of retinal ganglion cells - the output cells of the retina3,4.
A detailed understanding of visual processing in the retina requires an understanding of the signaling at each step in the pathway from photoreceptor to retinal ganglion cell. However, many retinal cell types are buried deep in the tissue and therefore relatively inaccessible for electrophysiological recording. This limitation can be overcome by working with vertical slices, in which cells residing within each of the retinal layers are clearly visible and accessible for electrophysiological recording.
Here, we describe a method for making vertical sections of retinas from larval tiger salamanders (Ambystoma tigrinum). While this preparation was originally developed for recordings with sharp microelectrodes5,6, we describe a method for dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells in which we manipulate the photoreceptor's membrane potential while simultaneously recording post-synaptic responses in horizontal or bipolar cells. The photoreceptors of the tiger salamander are considerably larger than those of mammalian species, making this an ideal preparation in which to undertake this technically challenging experimental approach. These experiments are described with an eye toward probing the signaling properties of the synaptic ribbon - a specialized synaptic structure found in a only a handful of neurons, including rod and cone photoreceptors, that is well suited for maintaining a high rate of tonic neurotransmitter release7,8 - and how it contributes to the unique signaling properties of this first retinal synapse.

We describe the preparation of thin retinal slices from aquatic tiger salamanders (Ambystoma tigrinum) and explain how we use these slices to study synaptic processing in the retina by obtaining dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells.

Les enregistrements de cellules entières simultanées de photorécepteurs et les neurones de second ordre dans une préparation de tranches rétine Amphibian

One of the central tasks in retinal neuroscience  is to understand the circuitry of retinal neurons and how those connections are  responsible for shaping ...

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 ...

Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. Retinal neurons in the network comprise of numerous subtypes. More than 10 subtypes of bipolar cells, ganglion cells, and amacrine cells have been identified by morphological studies. Multiple subtypes of retinal neurons are thought to encode distinct features of visual signaling, such as motion and color, and form multiple neural pathways. However, the functional roles of each neuron in visual signal processing are not fully understood. The patch clamp method is useful to address this fundamental question. Here, a protocol to record light-evoked synaptic responses in mouse retinal neurons using patch clamp recordings in dark-adapted conditions is provided. The mouse eyes are dark-adapted O/N, and retinal slice preparations are dissected in a dark room using infrared illumination and viewers. Infrared light does not activate mouse photoreceptors and thus preserves their light responsiveness. Patch clamp is used to record light-evoked responses in retinal neurons. A fluorescent dye is injected during recordings to characterize neuronal morphological subtypes. This procedure enables us to determine the physiological functions of each neuron in the mouse retina.

We will demonstrate how to prepare retinal slices from the mouse eye and record light responses in retinal neurons. The entire procedure is conducted in dark-adapted conditions.

Enregistrement Light-réponses évoquées dans les neurones post-synaptiques dans Dark-adaptés, Préparations rétine de la souris tranche en utilisant des techniques de patch clamp

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. ...

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 ...