Le financement fourni par la National Science Foundation (IOS-1050701 à BAC), le National Institutes of Health (NS54174 à la SM et F30DC0111907 à AML-W.), Le Uehara Memorial Foundation et la Société japonaise pour la promotion de la science (G2205 aux savoirs traditionnels ). Nous remercions Meeks Julian pour son soutien et des conseils sur le sujet des enregistrements axonales extracellulaires. Nous remercions Carl Hopkins pour fournir une chambre d&#39;enregistrement de prototype.

<ol>
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Aucun conflit d&#39;intérêt déclaré.

Une fois maîtrisée, cette technique permettra de cibler une neurones identifiés, y compris les axones individuels, pour les enregistrements in vivo dans de nombreux systèmes modèles. En outre, cette technique permet d&#39;enregistrer de manière fiable sortie pic de neurones présentant des caractéristiques anatomiques uniques qui font traditionnelle dans les méthodes d&#39;enregistrement in vivo difficile. Nous avons utilisé cette technique pour enregistrer à partir d&#39;ELA »petites cellules» dans mormyrid poissons faiblement électriques. Les tentatives précédentes pour étudier les propriétés de réglage de &quot;petites cellules&quot; ont échoué en raison des conditions d&#39;enregistrement difficiles 11, 14. Caractéristiques anatomiques similaires créent des obstacles à l&#39;obtention des enregistrements mono-unité d&#39;un grand nombre de vertébrés auditif différent et les neurones électrosensoriel 8-10. Pour relever ces défis dans notre système, nous avons profité du fait que les &quot;petites cellules&quot; sont les seules cellules dans ELA projet de PEL. Ainsi, le transport rétrograde de colorant placé dans PEL limite étiquetage sein d&#39;ELA pour &quot;petit&quot;soma et les axones cellulaires. Marquage fluorescent des axones ont permis le placement des électrodes précise à côté axones marqués, des enregistrements seule unité de cellules identifiées possibles malgré somas inaccessibles. Nous avons tenté enregistrements somatiques, mais sans succès, probablement due aux alentours synapses avalement 11 , 17. Cependant, somatique étiquetage était clairement visible ce qui suggère que cette technique pourrait être utilisée pour cibler les enregistrements somatiques dans d&#39;autres types cellulaires et d&#39;autres circuits. Marquage fluorescent des neurones par le transport rétrograde in vivo a été utilisé pour guider les enregistrements cibles in vitro 19-21 . Une technique similaire a été utilisée pour cible enregistrements in vivo des neurones moteurs chez le poisson zèbre moelle épinière 22. Notre travail représente une nouvelle extension de cette approche, dans laquelle les deux étiquetage et d&#39;enregistrement sont réalisées in vivo dans le cerveau. Notre méthode démontre que in vivo l&#39;étiquetage des zones du système nerveux central avec un rétrograde tcoureur peut être étendue à l&#39;étude d&#39;autres circuits intacts avec les neurones de projection similaire sélectifs. Par exemple, dans le traitement auditif des mammifères, le colliculus inférieur (IC) sert de centre de relais important pour les apports de multiples structures rhombencephalic 23. Injection de colorant dans le circuit intégré serait marquer sélectivement les cellules de projection de chacun de ces noyaux. Le colliculus supérieur (CS) a une fonction similaire à la vision 24. Les préparatifs de la moelle épinière sont particulièrement bien adaptés à cette technique, comme la moelle épinière est facilement accessible, injection de colorant peut se produire loin du site d&#39;enregistrement, et il peut être combiné avec l&#39;enregistrement intracellulaire et le remplissage de certains neurones d&#39;acquérir des informations anatomiques détaillées 25. Enfin, des voies de traçage est une technique bien établie utilisée dans le système nerveux central pour cartographier circuits complexes 26. Notre méthode peut être utilisée pour ajouter des informations fonctionnelles à ces études comme cela a été fait avecindicateurs sensibles au calcium Ith cortex visuel du chat 27. La chirurgie, qui est bien établi, fiable et régulièrement utilisé pour les aveugles in vivo des enregistrements 16, doit être complété par des saignements minimes et aucun dommage à la surface du cerveau pour permettre à l&#39;animal et le tissu pour survivre. Avec la pratique, l&#39;application de la chirurgie et de teinture peut être complété en 30-45 minutes. Nous avons étiqueté avec succès &quot;à petites cellules&quot; axones dans 67% des préparations. La plupart des préparations n&#39;ont que de 1 ou 2 axones marqués visibles, mais certains en avoir autant que 8. Sur les 119 unités marquées tentatives, nous avons obtenu des enregistrements-ensemble de 26 logements répartis sur 12 préparations (tableau 2). Ainsi, les données ont été recueillies auprès de 41% des préparations à axones marqués pour un taux de réussite global de 28%. L&#39;aspect critique de l&#39;application de teinture est l&#39;étiquetage profondeur. Faible insertion du fil de tungstène se traduira, dans le colorant étant aw lavéay. Toutefois, si la pénétration est trop profonde, les axones marqués ne seront pas visibles pour le ciblage. En outre, certains dommages mécaniques à la cellule doit se produire pour que le colorant soit dûment pris en hausse de 25, 28. Cependant, trop de dégâts va tuer les cellules. Nous avons tenté d&#39;étiquetage avec d&#39;autres colorants et d&#39;autres méthodes (tableau 2), y compris marquage antérograde par injection de colorant dans ELa jumelé avec l&#39;enregistrement à partir des axones marqués dans PEL (tableau 3). Nous émettons l&#39;hypothèse que l&#39;étiquetage antérograde n&#39;a pas réussi parce que l&#39;étiquetage a été limitée aux cellules avec des dommages somatiques, ce qui les rend insensibles. En outre, les bornes de pré-synaptiques peuvent avoir été dérangé. En revanche, l&#39;étiquetage rétrograde minimise ces deux préoccupations. La quantité et le lieu d&#39;application de teinture peuvent être modifiés en fonction du circuit particulier à l&#39;étude. Étiquetage maximale se produit avec la plus grande concentration de colorant, ce qui nous avons obtenu l&#39;aide de fils de tungstène enrobées. Toutefois, pour l&#39;étiquetage des axones avec dedes projections de PE, colorant peuvent se détacher de l&#39;aiguille de tungstène comme il est avancé. Dans ces cas, l&#39;injection sous pression serait plus approprié. Absorption de colorant et le transport sont rapides, avec des axones marqués dans notre préparation étant visible dès 2 heures après l&#39;injection et les axones marqués supplémentaires apparaissant le plus tard 6 heures après l&#39;injection. Ainsi, l&#39;étiquetage et l&#39;enregistrement peut être accompli en une seule journée, en éliminant les difficultés techniques liées à la chirurgie de survie. Timing variera pour chaque application en fonction de la distance nécessaire pour le transport colorant. Un autre aspect essentiel est le placement des électrodes. Il est important pour entrer dans le tissu à proximité du site de l&#39;axone marquée pour éviter le colmatage de la pointe. Pour les tissus denses tels que dans ELA une longue tige mince sur l&#39;électrode d&#39;enregistrement minimise l&#39;excès mouvement du tissu environnant. Si un enregistrement réussi n&#39;est pas atteint à la première tentative, répétée avec des électrodes frais jusqu&#39;à ce que l&#39;enregistrement soit obtenu ou la tissue est interrompu à l&#39;endroit où l&#39;axone n&#39;est plus visible. Cependant, il est également important de placer rapidement l&#39;électrode à côté de l&#39;axone pour minimiser la quantité d&#39;exposition fluorescent, ce qui peut provoquer la phototoxicité, le blanchiment et peuvent affecter les propriétés physiologiques de la cellule 29-31. Une fois un segment de l&#39;axone est aspiré avec succès dans l&#39;électrode d&#39;enregistrement, les enregistrements peuvent être obtenues pendant plusieurs heures. Si les unités sont toujours perdus en moins de 1 heure, envisager de faire des petites pointes d&#39;électrodes pour empêcher l&#39;axone de glisser. D&#39;autre part, trop petit d&#39;une pointe peut entraîner l&#39;obstruction, à faible rapport signal-bruit, ou des dommages à l&#39;axone. Une baisse constante pic d&#39;amplitude et le «retour» d&#39;une unité avec aspiration supplémentaire est une indication que la pointe est trop grand. Trop aspiration peut causer des dommages irréparables à l&#39;axone. Une solution consiste à permettre à une fuite dans la conduite d&#39;air de sorte que la pression revient lentement à zéro. Égaliser rapidement èmepression e se traduira par un parent-aller &#39;push&#39; transitoire qui peut expulser l&#39;axone. Bien que cette technique représente un atout majeur pour obtenir des enregistrements ciblées de neurones de projection identifiés, il ne sera pas utile pour distinguer les interneurones locaux, comme colorant sera repris par tous les types de cellules au site d&#39;injection. Théoriquement, l&#39;utilisation de plusieurs fluorophores avec des sites d&#39;injection séparés peut permettre que cette méthode soit élargi. Par exemple, la comparaison de simple et d&#39;une double-étiquetage pourrait être utilisée pour distinguer les interneurones de neurones de projection suite à deux injections à deux points dans le circuit 32. De même, les traceurs rétrogrades peuvent être combinés avec d&#39;autres techniques d&#39;imagerie avancées, telles que l&#39;imagerie à deux photons, comme l&#39;a fait récemment en diamant mandarin Haute centre Vocal (HVc) 32. En outre, des régions d&#39;enregistrement sont limités à ceux près de la surface lors de l&#39;utilisation des microscopes à épifluorescence, car nous n&#39;étions en mesure de résoudre 1 &amp;mu, les structures de m dans les 30 premiers um de tissu. Toutefois, cette profondeur pourrait être étendu grâce à l&#39;utilisation d&#39;autres techniques de microscopie, telles que la microscopie à deux photons 33 ou objectif couplé microscopie d&#39;éclairage planaire 34. Dans l&#39;ensemble, cette technique représente un progrès important dans l&#39;étude des circuits neuronaux in vivo, car il peut être utilisé pour enregistrer des neurones simples dans de nombreux circuits dans une variété de systèmes modèles - y compris ceux qui sont relativement inaccessibles.

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 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Tricaine Methanesulfonate</td>
 <td>Sigma-Aldrich</td>
 <td>E10521</td>
 <td>MS-222</td>
 </tr>
 <tr>
 <td>Gallamine Triethiodide </td>
 <td>Sigma-Aldrich</td>
 <td>G8134</td>
 <td>Flaxedil</td>
 </tr>
 <tr>
 <td>Lidocaine Hydrochloride</td>
 <td>Sigma-Aldrich</td>
 <td>L5647</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Hickman's Ringer Solution </td>
 <td>NA</td>
 <td>NA</td>
 <td>NaCl (6.48 g/l), KCl (0.15 g/l), CaCl2&bull;2H20 (0.29 g/l), MgSO4 (0.12 g/l), NaHCO3 (0.084 g/l), NaH2PO4 (0.06 g/l)</td>
 </tr>
 <tr>
 <td>Peristaltic Pump</td>
 <td>Gilson or Rainin</td>
 <td>Minipulse 3 Model 312; RP-1</td>
 <td>10.0 &plusmn; 1.0 rpm, Alternatively, a gravity-fed line with a flow-meter</td>
 </tr>
 <tr>
 <td>Dissection Microscope </td>
 <td>Nikon</td>
 <td>SM2645</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Super Glue</td>
 <td>Super Glue Corporation</td>
 <td>SGH2</td>
 <td>2g tube</td>
 </tr>
 <tr>
 <td>Omni Drill 35</td>
 <td>World Precision Instruments</td>
 <td>503-598</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ball Mill Carbide Drill Bit #1/4</td>
 <td>World Precision Instruments</td>
 <td>501860</td>
 <td>0.19 DIA (bit diameter = 0.48 mm) </td>
 </tr>
 <tr>
 <td>Low Temp Cautery Kit</td>
 <td>World Precision Instruments</td>
 <td>500391</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Manual Micromanipulator</td>
 <td>World Precision Instruments</td>
 <td>M3301R</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dextran Conjugated Alexa Fluor 568</td>
 <td>Invitrogen</td>
 <td>D-22912</td>
 <td>2mM concentration; 10,000MW The choice of dye wavelength should be selected to match the microscope filter settings</td>
 </tr>
 <tr>
 <td>Epifluorescent Microscope</td>
 <td>Nikon </td>
 <td>E600 FN</td>
 <td>A remote focus accessory is helpful for fine focus TRITC filter - or appropriate filter to match focal planes for different dye wavelengths</td>
 </tr>
 <tr>
 <td>Low Power Objective</td>
 <td>Nikon</td>
 <td>93182</td>
 <td>CFI Plan Achromat Series, 4X N.A. 0.1, W.D. 30mm</td>
 </tr>
 <tr>
 <td>High Power Objective</td>
 <td>Nikon</td>
 <td>93148</td>
 <td>CFI Fluor Series, 40X WI N.A. 0.8, W.D. 2.0 mm</td>
 </tr>
 <tr>
 <td>White Light Source</td>
 <td>Dolan-Jenner</td>
 <td>Model 190</td>
 <td>Fiber Optic Illuminator</td>
 </tr>
 <tr>
 <td>Fluorescent Light Source </td>
 <td>Lumen Dynamics</td>
 <td>X-Cite 120Q</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Low-light Level Camera</td>
 <td>Photometrics</td>
 <td>CoolSnap ES</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Digital Manometer</td>
 <td>Omega Engineering</td>
 <td>HHP-201</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Motorized Micromanipulator</td>
 <td>Sutter Instruments</td>
 <td>MP-285</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Headstage</td>
 <td>Molecular Devices</td>
 <td>CV-7B</td>
 <td>Axon Instruments</td>
 </tr>
 <tr>
 <td>Amplifier</td>
 <td>Molecular Devices</td>
 <td>MultiClamp 700B</td>
 <td>Axon Instruments</td>
 </tr>
 <tr>
 <td>Data Acquisition System</td>
 <td>Molecular Devices</td>
 <td>Digidata 1322A</td>
 <td>Axon Instruments</td>
 </tr>
 <tr>
 <td>Isolated Pulse Stimulator</td>
 <td>A-M Systems</td>
 <td>2100</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Flaming/Brown Micropipette Puller</td>
 <td>Sutter Instruments</td>
 <td>P-97</td>
 <td>Program settings for our application: Heat: ramp + 1, Pull: '0', Velocity: 60, Time: 90 Box filament</td>
 </tr>
 <tr>
 <td>Pipette Glass</td>
 <td>World Precision Instruments</td>
 <td>1B100F-4</td>
 <td>Borosilicate capillary glass with filament (1 mm OD, 0.58 mm ID) </td>
 </tr>
 </tbody>
</table>

retrograde fluorescent labeling allows for targeted extracellular single-unit recording from identified neurons in vivo

L&#39;objectif global de cette méthode consiste à enregistrer les réponses-ensemble d&#39;une population identifiée de neurones. Dans enregistrements électrophysiologiques in vivo des neurones individuels sont essentielles pour comprendre comment les circuits neuronaux fonctionnent dans des conditions naturelles. Traditionnellement, ces enregistrements ont été effectués «en aveugle», ce qui signifie l&#39;identité de la cellule enregistrée est inconnu au début de l&#39;enregistrement. Identité cellulaire peut être déterminé par la suite via intracellulaire 1, 2 ou juxtacellulaire lâche-patch 3 iontophorèse de colorant, mais ces enregistrements ne peut pas être pré-destiné aux neurones spécifiques dans les régions où les types de cellules fonctionnellement hétérogènes. Les protéines fluorescentes peuvent être exprimés de manière spécifique du type cellulaire permettant visuellement guidé par électrophysiologie unicellulaire 4-6. Cependant, il ya beaucoup de systèmes modèles pour lesquels ces outils génétiques ne sont pas disponibles. Même dans des systèmes modèles génétiquement accessibles, le désiré propromoteur peut être inconnu ou neurones génétiquement homogènes peut avoir différents modèles de projection. De même, les vecteurs viraux ont été utilisés pour étiqueter les sous-groupes spécifiques de neurones de projection 7, mais l&#39;utilisation de cette méthode est limitée par la toxicité et le manque de spécificité trans-synaptique. Ainsi, les techniques supplémentaires qui offrent des pré-visualisation spécifique pour enregistrer des neurones individuels identifiés in vivo sont nécessaires. Pré-visualisation du neurone cible est particulièrement utile pour des conditions d&#39;enregistrement difficiles, pour lesquels les enregistrements mono-cellulaires classiques sont souvent excessivement difficile 8-11. La nouvelle technique décrite dans cet article utilise le transport rétrograde d&#39;un colorant fluorescent appliqué en utilisant des aiguilles de tungstène pour étiqueter rapidement et sélectivement un sous-ensemble spécifique de cellules dans une région particulière du cerveau en fonction de leurs projections axonales uniques, offrant ainsi un repère visuel pour obtenir des enregistrements électrophysiologiques ciblées à partir de neurones identifiés dans un circuit intact withina vertébré CNS. Le roman progrès le plus significatif de notre méthode est l&#39;utilisation d&#39;un marquage fluorescent pour cibler des cellules spécifiques dans un système de modèle accessible non génétiquement. Poissons faiblement électriques sont un excellent modèle pour l&#39;étude des circuits neuronaux en éveil, se comportant 12 animaux. Nous avons utilisé cette technique pour étudier le traitement sensoriel par des «petites cellules» dans le noyau exterolateral antérieure (ELA) de poissons mormyrid faiblement électrique. «Les petites cellules&quot; sont suppose être temps neurones de comparaison importants pour détecter des différences milliseconde dans les temps d&#39;arrivée des pointes présynaptiques 13. Cependant, les caractéristiques anatomiques telles que la myéline dense, engloutissant synapses et les organismes à petites cellules ont rendu extrêmement difficile d&#39;enregistrer à partir de ces cellules en utilisant des méthodes traditionnelles 11, 14. Nous démontrons ici que notre méthode de nouveaux marqueurs de manière sélective ces cellules dans 28% des préparations, permettant des enregistrements, fiables et robustes et caracrisation des réponses à la stimulation électrosensoriel.

Watch this Scientific Journal Video about Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo at JoVE.com

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Transport rétrograde de colorant fluorescent marque une sous-population de neurones en fonction de projection anatomique. Axones marqués peuvent être ciblés visuellement<em&gt; In vivo</em&gt;, Permettant l&#39;enregistrement extracellulaire axones identifiés. Cette technique facilite l&#39;enregistrement lorsque les neurones ne peuvent pas être étiquetés par manipulation génétique ou sont difficiles à isoler en utilisant «aveugle»<em&gt; In vivo</em&gt; Approches.

Marquage fluorescent rétrograde Permet ciblée extracellulaire seule unité d&#39;enregistrement de neurones identifiés<em&gt; In vivo</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

retrograde-fluorescent-labeling-allows-for-targeted-extracellular

Research

JoVE Journal

Neuroscience

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

19.8K vues.  Washington University in St. Louis. Transport rétrograde de colorant fluorescent marque une sous-population de neurones en fonction de projection anatomique. Axones marqués peuvent être ciblés visuellement In vivo, Permettant l'enregistrement extracellulaire axones identifiés. Cette technique facilite l'enregistrement lorsque les neurones ne peuvent pas être étiquetés par manipulation génétique ou sont difficiles à isoler en utilisant «aveugle» In vivo Appr...

Vidéo: Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Dual Somatic Recordings from Gonadotropin-Releasing Hormone (GnRH) Neurons Identified by Green Fluorescent Protein (GFP) in Hypothalamic Slices

Gonadotropin-Releasing Hormone (GnRH) is a small neuropeptide that regulates pituitary release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). These gonadotropins are essential for the regulation of reproductive function. The GnRH-containing neurons are distributed diffusely throughout the hypothalamus and project to the median eminence where they release GnRH from their axon terminals into the hypophysiotropic portal system (1). In the portal capillaries, GnRH travels to the anterior pituitary gland to stimulate release of gonadotropins into systemic circulation. GnRH release is not continuous but rather occurs in episodic pulses. It is well established that the intermittent manner of GnRH release is essential for reproduction (2, 3).
Coordination of activity of multiple GnRH neurons probably underlies GnRH pulses. Total peptide content in GnRH neurons is approximately 1.0 pg/cell (4), of which 30% likely comprises the releasable pool. Levels of GnRH during a pulse (5, 6), suggest multiple GnRH neurons are probably involved in neurosecretion. Likewise, single unit activity extracted from hypothalamic multi-unit recordings during LH release indicates changes in activity of multiple neurons (7). The electrodes with recorded activity during LH pulses are associated with either GnRH somata or fibers (8). Therefore, at least some of this activity arises from GnRH neurons.
The mechanisms that result in synchronized firing in hypothalamic GnRH neurons are unknown. Elucidating the mechanisms that coordinate firing in GnRH neurons is a complex problem. First, the GnRH neurons are relatively few in number. In rodents, there are 800-2500 GnRH neurons. It is not clear that all GnRH neurons are involved in episodic GnRH release. Moreover, GnRH neurons are diffusely distributed (1). This has complicated our understanding of coordination of firing and has made many technical approaches intractable. We have optimized loose cell-attached recordings in current-clamp mode for the direct detection of action potentials and developed a recording approach that allows for simultaneous recordings from pairs of GnRH neurons.

Dual Somatic Recordings from Gonadotropin-Releasing Hormone GnRH Neurons Identified by Green Fluorescent Protein GFP in Hypothalamic Slices

Activity in neuronal systems often requires synchronous action potential discharges from neurons within a specific population. For example, pulses of gonadotropin-releasing hormone (GnRH) likely require coordinated activity between GnRH neurons. We present our methodological approach for reliably obtaining simultaneous electrophysiological recordings from the diffusely distributed GnRH neurons.

Double Recordings somatiques de la gonadolibérine (GnRH) neurones identifiés par Green Fluorescent Protein (GFP) en tranches hypothalamiques

Gonadotropin-Releasing Hormone (GnRH) is a small neuropeptide that regulates pituitary release of luteinizing hormone (LH) and follicle-stimulating ...

Recherche

Neurosciences

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic nerve. In vivo recordings of ganglion cell spike trains in several animal models have revealed much of what is known about how the early visual system processes and encodes visual information. However, such recordings have been rare in one of the most common animal models, the rat, possibly owing to difficulty in detecting spikes fired by small diameter axons. The many retinal disease models involving rats motivate a need for characterizing the functional properties of ganglion cells without disturbing the eye, as with intraocular or in vitro recordings. Here, we demonstrate a method for recording ganglion cell spike trains from the optic chiasm of the anesthetized rat. We first show how to fabricate tungsten-in-glass electrodes that can pick up electrical activity from single ganglion cell axons in rat. The electrodes outperform all commercial ones that we have tried. We then illustrate our custom-designed stereotaxic system for in vivo visual neurophysiology experiments and our procedures for animal preparation and reliable and stable electrode placement in the optic chiasm.

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Retinal ganglion cells transmit visual information from the eye to the brain with sequences of action potentials. Here, we demonstrate how to record the action potentials of single ganglion cells in vivo from anesthetized rats.

Une seule unité In vivo Enregistrements à partir du chiasma optique du Rat

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic ...

Surgical Implantation of Chronic Neural Electrodes for Recording Single Unit Activity and Electrocorticographic Signals

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery. Here we provide useful information for surgeons who are learning the process of implanting electrode systems. We demonstrate the implantation procedure of both a penetrating and a surface electrode. The surgical process is described from start to finish, including detailed descriptions of each step throughout the procedure. It should also be noted that this video guide is focused towards procedures conducted in rodent models and other small animal models. Modifications of the described procedures are feasible for other animal models.

We provide useful information for surgeons who are learning the process of implanting chronic neural recording electrodes. Techniques for both penetrating and surface electrode systems are described in a rodent animal model.

L&#39;implantation chirurgicale d&#39;électrodes neurales chroniques pour l&#39;enregistrement d&#39;activité Unité unique et Signaux Electrocorticographic

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery.  Here we provide useful information for ...

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

Enregistrement simultané des signaux calciques de neurones identifiés et le comportement alimentaire des Drosophila melanogaster</em

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

Retrograde Loading of Nerves, Tracts, and Spinal Roots with Fluorescent Dyes

Retrograde labeling of neurons is a standard anatomical method1,2 that has also been used to load calcium and voltage-sensitive dyes into neurons3-6. Generally, the dyes are applied as solid crystals or by local pressure injection using glass pipettes. However, this can result in dilution of the dye and reduced labeling intensity, particularly when several hours are required for dye diffusion. Here we demonstrate a simple and low-cost technique for introducing fluorescent and ion-sensitive dyes into neurons using a polyethylene suction pipette filled with the dye solution. This method offers a reliable way for maintaining a high concentration of the dye in contact with axons throughout the loading procedure.

We describe a simple and low cost technique for introducing high concentration of fluorescent and calcium-sensitive dyes into neurons or any neuronal tract using a polyethylene suction pipette.

Chargement rétrograde des nerfs, des tracts, et les racines spinales avec des colorants fluorescents

Retrograde labeling of neurons is a standard anatomical method1,2 that has also been used to load calcium and voltage-sensitive dyes into neurons3-6. ...

Recording Electrical Activity from Identified Neurons in the Intact Brain of Transgenic Fish

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be performed in an intact brain preparation where the neural circuitry of the CNS remains intact. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with green fluorescent protein for identification in the intact brain. Fish have multiple populations of GnRH neurons, and their functions are dependent on their location in the brain and the GnRH gene that they express1 . We have focused our demonstration on GnRH3 neurons located in the terminal nerves (TN) associated with the olfactory bulbs using the intact brain of transgenic medaka fish (Figure 1B and C). Studies suggest that medaka TN-GnRH3 neurons are neuromodulatory, acting as a transmitter of information from the external environment to the central nervous system; they do not play a direct role in regulating pituitary-gonadal functions, as do the well-known hypothalamic GnRH1 neurons2, 3 .The tonic pattern of spontaneous action potential firing of TN-GnRH3 neurons is an intrinsic property4-6, the frequency of which is modulated by visual cues from conspecifics2 and the neuropeptide kisspeptin 15. In this video, we use a stable line of transgenic medaka in which TN-GnRH3 neurons express a transgene containing the promoter region of Gnrh3 linked to enhanced green fluorescent protein7 to show you how to identify neurons and monitor their electrical activity in the whole brain preparation6.

In this video, we will demonstrate how to record electrical activity from identified single neurons in a whole brain preparation, which preserves complex neural circuits. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with a fluorescent protein for identification in the intact brain preparation.

Enregistrement de l&#39;activité électrique de neurones identifiés dans le cerveau intact des poissons transgéniques

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be ...

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of neurophysiology, neuroethology, and developmental biology. The goal of this report is to consolidate experimental techniques from the leech system into a single article that will be of use to physiologists with expertise in other nervous system preparations, or to biology students with little or no electrophysiology experience. We demonstrate how to dissect the leech for recording intracellularly from identified neural circuits in the ganglion. Next we show how individual cells of known function can be removed from the ganglion to be cultured in a Petri dish, and how to record from those neurons in culture. Then we demonstrate how to prepare a patch of innervated skin to be used for mapping sensory or motor fields. These leech preparations are still widely used to address basic electrical properties of neural networks, behavior, synaptogenesis, and development. They are also an appropriate training module for neuroscience or physiology teaching laboratories.

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

This article describes three nervous system preparations using leeches: intracellular recording from neurons in ventral ganglia, culturing neurons from ventral ganglia, and recording from a patch of innervated skin to map sensory fields.

Intracellulaire enregistrement, le mappage des champs sensorielle, et la culture de neurones identifiés dans le Leech, Hirudo medicinalis</i

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of ...

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

Marquage rétrograde des cellules ganglionnaires de la rétine à l&#39;âge adulte de poisson zèbre avec des colorants fluorescents

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral cortex. We here share our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex using a method developed by Lima and colleagues1. Recordings are performed in mice expressing Channelrhodopsin-2 (ChR2) in specific neuronal subpopulations. Members of the population are identified by their response to a brief flash of blue light. This technique &#8211; termed &#8220;PINP&#8221;, or Photostimulation-assisted Identification of Neuronal Populations &#8211; can be implemented with standard extracellular recording equipment. It can serve as an inexpensive and accessible alternative to calcium imaging or visually-guided patching, for the purpose of targeting extracellular recordings to genetically-identified cells. Here we provide a set of guidelines for optimizing the method in everyday practice. We refined our strategy specifically for targeting parvalbumin-positive (PV+) cells, but have found that it works for other interneuron types as well, such as somatostatin-expressing (SOM+) and calretinin-expressing (CR+) interneurons.

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

Here we describe our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. Neurons expressing ChR2 are identified by their response to blue light. The method uses standard extracellular recording equipment, and serves as an inexpensive alternative to calcium imaging or visually-guided patching.

Un guide pour<em&gt; In vivo</em&gt; Simple unité d&#39;enregistrement de Optogenetically identifié corticale inhibitrice Interneurones

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral ...

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

Perforé patch-clamp enregistrement des souris neurones sensoriels olfactifs dans neuroépithélium Intact: Analyse fonctionnelle des neurones exprimant un récepteur odorant Identifié

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...