Nous remercions Manfred Heckmann pour des discussions utiles lors de l&#39;établissement de la méthode. De plus, nous remercions Claudia Sommer pour l&#39;assistance technique et Frank Schubert pour support la production de la vidéo. Le travail a été soutenu par le Ministère fédéral de l&#39;Education et de la Recherche (BMBF), Allemagne, FKZ: 01EO1002 et le Centre interdisciplinaire pour la recherche clinique (IZKF) de l&#39;hôpital universitaire de Iéna.

Toutes les procédures expérimentales mentionnées dans le protocole suivant a été approuvé par les autorités de l&#39;Etat de Thuringe (Thüringer LANDESAMT für Verbraucherschutz, Reg.-Nr. 02-044/12). Une. Préparatifs pour l&#39;expérience <ol><li> La fabrication des électrodes d&#39;aspiration <ol><li> Tirez une micropipette en utilisant un capillaire en verre borosilicate standard avec un extracteur micropipette, par exemple une électrode de raccordement standard. </li><li> Frein l&#39;électrode à la pointe diamètre de 0,5 à 1 mm (légèrement plus grand que le diamètre des racines dorsales) à l&#39;aide d&#39;un fichier de diamant. </li><li> vernis à chaleur de la pointe afin de ne pas nuire à la racine dorsale quand il est aspiré po Une torche de laboratoire standard convenir. </li><li> Monter le filament de verre sur un support d&#39;électrode connecté à une seringue à travers lequel la pression négative peut être appliquée. </li></ol></li><li> Préparation des solutions <ol><li> anesthésie par injection chez la souris: Mélanger 0,75 ml de kétamine 10%, 0,24 ml de xylazine 2%e 5 ml saline à 0,9%. 10 ul / g de poids corporel (BW) de la solution sera injectée par voie intrapéritonéale (ip). </li><li> Liquide céphalo-rachidien artificiel (aCSF): Diluer dans de l&#39;eau distillée deux fois les sels suivants (en mm): NaCl 134; KCl 3; KH 2 PO 4 1,25; MgSO 4 • H 2 O 2; NaHCO 3 25; CaCl2 2, D- glucose 10. Ajouter H 2 O 2 à une concentration finale de 0,003%. Utiliser toujours solution fraîchement préparée. </li></ol></li><li> Préparation de la configuration d&#39;enregistrement (figure 2) <ol><li> Connectez l&#39;amplificateur à une interface PC pour numériser les données. </li><li> Utilisez un programme basé sur PC ou une minuterie analogique pour déclencher un stimulateur de créneau et d&#39;acquisition de données sur le disque dur du PC. </li><li> Utilisez silverwires chlorée connectés au porte-électrodes de verre standards pour la stimulation et l&#39;enregistrement. </li><li> Assemblez trois manipulateurs pour la stimulation, l&#39;enregistrement et l&#39;électrode de référence respectivement autour d&#39;un stèreotactic trame pour les souris, de sorte que toutes les électrodes ont accès à la moelle épinière préparé par la suite. </li><li> Connectez tuyaux et des seringues pour les porte-électrodes à être pomme pour appliquer une pression négative. </li></ol></li></ol> 2. Observations générales pour l&#39;expérimentation animale et la préparation des animaux pour la procédure d&#39;enregistrement <ol><li> Effectuez toutes les expériences utilisant des souris selon les directives du comité de protection des animaux et l&#39;utilisation respective. Effectuer toutes les interventions chirurgicales et les enregistrements sous anesthésie profonde assurant que la souffrance des animaux est réduite au minimum. </li><li> Anesthésier l&#39;animal par injection intrapéritonéale de kétamine / xylazine (125 mg et 8 mg par g BW de kétamine et de xylazine, respectivement, 10 ul / g BW de la solution préparée comme décrit ci-dessus). Si nécessaire pendant les enregistrements à long terme, des injections supplémentaires peuvent être faites ip ou im Remarque: répétitives im injections de 0,05-0,1 ml de solution de kétamine / xylazine dans le haut des cuisses se sont révélés être adaptépour garder l&#39;animal en anesthésie profonde jusqu&#39;à 3 h. </li><li> Utilisez vétérinaire pommade sur les yeux pour prévenir la sécheresse tandis que sous anesthésie. </li><li> Fixer la tête de l&#39;animal dans un cadre stéréotaxique et d&#39;utiliser un coussin chauffant avec sonde rectale et boucle réflexe de contrôler la température du corps de l&#39;animal lors de l&#39;expérience. Fixation de la moelle épinière dans un cadre stéréotaxique n&#39;est pas nécessaire. </li><li> Avant de commencer la préparation, vérifiez la profondeur de la narcose par réflexe du clignement de paupières et secousses entre les orteils des souris. Réflexes devraient être abolies. </li><li> Ouvrez la peau le long de la ligne médiane au-dessus de la moelle épinière au niveau thoracique haut en bas la région lombaire pour obtenir un champ opérationnel clair à l&#39;aide d&#39;un scalpel. Ne pas utiliser des ciseaux pour faire des incisions cutanées. Perdre soigneusement la peau à partir du tissu sous-jacent. Au cours des étapes ultérieures de garder la plaie humide de 0,9% de solution saline. </li><li> Couper les tendons et les tissus conjonctifs de chaque côté des vertèbres lombaires à partir de niveaux thoraciques en utilisant un scalpel et ciseaux. </li&gt; <li> Retirez les apophyses épineuses et le reste du tissu conjonctif autour des vertèbres à l&#39;aide d&#39;une petite pince. </li><li> Casser délicatement vertèbres avec la pince à partir de niveaux lombaires (L4/L5) de sous un microscope à dissection. Shove la pointe de la pince dans l&#39;espace entre les vertèbres et la moelle épinière et soulever des morceaux d&#39;os en dehors. Ne pas nuire à la mère et éviter la pression de la moelle épinière. Les deux sont essentiels pour le succès. Gardez la moelle épinière humide durant toute la procédure. Procéder à des niveaux mi-thoraciques. </li></ol> 3. Séparation des racines dorsales et DRP enregistrement (Figure 2) <ol><li> Ouvrez la dure-mère soigneusement à l&#39;aide d&#39;une aiguille fine (30 G) avec une pointe recourbée. Utilisez aCSF pour humidifier à partir de maintenant. </li><li> Séparer les racines dorsales, autant que possible en utilisant le gabarit et couper deux racines adjacentes distale que possible. Vigoureuse traction sur les racines au cours de la séparation affecte le succès de l&#39;expérience. </li><li> Abaisser l&#39;électrode d&#39;aspiration vers le doRSAL racines. Ajouter autant aCSF à la moelle épinière que possible parce que le liquide permet d&#39;aspirer les racines dorsales. </li><li> Aspirer l&#39;extrémité d&#39;une racine dorsale coupée dans une pipettes en verre par application d&#39;une pression négative par l&#39;intermédiaire d&#39;une seringue. Si nécessaire, déplacer la racine dorsale en avant de l&#39;ouverture de l&#39;électrode à l&#39;aide d&#39;une aiguille fine. </li><li> Si la racine dorsale est sec à l&#39;intérieur de l&#39;électrode d&#39;aspiration, ajouter un peu de aCSF à la pointe tout en suçant soigneusement jusqu&#39;à ce que la pipette est suffisamment rempli aCSF. </li><li> Après la racine dorsale est aspiré, puis soulever la pointe de l&#39;électrode à partir de la moelle épinière. Veillez à ce que non &quot;pont d&#39;eau&quot; entre la pointe de la pipette et la moelle épinière de court-circuit l&#39;électrode d&#39;enregistrement / stimulation. </li><li> Répétez les étapes 3.3 à 3.6 pour une racine adjacente ipsilatéral. </li><li> Placez l&#39;électrode de référence aussi proche que possible de la racine dorsale et le garder humide en appliquant aCSF. </li><li> En établissant la configuration de l&#39;enregistrement, une racine est déjà choisi pour la stimulation, lal&#39;autre pour l&#39;enregistrement. Augmenter la tension par étapes lors de l&#39;enregistrement de la deuxième racine dorsale se fait en mode pince de courant. On peut remarquer une déviation courte vers le bas qui est suivie d&#39;une lente durable déviation, vers le haut représentant de la DRP. </li><li> Régler la tension de relance pour supramaximal niveaux et enregistrer plusieurs balayages (au moins 20 - 30 balayages / racine dorsale à 0,1 Hz, filtre entre 0,3 Hz à 3 kHz). </li><li> Enregistrez trains courts de trois stimuli (100 Hz) pour obtenir des informations sur la somme en fonction du temps de la DRP. </li><li> Mettez l&#39;enregistrement et le site de stimulation pour les enregistrements controlatérale supplémentaires. </li><li> Les animaux ne sont pas destinés à survivre à l&#39;opération. Sacrifier des animaux après le dernier enregistrement par décapitation tout en restant sous anesthésie profonde (kétamine / xylazine, voir ci-dessus, l&#39;étape 2.2). </li></ol> 4. Analyse des données <ol><li> Transférer des données à un programme d&#39;analyse (par exemple Sigma Plot, Igor Pro, ou MATLAB). </li><li> Calculer traces moyens frOM 20-30 balayages. </li><li> Prendre l&#39;amplitude maximale de la déviation de la tension (à partir de la ligne de base; DRP pic d&#39;amplitude) pour une analyse ultérieure (figure 3). </li><li> Calculer le rapport de l&#39;amplitude crête DRP après trois impulsions et une seule impulsion pour obtenir une mesure de la somme dépend des potentiels ultérieurs de temps. </li></ol>

<ol>
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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depolarization+of+central+terminals+of+Group+I+afferent+fibres+from+muscle.">Depolarization of central terminals of Group I afferent fibres from muscle.</a> J. Physiol. 160, 62-93 (1962).</li><li>Eccles, J. C., Schmidt, R., Willis, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+Studies+on+Presynaptic+Inhibition.">Pharmacological Studies on Presynaptic Inhibition.</a> J. Physiol. 168, 500-530 (1963).</li><li>Maxwell, D. J., Bannatyne, B. 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Enregistrements électrophysiologiques extra-et intracellulaires de l&#39;activité neuronale et potentiels synaptiques in vivo sont l&#39;état de l&#39;art des techniques pour enquêter sur les fonctions neuronales du système nerveux central et la physiopathologie. Spinal intégration est critique pour la fonction motrice, par exemple, les mouvements des membres et de la perception sensorielle multimodale. Inhibition présynaptique est un mécanisme essentiel dans ce processus de calcul assurer des ...

Inhibition présynaptique est l&#39;un des mécanismes inhibiteurs les plus puissants de la moelle épinière. Il inhibe potentiels post-synaptiques excitateurs (EPSPS) dans motoneurones monosynaptique excités sans modifier le potentiel de membrane post-synaptique et l&#39;excitabilité des motoneurones 1-3. Dépolarisation afférente primaire (PAD) induite par GABAergiques synapses axo-axonale sur des fibres présynaptiques sensorielles est le mécanisme sous-jacent 4-7 (voir aussi Figure1a). Ces synapses contiennent GABA A et GABA B-récepteurs GABA A (R et GABA B R). Activité GABA A R conduit à une augmentation de la conductance du chlorure PAD qui provoque en raison de la distribution des ions local. Cette dépolarisation bloque la propagation des potentiels d&#39;action dans les terminaisons axonales et réduit leur force conduisant à une diminution de Ca 2 + afflux et une réduction de la libération du transmetteur. L&#39;activation des récepteurs GABA B ne fait past contribuent au PAD mais conduit à une diminution de Ca 2 +-afflux améliorant ainsi l&#39;inhibition présynaptique. Bien que l&#39;activation de GABA A R semble être impliquée dans l&#39;inhibition à court terme, le GABA B R sont impliqués dans la modulation à long terme 8-10. En plus de GABA, qui représente la majeure partie de la MAP et l&#39;inhibition présynaptique, d&#39;autres systèmes de transmetteurs peuvent également moduler et à contribuer à ce mécanisme 11,12. Des changements pathologiques dans l&#39;inhibition présynaptique semblent être crucial dans plusieurs états pathologiques par exemple inflammation périphérique et la douleur neuropathique 13,14, ainsi que le traitement de la douleur centrale anormale 15, lésions de la moelle épinière 16, et les maladies du système nerveux central avec une hyperexcitabilité du moteur médiée par défaut la transmission GABAergique 17, 18. Ainsi, l&#39;estimation de l&#39;inhibition présynaptique est utile d&#39;étudier les conditions pathologiques expérimentales sur le niveau de la moelle épinière in vivo </em&gt;. PAD donne lieu à volumes menée potentiels fournissant une mesure directe de l&#39;inhibition présynaptique dans la moelle épinière. Ces potentiels sont appelés potentiels de racine dorsale (DRP), et peuvent être mesurées à partir de la colonne vertébrale racines cordon dorsales, après stimulation des racines dorsales adjacentes 7. Premières mesures de DRP ont été rapportés chez les chats et les grenouilles 19 et ont été intensément étudié chez les chats par Eccles, Schmidt, et d&#39;autres dans le début des années 1970 3,4,20,21. Alors que les enregistrements in vivo de DRP chez les chats et les rats 22 23 ont été largement utilisés, les mesures chez la souris ont été réalisées presque exclusivement dans les préparations isolées ex vivo de la moelle épinière 15,24. Ici, nous décrivons une méthode pour enregistrer DRP chez la souris in vivo anesthésié permettant une mesure directe de l&#39;inhibition pré-synaptique dans l&#39;organisme intact.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Glass tubing (inner diameter 1.16 mm)</td>
			<td>Science Products (Hofheim, Germany)</td>
			<td>GB200F-10</td>
			<td>Other glass tubing might also be suitable</td>
		</tr>
		<tr>
			<td>Superfusion solution (sterile, 0,9% NaCl)</td>
			<td>Braun Melsungen AG&#160;</td>
			<td>3570350</td>
			<td></td>
		</tr>
		<tr>
			<td>(Melsungen, Germany)</td>
		</tr>
		<tr>
			<td>Rompun 2% (Xylazine)</td>
			<td>Bayer Animal Health GmbH (Leverkusen, Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamin 10%</td>
			<td>Medistar GmbH (Ascheberg, Germany)</td>
			<td>KETAMIN 10%</td>
			<td></td>
		</tr>
		<tr>
			<td>30G micro needle/ Sterican</td>
			<td>Braun Melsungen AG&#160;</td>
			<td>4656300</td>
			<td></td>
		</tr>
		<tr>
			<td>(Melsungen, Geramny)</td>
		</tr>
		<tr>
			<td>Salts for aCSF</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>Diverse</td>
			<td></td>
		</tr>
		<tr>
			<td>S88 Dual Output Square Pulse</td>
			<td>Grass Technologies (Warwick, USA)</td>
			<td>S88X</td>
			<td></td>
		</tr>
		<tr>
			<td>Stimulator</td>
		</tr>
		<tr>
			<td>SIU5 RF Transformer Isolation Unit</td>
			<td>Grass Technologies (Warwick, USA)</td>
			<td>SIU-V</td>
			<td></td>
		</tr>
		<tr>
			<td>InstruTECH LIH 8+8</td>
			<td>HEKA (Lambrecht, Deutschland)</td>
			<td>LIH 8+8 + Patchmaster software</td>
			<td></td>
		</tr>
		<tr>
			<td>Data acquisition&#160;</td>
		</tr>
		<tr>
			<td>Universal amplifier</td>
			<td>npi (Tamm, Deutschland)</td>
			<td>ELC-03X</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter Instruments (Novato, USA)</td>
			<td>P-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Olympus (Tokyo, Japan)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Sutter Instruments (Novato, USA)</td>
			<td>MPC-200/MPC-325</td>
			<td>Mechanical micromanipulators also possible</td>
		</tr>
		<tr>
			<td>Homeothermic Blanket System</td>
			<td>Stoelting (Wood Dale, USA)</td>
			<td>50300V</td>
			<td></td>
		</tr>
		<tr>
			<td>Intra-/extracellular recording electrode holder</td>
			<td>Harvard Apparatus (Holliston, USA)</td>
			<td>641227</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring spinal presynaptic inhibition in mice by dorsal root potential recording in vivo

Inhibition présynaptique est l&#39;un des mécanismes inhibiteurs les plus puissants de la moelle épinière. Le mécanisme physiologique sous-jacent est une dépolarisation des fibres afférentes primaires médiées par GABAergiques synapses axo-axonale (dépolarisation afférente primaire). La force de la dépolarisation afférente primaire peut être mesurée par l&#39;enregistrement des potentiels de volume menée à la racine dorsale (potentiels de la racine dorsale, DRP). Les modifications pathologiques de l&#39;inhibition présynaptique sont cruciales dans la centrale anormale de certaines conditions de la douleur et dans certains troubles de moteur hyperexcitabilité. Ici, nous décrivons un procédé d&#39;enregistrement DRP in vivo chez la souris. La préparation de la moelle épinière racines dorsales chez l&#39;animal anesthésié et la procédure d&#39;enregistrement à l&#39;aide des électrodes d&#39;aspiration sont expliqués. Cette méthode permet de mesurer GABAergique DRP et ainsi estimer inhibition présynaptique de la moelle chez la souris vivante. En combinaison avec des modèles de souris transgéniques, l&#39;enregistrement peut se DRPrve comme un outil puissant pour étudier la maladie associée à la moelle physiopathologie. enregistrement in vivo a plusieurs avantages par rapport aux ex vivo des préparations isolées de la moelle épinière, par exemple la possibilité d&#39;enregistrer ou de manipulation de réseaux supraspinales et induction de DRP par la stimulation des nerfs périphériques simultanément.

Traces DRP typiques sont présentés dans la figure 3. L&#39;artefact de stimulation importante est généralement suivie d&#39;une déviation vers le bas à court. Par la suite, une longue durée de déviation vers le haut lent, représentant le DRP se distingue nettement. Dans un sous-ensemble d&#39;enregistrements, dorsales réflexes profondes sont visibles sous forme de petites pointes sur le dessus de la DRP. Chez les souris de type sauvage normales, réflexes racines dorsales apparaissent le plus ...

Watch this Scientific Journal Video about Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo at JoVE.com

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

GABAergique inhibition présynaptique est un mécanisme inhibiteur puissant de la moelle épinière et le moteur important pour l&#39;intégration du signal sensoriel dans les réseaux de la moelle épinière. Sous-jacent afférent primaire dépolarisation peut être mesurée par l&#39;enregistrement des potentiels de la racine dorsale (DRP). Ici, nous démontrons une méthode d&#39;enregistrement de DRP in vivo chez la souris.

Mesure vertébrale présynaptique inhibition chez la souris par la racine dorsale enregistrement potentiel<em&gt; In Vivo</em

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of ...

measuring-spinal-presynaptic-inhibition-mice-dorsal-root-potential

Research

JoVE Journal

Neuroscience

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

13.9K vues.  Jena University Hospital, Jena, Germany.  GABAergique inhibition présynaptique est un mécanisme inhibiteur puissant de la moelle épinière et le moteur important pour l'intégration du signal sensoriel dans les réseaux de la moelle épinière. Sous-jacent afférent primaire dépolarisation peut être mesurée par l'enregistrement des potentiels de la racine dorsale (DRP). Ici, nous démontrons une méthode d'enregistrement de DRP in vivo chez la souris. 

Vidéo: Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

La transfection des cellules ganglionnaires de la rétine de la souris par In vivo L&#39;électroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

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Neurosciences

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

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

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

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

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 ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

Simultané<em&gt; Ex vivo</em&gt; Tests fonctionnels de deux rétines par<em&gt; In vivo</em&gt; Système Électrorétinogramme

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Polygraphic Recording Procedure for Measuring Sleep in Mice

Recording of the epidural electroencephalogram (EEG) and electromyogram (EMG) in small animals, like mice and rats, has been pivotal to study the homeodynamics and circuitry of sleep-wake regulation. In many laboratories, a cable-based sleep recording system is used to monitor the EEG and EMG in freely behaving mice in combination with computer software for automatic scoring of the vigilance states on the basis of power spectrum analysis of EEG data. A description of this system is detailed herein. Steel screws are implanted over the frontal cortical area and the parietal area of 1 hemisphere for monitoring EEG signals. In addition, EMG activity is monitored by the bilateral placement of wires in both neck muscles. Non-rapid eye movement (Non-REM; NREM) sleep is characterized by large, slow brain waves with delta activity below 4 Hz in the EEG, whereas a shift from low-frequency delta activity to a rapid low-voltage EEG in the theta range between 6 and 10 Hz can be observed at the transition from NREM to REM sleep. By contrast, wakefulness is identified by low- to moderate-voltage brain waves in the EEG trace and significant EMG activity.

The recording of electroencephalogram (EEG) and electromyogram (EMG) in freely behaving mice is a critical step to correlate behavior and physiology with sleep and wakefulness. The experimental protocol described herein provides a cable-based system for acquiring EEG and EMG recordings in mice.

Procédure d&#39;enregistrement polygraphique pour la mesure du sommeil chez la souris

Recording of the epidural electroencephalogram (EEG) and electromyogram (EMG) in small animals, like mice and rats, has been pivotal to study the ...

Hydraulic Extrusion of the Spinal Cord and Isolation of Dorsal Root Ganglia in Rodents

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may result in damage to the spinal cord caused by the dissection process. Here, we show how the spinal cord can be extruded using hydraulic pressure. Handling time is significantly reduced to only a few minutes, likely decreasing protein damage. The low risk of damage to the spinal cord tissue improves subsequent immunohistochemical analysis. By performing hydraulic spinal cord extrusion instead of traditional laminectomy, the rodents can further be used for DRG isolation, thereby lowering the number of animals and allowing analysis across tissues from the same rodent. We demonstrate a consistent method to identify and isolate the DRGs according to their localization relative to the costae. It is, however, important to adjust this method to the particular animal used, as the number of spinal cord segments, both thoracic and lumbar, may vary according to animal type and strain. In addition, we illustrate further processing examples of the isolated tissues.

Here, we present a protocol for hydraulic extrusion of the spinal cord as well as identification and isolation of specific dorsal root ganglia (DRGs) in the same rodent. Compared to standard spinal cord isolation methods, this method is significantly faster and reduces the risk of tissue damage.

Extrusion hydraulique de la moelle épinière et isolement de ganglions rachidiens en Rongeurs

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may ...

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

Racine Dorsale Ganglion Injection et Racine Dorsale Crush blessures comme modèle pour la régénération sensorielle Axon

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

Laminotomy for Lumbar Dorsal Root Ganglion Access and Injection in Swine

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive targets for injection of novel therapeutics aimed at treating chronic pain. In small animal models, laminectomy has been used to facilitate DRG injection because it involves surgical removal of the vertebral bone surrounding each DRG. We demonstrate a technique for intraganglionic injection of lumbar DRG in a large animal species, namely, swine. Laminotomy is performed to allow direct access to DRG using standard neurosurgical techniques, instruments, and materials. Compared with more extensive bone removal via laminectomy, we implement laminotomy to conserve spinal anatomy while achieving sufficient DRG access. Intraoperative progress of DRG injection is monitored using a non-toxic dye. Following euthanasia on post-operative day 21, the success of injection is determined by histology for intraganglionic distribution of 4&#39;,6-diamidino-2-phenylindole (DAPI). We inject a biologically inactive solution to demonstrate the protocol. This method could be applied in future preclinical studies to target therapeutic solutions to DRG. Our methodology should facilitate testing the translatability of intraganglionic small animal paradigms in a large animal species. Additionally, this protocol may serve as a key resource for those planning preclinical studies of DRG injection in swine.

We describe a method for laminotomy in swine that provides access to lumbar dorsal root ganglia (DRG) for intraganglionic injection. Injection progress is monitored intraoperatively and histologically confirmed up to 21 days after surgery. This protocol could be used for future preclinical studies involving DRG injection.

Laminotomy pour accès de Ganglion de la racine dorsale lombaire et Injection chez le porc

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive ...

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the dorsal root ganglion (DRGs) extends a single axon with two branches. One branch goes to the peripheral nerve (peripheral branch), and the other branch enters the spinal cord through the dorsal root (central branch). After the neural injury, the peripheral branch regenerates robustly whereas the central branch does not regenerate. Due to the high regenerative capacity, sensory axon regeneration has been widely used as a model system to study mammalian axon regeneration in both the peripheral nervous system (PNS) and the central nervous system (CNS). Here, we describe a previously established approach protocol to manipulate gene expression in mature sensory neurons in vivo via electroporation. Based on transfection with plasmids or small RNA oligos (siRNAs or microRNAs), the approach allows for both loss- and gain-of-function experiments to study the roles of genes-of-interests or microRNAs in regulation of axon regeneration in vivo. In addition, the manipulation of gene expression in vivo can be controlled both spatially and temporally within a relatively short time course. This model system provides a unique tool to investigate the molecular mechanisms by which mammalian axon regeneration is regulated in vivo.

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an effective approach to deliver genes of interests into cells. By applying this approach in vivo on the neurons of adult mouse dorsal root ganglion (DRG), we describe a model to study axon regeneration in vivo.

Chargée de l’enquête la régénération axonale mammifères : In Vivo électroporation du Ganglion de la racine dorsale de souris adulte

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...

Use of In Vivo Single-fiber Recording and Intact Dorsal Root Ganglion with Attached Sciatic Nerve to Examine the Mechanism of Conduction Failure

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for nerve fibers in the central and peripheral nervous systems. This method is particularly applicable to dorsal root ganglia (DRG), which are primary sensory neurons that exhibit a pseudo-unipolar structure of nervous processes. The patterns and features of the action potentials passed along axons are recordable in these neurons. The present study uses in vivo single-fiber recordings to observe the conduction failure of sciatic nerves in complete Freund&#8217;s adjuvant (CFA)-treated rats. As the underlying mechanism cannot be studied using in vivo single-fiber recordings, patch-clamp-recordings of DRG neurons are performed on preparations of intact DRG with the attached sciatic nerve. These recordings reveal a positive correlation between conduction failure and the rising slope of the after-hyperpolarization potential (AHP) of DRG neurons in CFA-treated animals. The protocol for in vivo single fiber-recordings allows the classification of nerve fibers via the measurement of conduction velocity and monitoring of abnormal conditions in nerve fibers in certain diseases. Intact DRG with attached peripheral nerve allows observation of the activity of DRG neurons in most physiological conditions. Conclusively, single-fiber recording combined with electrophysiological recording of intact DRGs is an effective method to examine the role of conduction failure during the analgesic process.

Single-fiber recording is an effective electrophysiological technique that is applicable to the central and peripheral nervous systems. Along with the preparation of intact DRG with the attached sciatic nerve, the mechanism of conduction failure is examined. Both protocols improve the understanding of the peripheral nervous system&#39;s relationship with pain.

Utilisation de l'enregistrement in vivo à fibre unique et du ganglion de racine dorsal intact avec le nerf sciatique attaché pour examiner le mécanisme de l'échec de conduction

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for ...

Rapid Isolation of Dorsal Root Ganglion Macrophages

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after peripheral nerve injury. Peripheral monocytic cells, including macrophages, are known to respond to a tissue injury through phagocytosis, antigen presentation, and cytokine release. Emerging evidence has implicated the contribution of dorsal root ganglia macrophages to neuropathic pain development and axonal repair in the context of nerve injury. Rapidly phenotyping (or &#8220;rapid isolation of&#8221;) the response of dorsal root ganglia macrophages in the context of nerve injury is desired to identify the unknown neuroimmune factors. Here we demonstrate how our lab rapidly and effectively isolates macrophages from the dorsal root ganglia using an enzyme-free mechanical dissociation protocol. The samples are kept on ice throughout to limit cellular stress. This protocol is far less time consuming compared to the standard enzymatic protocol and has been routinely used for our Fluorescence-activated Cell Sorting analysis.

Here we present a mechanical dissociation protocol to rapidly isolate macrophages from the dorsal root ganglion for phenotyping and functional analysis.

Isolement rapide des macrophages de ganglion de racine dorsal

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after ...