Supported by grants from CIHR (MOP231122) and NSERC (RGPIN-2014-06503). We would like to thank Genevi&#232;ve Cyr for her help preparing experiments and supervising laboratory work. MAL received a scholarship from The Natural Sciences and Engineering Research Council of Canada (NSERC).

NOTE: Toutes les procédures ont été effectuées en conformité avec les directives du Conseil canadien pour la protection des animaux et de la commission d&#39;examen d&#39;éthique de l&#39;Université de Montréal. 1. Assemblée de la pipette d&#39;enregistrement injection <ol><li> Tirer un capillaire en verre d&#39;environ 7 cm de long (diamètre externe 1 mm) à l&#39;aide d&#39;un extracteur de pipette. </li><li> Casser l&#39;embout du capillaire et vérifier l&#39;ouverture sous un microscope optique. Assurez-vous que le diamètre interne est compris entre 30 um à 40 um. </li><li> Insérer un long fil d&#39;argent de 7 cm dans le capillaire de verre avec environ 1 cm en saillie à partir de l&#39;extrémité non effilée de la pipette en verre. </li><li> Plier l&#39;excès filament perpendiculairement au capillaire de verre. </li><li> Appliquer une goutte de colle de matière plastique souple sur l&#39;arbre d&#39;une aiguille hypodermique 30 G. </li><li> Insérer une aiguille hypodermique 30 G dans la pipette en verre selon les schémas présentés sur la figure 1 </strong&gt;. </li><li> Ajouter un deuxième revêtement de colle pour assurer une bonne étanchéité de la jonction entre la pipette en verre et l&#39;aiguille hypodermique. </li><li> Laissez sécher la pipette avec la pointe vers le haut pendant environ 12 heures pour assurer le bon durcissement de la colle. Le résultat final est représenté sur la Figure 2. </li></ol> NOTE: Cette procédure est effectuée sur des expériences et la stérilisation de la pointe de la pipette aigus est pas nécessaire. 2. Préparation des animaux <ol><li> Placez le rat dans une boîte d&#39;anesthésie. </li><li> Induire une anesthésie à l&#39;aide de 4% d&#39;isoflurane pendant 5 à 10 min. </li><li> Placez l&#39;animal sur une table stéréotaxique avec coussin chauffant et sonde rectale pour maintenir une température corporelle de 37 ° C. Utilisez un cône de nez pour maintenir l&#39;anesthésie avec 2% d&#39;isoflurane. Fixez la tête du rat en utilisant des barres de l&#39;oreille et porte-dents. </li><li> Appliquer une pommade ou collyres ophtalmiques, qui comprennent 1% gouttes d&#39;atropine pour aider à la dilatation de la pupille. Pour prévenir la sécheresse, appliquer baisse de lubrifications environ toutes les 30 min. </li><li> Raser la tête et le nettoyer avec 10% de povidone-iode. </li><li> Pour l&#39;anesthésie locale, d&#39;injecter 0,5 ml de lidocaïne 2% sous le cuir chevelu en 2-3 endroits en soulevant la peau et l&#39;insertion de la pointe de l&#39;aiguille. </li><li> Confirmez niveau adéquat d&#39;anesthésie en effectuant une pincée d&#39;orteil et en observant l&#39;absence de mouvement. En outre, surveiller la fréquence cardiaque à assurer qu&#39;elle est à l&#39;intérieur des valeurs normales (300 à 400 battements / min). </li><li> Inciser le cuir chevelu en ligne droite le long de la médiane avec un n ° 10 lame de scalpel pour exposer à la fois les sutures frontal et sagittal. </li><li> Révéler les points de Lambda et Bregma en écartant le tissu qui recouvre le crâne avec une spatule chirurgicale. </li><li> Niveau de sorte que le crâne positions Bregma et lambda sont sur le même plan. </li><li> Pour définir le point de référence, utiliser un appareil de stéréotaxie avec un tube de verre monté pour le mettre juste au-dessus Bregma. Ce sera le «zéro» pour l&#39;antéro-postérieur et medcoordonne les mesures ial-latérales. </li><li> Réglez le point d&#39;intérêt en déplaçant la monture stéréotaxique les coordonnées nécessaires, notez les coordonnées stéréotaxiques et dessiner un carré autour de la zone cible qui marque où le craniotomie sera effectuée. </li><li> Utilisez une perceuse chirurgicale avec une perceuse stérilisée long de la place marquée lentement sans pression pour retirer lentement la matière osseuse. Veillez à ne pas percer trop longtemps dans la même zone, car il va produire de la chaleur et provoquer des lésions sur le cortex. </li><li> Lorsque l&#39;os délimitant la craniotomie est devenue suffisamment mince, retirez soigneusement la section crânienne avec des pincettes pour exposer le cortex. </li><li> Foire irriguer le cortex exposée avec du liquide céphalo-rachidien artificiel pour éviter la dessiccation du tissu. NOTE: mater élimination Dura est inutile sur le rat que la pointe de l&#39;injectrode est suffisamment solide pour pénétrer. </li></ol> 3. Remplir et le montage du système d&#39;injection <ol><li> Remplissez le5-10 ul microseringue par aspiration avec de l&#39;huile minérale. </li><li> Remplissez l&#39;aiguille hypodermique avec la pipette en verre fixe avec une solution de 0,5% de Chicago Blue Sky (CSB) et 300 um γ-aminobutyrique (GABA) ou une solution de lidocaïne 2% avec 0,5% CSB 9. Diluer toutes les solutions avec une solution saline. <ol><li> Dans le cas d&#39;une substance abondante, remplissez aide d&#39;une seringue régulière et utiliser les techniques habituelles de précaution pour éviter la formation de bulles d&#39;air. </li><li> Dans le cas des substances les plus chers, utiliser de l&#39;huile minérale pour remplir le injectrode et l&#39;agent chimique peut alors être introduit par aspiration. Comme la différence de densité entre l&#39;huile minérale et de l&#39;eau est relativement élevée, cette substance est un bon candidat pour l&#39;injection de solutions aqueuses. REMARQUE: Un colorant peut être ajouté à confirmer la séparation entre les deux liquides. </li></ol></li><li> Pour remplir la pipette d&#39;injection, de remplir une seringue de 1 ml avec la solution par aspiration, puis injecter lentement la solutiondans la pipette d&#39;injection. </li><li> Prêtez une attention particulière pour les fuites dans les régions indiquées dans la figure 1 en frottant ces lieux propres et en observant les fuites en injectant plus lentement la solution avec la seringue. </li><li> Retirez la seringue de 1 ml. Ce faisant, être sûr de garder une légère pression sur le piston de sorte que le vide ne supprime pas la solution de la pipette d&#39;injection. </li><li> Remplissez la microseringue avec de l&#39;huile minérale par aspiration et l&#39;attacher fermement à la pipette d&#39;injection remplie, puis essuyer soigneusement tout excès de solution de la injectrode assemblé avec de la gaze. </li><li> Vérifiez que la pointe ne soit pas bloqué par injection d&#39;un volume très faible, assez pour voir une petite goutte de formation à la pointe de la pipette de verre. </li><li> Montez le injectrode sur le système de micro-pompe et vérifier qu&#39;il est bien fixé. </li><li> Positionner soigneusement la pointe de injectrode les coordonnées de la cible et de réduire la pointe à la surface du cortex. </li><li> Lentement abaisser le injectrOde à l&#39;aide de l&#39;appareil de stéréotaxie à la structure cible (colliculus supérieur dans ce cas) en utilisant les coordonnées anatomiques appropriées. </li><li> Couvrir le cortex exposée avec de l&#39;agar chaud pour éviter la dessiccation du tissu. </li></ol> 4. Injection et inactivation réversible <ol><li> Régler la pompe de micro-injection pour injecter 400 à 800 nl à 40 nl / min et appuyez sur Exécuter pour démarrer l&#39;injection. Notez que le taux de pic connaîtront une réduction lors de l&#39;injection. NOTE: Dans la configuration expérimentale, l&#39;activité neuronale récupéré moins d&#39;une heure après la fin de la prestation de GABA. Tout dispositif mécanique calibré peut être utilisé pour appliquer une pression sur la seringue de micro-injection afin de procéder à l&#39;injection. </li><li> Après l&#39;acquisition des données électrophysiologiques, euthanasier en utilisant une méthode approuvée par la Communauté locale éthique animale. </li></ol>

<ol>
	<li>Martin, J. H., Ghez, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+inactivation+in+the+analysis+of+the+central+control+of+movement.">Pharmacological inactivation in the analysis of the central control of movement.</a> Journal Of Neuroscience Methods. 86 (2), 145-159 (1999).</li><li>Ponce, C. R., Hunter, J. N., Pack, C. C., Lomber, S. G., Born, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contributions+of+indirect+pathways+to+visual+response+properties+in+macaque+middle+temporal+area+MT.">Contributions of indirect pathways to visual response properties in macaque middle temporal area MT.</a> The Journal Of Neuroscience The Official Journal Of The Society For Neuroscience. 31 (10), 3894-3903 (2011).</li><li>Lomber, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+advantages+and+limitations+of+permanent+or+reversible+deactivation+techniques+in+the+assessment+of+neural+function.">The advantages and limitations of permanent or reversible deactivation techniques in the assessment of neural function.</a> Journal Of Neuroscience Methods. 86 (2), 109-117 (1999).</li><li>Malpeli, J., Schiller, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+of+reversible+inactivation+of+small+regions+of+brain+tissue.">A method of reversible inactivation of small regions of brain tissue.</a> Journal Of Neuroscience Methods. 1 (2), 143-151 (1979).</li><li>Lomber, S. G., Payne, B. R., Horel, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cryoloop:+an+adaptable+reversible+cooling+deactivation+method+for+behavioral+or+electrophysiological+assessment+of+neural+function.">The cryoloop: an adaptable reversible cooling deactivation method for behavioral or electrophysiological assessment of neural function.</a> Journal Of Neuroscience Methods. 86 (2), 179-194 (1999).</li><li>Gonzalez-Perez, O., Guerrero-Cazares, H., Qui&#241;ones-Hinojosa, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+of+deep+brain+structures+with+microinjections+for+delivery+of+drugs,+viral+vectors,+or+cell+transplants.">Targeting of deep brain structures with microinjections for delivery of drugs, viral vectors, or cell transplants.</a> Journal Of Visualized Experiments. (46), (2010).</li><li>Hup&#233;, J., Chouvet, G., Bullier, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+temporal+parameters+of+cortical+inactivation+by+GABA.">Spatial and temporal parameters of cortical inactivation by GABA.</a> Journal Of Neuroscience Methods. 86 (2), 129-143 (1999).</li><li>Casanova, C., McKinley, P., Molotchnikofff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responsiveness+of+Reorganized+Primary+Somatosensory+(SI)+Cortex+after+Local+Inactivation+of+Normal+SI+Cortex+in+Chronic+Spinal+Cats.">Responsiveness of Reorganized Primary Somatosensory (SI) Cortex after Local Inactivation of Normal SI Cortex in Chronic Spinal Cats.</a> Somatosensory &amp; Motor Research. 8 (1), 65-76 (1991).</li><li>Malpeli, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+inactivation+of+subcortical+sites+by+drug+injection.">Reversible inactivation of subcortical sites by drug injection.</a> Journal Of Neuroscience Methods. 86 (2), 119-128 (1999).</li><li>Minville, K., Casanova, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+frequency+processing+in+posteromedial+lateral+suprasylvian+cortex+does+not+depend+on+the+projections+from+the+striate-recipient+zone+of+the+cat&#8217;s+lateral+posterior-pulvinar+complex.">Spatial frequency processing in posteromedial lateral suprasylvian cortex does not depend on the projections from the striate-recipient zone of the cat&#8217;s lateral posterior-pulvinar complex.</a> Neuroscience. 84 (3), 699-711 (1998).</li><li>Diao, Y., Wang, Y., Xiao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Representation+of+the+binocular+visual+field+in+the+superior+colliculus+of+the+albino+rat.">Representation of the binocular visual field in the superior colliculus of the albino rat.</a> Experimental Brain Research. 52 (1), 67-72 (1983).</li></ol>

We have nothing to disclose.

Le protocole proposé a été conçu pour résoudre les défis découlant de méthodes d&#39;inactivation réversible actuelles. Plus précisément, ce projet vise à affiner les méthodes utilisées pour les micro-injections de substances chimiques modulant l&#39;activité neuronale, en particulier dans les structures profondes du cerveau. Un défi technique émergente de ce type de configuration est la nécessité pour les deux sondes pour être colocalisés dans le même espace restreint in vivo afin d&#39;e...

L&#39;inactivation des aires corticales et les noyaux sous-corticales est important dans l&#39;étude des relations fonctionnelles entre les différentes structures cérébrales 4.2. La littérature récente a employé des techniques cryogéniques chimique ou la perte de fonction d&#39;étudier le rôle des structures cérébrales 2,5. En ce qui concerne les micro-injections pharmacologiques, de petites quantités de médicaments peuvent être administrés dans une région du cerveau à une vitesse contrôlée, tout en minimisant les dommages collatéraux au tissu environnant de 6,7. Cette technique peut être utilisée pour délivrer des agonistes spécifiques, agonistes inverses ou antagonistes pour étudier l&#39;effet des différentes cibles pharmacologiques sur l&#39;activité neuronale. Ces effets peuvent également être étudiés en mesurant les changements dans les réponses neuronales à partir d&#39;emplacements éloignés, permettant aux chercheurs d&#39;étudier les relations entre les différentes structures corticales et sous-corticales. Ici, nous démontrons l&#39;assemblage d&#39;un appareil, le injectrode, capable de boe l&#39;enregistrement de signaux électrophysiologiques et délivrer de petites quantités de drogues à l&#39;emplacement cible. Nous démontrons les capacités de ce système en injectant GABA, un inhibiteur de l&#39;activité neuronale commune, chez le rat colliculus supérieur. Cette région est sensible à la stimulation visuelle, qui nous a permis d&#39;utiliser l&#39;activité multiunit évoqué visuellement pour confirmer injectrode localisation. La réversibilité de l&#39;inactivation a été évaluée par la récupération de l&#39;activité neuronale normale après la fin de l&#39;injection GABA. La capacité de surveiller l&#39;activité multi-unité à partir du site d&#39;injection permet le réglage fin des débits d&#39;injection et les volumes nécessaires pour atteindre la réponse pharmacodynamique souhaitée. Par conséquent, un avantage de cette technique est le potentiel de limitation de dommages aux tissus causés par microperfusion, étant donné que les volumes effectifs plus petits sont injectés. Le protocole proposé offre une méthode rentable pour générer le nécessa matériel jetabley pour les expériences conducteurs où la livraison de la drogue et de l&#39;enregistrement de l&#39;activité neuronale locale est souhaitée.

<table border="0" cellpadding="0" cellspacing="0" width="981">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:280px;">Name of the reagent</td>
			<td style="width:209px;">Company</td>
			<td style="width:184px;">Catalogue number</td>
			<td style="width:308px;">Comments (optional)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:280px;">Injection pump (UltraMicroPump III)</td>
			<td style="width:209px;">WPI</td>
			<td style="width:184px;">#UMP3</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:280px;">Injection console (Micro4 Controller)</td>
			<td style="width:209px;">WPI</td>
			<td style="width:184px;">#SYS-MICRO4</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:280px;">Hamilton syringe</td>
			<td style="width:209px;">Hamliton</td>
			<td style="width:184px;">(80301) 701LT 10 &#181;L SYR</td>
			<td style="width:308px;">Syringes between 5 and 10 &#956;L used</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:280px;">Gel cyanoacrylate adhesive</td>
			<td style="width:209px;">Krazy Glue</td>
			<td style="width:184px;">KG86648R</td>
			<td style="width:308px;">The gel form is easier to apply on the shaft of the 30G hypodermic needle</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:280px;">Glass pipettes</td>
			<td style="width:209px;">WPI</td>
			<td style="width:184px;">#TW100F-4</td>
			<td style="width:308px;">Thin wall, 1mm OD, 0.75mm ID with filament pipettes used</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:280px;">720 Needle Pipette Puller</td>
			<td style="width:209px;">Kopf</td>
			<td style="width:184px;">720</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:280px;">Silver wire</td>
			<td style="width:209px;">A-M Systems, Inc.</td>
			<td style="width:184px;">782500</td>
			<td style="width:308px;">Bare 0.010&#8221;</td>
		</tr>
	</tbody>
</table>

simultaneous electrophysiological recording and micro-injections of inhibitory agents in the rodent brain

Nous décrivons ici une méthode pour la construction d&#39;un usage unique &quot;injectrode&quot; en utilisant des pièces commercialement accessibles et abordables. Un système de palpage est développé qui permet l&#39;injection d&#39;un médicament lors de l&#39;enregistrement de signaux électrophysiologiques à partir de la population neuronale affectée. Cette méthode offre une alternative simple et économique à des solutions commerciales. Une pipette de verre a été modifié en le combinant avec une aiguille hypodermique et un filament d&#39;argent. Le injectrode est attaché à la pompe de microseringue commerciale pour la livraison de la drogue. Cela se traduit par une technique qui fournit en temps réel par réaction de la pharmacodynamique à unités multiples signaux extracellulaires provenant du site d&#39;administration de médicament. Comme une preuve de concept, nous avons enregistré l&#39;activité neuronale du colliculus supérieur provoquée par des éclairs de lumière chez les rats, en concomitance avec la livraison de médicaments à travers la injectrode. La capacité d&#39;enregistrement d&#39;injectrode permet la caractérisation fonctionnelle de la injsite de réflexion favorisant un contrôle précis sur la localisation de l&#39;administration de médicaments. L&#39;application de cette méthode étend également bien au-delà ce qui est démontré ici, comme le choix de la substance chimique chargé dans le injectrode est vaste, y compris le tracé des marqueurs pour les expériences anatomiques.

La construction de la injectrode est illustré sur la figure 1. Un fil d&#39;argent (C) est introduit dans une pipette en verre (D) avec une portion pliée du fil et faisant saillie hors de l&#39;ouverture. Une aiguille de 30 G (B) est fixé et scellé à l&#39;ouverture de la pipette en verre avec de la colle. Après la pipette a été rempli avec la substance d&#39;injection, un micro-seringue en verre (A) est fixé à l&#39;aiguille. Il est important qu&#39;il y ait une bonne étanchéité lorsque le...

Watch this Scientific Journal Video about Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain at JoVE.com

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

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

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

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

simultaneous-electrophysiological-recording-micro-injections

Research

JoVE Journal

Neuroscience

8.0K vues.  Université de Montréal. Here, we craft a glass pipette with dual functions: inhibition of deep brain structures by microinjections of drugs and real-time monitoring of their effects through simultaneous electrophysiological recordings.

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

Simultaneous fMRI and Electrophysiology in the Rodent Brain

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in which functional MRI data and in vivo intracortical recording can be performed simultaneously. The combination of MRI and electrical recording is technically challenging because the electrodes used for recording distort the MRI images and the MRI acquisition induces noise in the electrical recording. To minimize the mutual interference of the two modalities, glass microelectrodes were used rather than metal and a noise removal algorithm was implemented for the electrophysiology data. In our studies, two microelectrodes were separately implanted in bilateral primary somatosensory cortices (SI) of the rat and fixed in place. One coronal slice covering the electrode tips was selected for functional MRI. Electrode shafts and fixation positions were not included in the image slice to avoid imaging artifacts. The removed scalp was replaced with toothpaste to reduce susceptibility mismatch and prevent Gibbs ringing artifacts in the images. The artifact structure induced in the electrical recordings by the rapidly-switching magnetic fields during image acquisition was characterized by averaging all cycles of scans for each run. The noise structure during imaging was then subtracted from original recordings. The denoised time courses were then used for further analysis in combination with the fMRI data. As an example, the simultaneous acquisition was used to determine the relationship between spontaneous fMRI BOLD signals and band-limited intracortical electrical activity. Simultaneous fMRI and electrophysiological recording in the rodent will provide a platform for many exciting applications in neuroscience in addition to elucidating the relationship between the fMRI BOLD signal and neuronal activity.

We have developed a method for simultaneous functional magnetic resonance imaging and electrophysiological recording in the rodent brain, providing a platform for the investigation of the relationship between neural activity and the blood oxygenation level dependent (BOLD) MRI signal.

IRMf simultanées et l&#39;électrophysiologie dans le cerveau de rongeurs

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in ...

Recherche

Neurosciences

Design and Construction of a Cost Effective Headstage for Simultaneous Neural Stimulation and Recording in the Water Maze

Headstage preamplifiers and source followers are commonly used to study neural activity in behavioral neurophysiology experiments. Available commercial products are often expensive, not easily customized, and not submersible. Here we describe a method to design and build a customized, integrated circuit headstage for simultaneous 4-channel neural recording and 2-channel simulation in awake, behaving animals. The headstage is designed using a free, commercially available CAD-type design package, and can be modified easily to accommodate different scales (e.g. to add channels). A customized printed circuit board is built using surface mount resistors, capacitors and operational amplifiers to construct the unity gain source follower circuit. The headstage is made water-proof with a combination of epoxy, parafilm and a synthetic rubber putty. We have successfully used this device to record local field potentials and stimulate different brain regions simultaneously via independent channels in rats swimming in a water maze. The total cost is &lt; $30/unit and can be manufactured readily.

We present a low-cost method to design and construct a light headstage pre-amplifier system with simultaneous neural recording and stimulation capability. This device can be waterproofed for use in swimming animals.

Conception et construction d&#39;un Headstage rentable pour la stimulation neuronale et enregistrement simultanés dans le labyrinthe de l&#39;eau

Headstage preamplifiers and source followers are commonly used to study neural activity in behavioral neurophysiology experiments. Available commercial ...

Forebrain Electrophysiological Recording in Larval Zebrafish

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked seizures, epilepsy can be acquired as a result of an insult to the brain or a genetic mutation. Efforts to model seizures in animals have primarily utilized acquired insults (convulsant drugs, stimulation or brain injury) and genetic manipulations (antisense knockdown, homologous recombination or transgenesis) in rodents. Zebrafish are a vertebrate model system1-3 that could provide a valuable alternative to rodent-based epilepsy research. Zebrafish are used extensively in the study of vertebrate genetics or development, exhibit a high degree of genetic similarity to mammals and express homologs for ~85% of known human single-gene epilepsy mutations. Because of their small size (4-6 mm in length), zebrafish larvae can be maintained in fluid volumes as low as 100 &mu;l during early development and arrayed in multi-well plates. Reagents can be added directly to the solution in which embryos develop, simplifying drug administration and enabling rapid in vivo screening of test compounds4. Synthetic oligonucleotides (morpholinos), mutagenesis, zinc finger nuclease and transgenic approaches can be used to rapidly generate gene knockdown or mutation in zebrafish5-7. These properties afford zebrafish studies an unprecedented statistical power analysis advantage over rodents in the study of neurological disorders such as epilepsy. Because the "gold standard" for epilepsy research is to monitor and analyze the abnormal electrical discharges that originate in a central brain structure (i.e., seizures), a method to efficiently record brain activity in larval zebrafish is described here. This method is an adaptation of conventional extracellular recording techniques and allows for stable long-term monitoring of brain activity in intact zebrafish larvae. Sample recordings are shown for acute seizures induced by bath application of convulsant drugs and spontaneous seizures recorded in a genetically modified fish.

A simple method to record extracellular field potentials in the larval zebrafish forebrain is described. The method provides a robust in vivo read-out of seizure-like activity. This technique can be used with genetically modified zebrafish larvae carrying epilepsy-related genes or seizures evoked by administration of convulsant drugs.

Prosencéphale enregistrement électrophysiologique des larves dans le poisson-zèbre

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

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

Much information about the coupling of presynaptic ionic currents with the release of neurotransmitter has been obtained from invertebrate preparations, most notably the squid giant synapse1. However, except for the preparation described here, few vertebrate preparations exist in which it is possible to make simultaneous measurements of neurotransmitter release and presynaptic ionic currents. Embryonic Xenopus motoneurons and muscle cells can be grown together in simple culture medium at room temperature; they will form functional synapses within twelve to twenty-four hours, and can be used to study nerve and muscle cell development and synaptic interactions for several days (until overgrowth occurs). Some advantages of these co-cultures over other vertebrate preparations include the simplicity of preparation, the ability to maintain the cultures and work at room temperature, and the ready accessibility of the synapses formed2-4. The preparation has been used widely to study the biophysical properties of presynaptic ion channels and the regulation of transmitter release5-8. In addition, the preparation has lent itself to other uses including the study of neurite outgrowth and synaptogenesis9-12, molecular mechanisms of neurotransmitter release13-15, the role of diffusible messengers in neuromodulation16,17, and in vitro synaptic plasticity18-19.

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

This video demonstrates the procedures used to grow primary cultures of embryonic Xenopus nerve and muscle cells and the usefulness of this preparation for making simultaneous pre- and post-synaptic patch clamp recordings.

Simultanée pré-et post-synaptique enregistrement électrophysiologique de<em&gt; Xenopus</em&gt; Nerf-muscle co-cultures

Much information about the coupling of  presynaptic ionic currents with the release of neurotransmitter has been obtained  from invertebrate preparations, ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

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

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

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

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

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

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

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

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

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

Enregistrement de la plasticité synaptique dans des tranches d’hippocampe aiguës maintenues dans un petit volume recyclage - Perfusion- et système de chambre de Submersion-type

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Recording Brain Electromagnetic Activity During the Administration of the Gaseous Anesthetic Agents Xenon and Nitrous Oxide in Healthy Volunteers

Anesthesia arguably provides one of the only systematic ways to study the neural correlates of global consciousness/unconsciousness. However to date most neuroimaging or neurophysiological investigations in humans have been confined to the study of &#947;-Amino-Butyric-Acid-(GABA)-receptor-agonist-based anesthetics, while the effects of dissociative N-Methyl-D-Aspartate-(NMDA)-receptor-antagonist-based anesthetics ketamine, nitrous oxide (N2O) and xenon (Xe) are largely unknown. This paper describes the methods underlying the simultaneous recording of magnetoencephalography (MEG) and electroencephalography (EEG) from healthy males during inhalation of the gaseous anesthetic agents N2O and Xe. Combining MEG and EEG data enables the assessment of electromagnetic brain activity during anesthesia at high temporal, and moderate spatial, resolution. Here we describe a detailed protocol, refined over multiple recording sessions, that includes subject recruitment, anesthesia equipment setup in the MEG scanner room, data collection and basic data analysis. In this protocol each participant is exposed to varying levels of Xe and N2O in a repeated measures cross-over design. Following relevant baseline recordings participants are exposed to step-wise increasing inspired concentrations of Xe and N2O of 8, 16, 24 and 42%, and 16, 32 and 47% respectively, during which their level of responsiveness is tracked with an auditory continuous performance task (aCPT). Results are presented for a number of recordings to highlight the sensor-level properties of the raw data, the spectral topography, the minimization of head movements, and the unequivocal level dependent effects on the auditory evoked responses. This paradigm describes a general approach to the recording of electromagnetic signals associated with the action of different kinds of gaseous anesthetics, which can be readily adapted to be used with volatile and intravenous anesthetic agents. It is expected that the method outlined can contribute to the understanding of the macro-scale mechanisms of anesthesia by enabling methodological extensions involving source space imaging and functional network analysis.

Simultaneous magnetoencephalography and electroencephalography provides a useful tool to search for common and distinct macro-scale mechanisms of reductions in consciousness induced by different anesthetics. This paper illustrates the empirical methods underlying the recording of such data from healthy humans during N-Methyl-D-Aspartate-(NMDA)-receptor-antagonist-based anesthesia during inhalation of nitrous oxide and xenon.

Enregistrement activité électromagnétique du cerveau au cours de l’Administration des Agents anesthésiques gazeux Xenon et l’oxyde nitreux chez des volontaires sains

Anesthesia arguably provides one of the only systematic ways to study the neural correlates of global consciousness/unconsciousness. However to date most ...

Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) is a promising approach when pharmacological treatment fails or neurosurgery is not recommended. Acute brain slices coupled to microelectrode arrays (MEAs) represent a valuable tool to study neuronal network interactions and their modulation by electrical stimulation. As compared to conventional extracellular recording techniques, they provide the added advantages of a greater number of observation points and a known inter-electrode distance, which allow studying the propagation path and speed of electrophysiological signals. However, tissue oxygenation may be greatly impaired during MEA recording, requiring a high perfusion rate, which comes at the cost of decreased signal-to-noise ratio and higher oscillations in the experimental temperature. Electrical stimulation further stresses the brain tissue, making it difficult to pursue prolonged recording/stimulation epochs. Moreover, electrical modulation of brain slice activity needs to target specific structures/pathways within the brain slice, requiring that electrode mapping be easily and quickly performed live during the experiment. Here, we illustrate how to perform the recording and electrical modulation of 4-aminopyridine (4AP)-induced epileptiform activity in rodent brain slices using planar MEAs. We show that the brain tissue obtained from mice outperforms rat brain tissue and is thus better suited for MEA experiments. This protocol guarantees the generation and maintenance of a stable epileptiform pattern that faithfully reproduces the electrophysiological features observed with conventional field potential recording, persists for several hours, and outlasts sustained electrical stimulation for prolonged epochs. Tissue viability throughout the experiment is achieved thanks to the use of a small-volume custom recording chamber allowing for laminar flow and quick solution exchange even at low (1 mL/min) perfusion rates. Quick MEA mapping for real-time monitoring and selection of stimulating electrodes is performed by a custom graphic user interface (GUI).

We illustrate how to perform recording and electrical modulation of 4-aminopyridine-induced epileptiform activity in rodent brain slices using microelectrode arrays. A custom recording chamber maintains tissue viability throughout prolonged experimental sessions. Live electrode mapping and selection of stimulating pairs are performed by a custom graphical user interface.

Enregistrement et Modulation de l’activité épileptiforme dans des tranches de cerveau rongeur couplé aux baies de microélectrodes

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) ...

Simultaneous Cryosectioning of Multiple Rodent Brains

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In neuroscience, as well as a plethora of other scientific fields, these techniques are used. Immunohistochemistry can be done on slide mounted tissue or free-floating sections. Preparing slide-mounted samples is a time intensive process. The following protocol for a technique, called the Megabrain, reduced the time taken to cryosection and mount brain tissue by up to 90% by combining multiple brains into a single frozen block. Furthermore, this technique reduced variability seen between staining rounds, in a large histochemical study. The current technique has been optimized for using rodent brain tissue in downstream immunohistochemical analyses; however, it can be applied to different scientific fields that use cryosectioning.

Here, we present a protocol to freeze and section brain tissue from multiple animals as a timesaving alternative to processing single brains. This reduces staining variability during immunohistochemistry and reduces time cryosectioning and imaging.

Cryosectioning simultanée de plusieurs cerveaux de rongeurs

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In ...