This work was funded by the Whitehall Foundation and the NIH. We thank Clifford Dax (University of Oregon Technical Support Administration) for his help and expertise in designing a circuit for light delivery.

REMARQUE: Le protocole suivant est en conformité avec les Instituts nationaux de la santé des lignes directrices approuvées par l&#39;Université de l&#39;Oregon de protection des animaux et l&#39;utilisation Comité. 1. Chirurgie aiguë <ol><li> Anesthésier l&#39;animal avec un cocktail de kétamine-médétomidine, par voie intrapéritonéale (ip) injection (tableau 1). REMARQUE: Les souris utilisées dans ces expériences sont générées par croisement d&#39;une CHR2-EYFP transgénique ligne 10 cre dépendant de interneurone lignes d&#39;attaque (Pvalb-iCre11, PV +; Sst-iCre12, SOM +; Cr-iCre12, CR +). Livraison virale de ChR2 ou opsins connexes doit fonctionner aussi bien, en supposant que les niveaux d&#39;expression similaires sont obtenus. </li><li> Avant de commencer la chirurgie, veiller à ce que les expositions d&#39;animaux pas de réponse à un pincement de l&#39;orteil douce. Re-administrer des anesthésiques pendant toute l&#39;expérience nécessaire pour maintenir cette profondeur de l&#39;anesthésie. Si l&#39;utilisation d&#39;anesthésiques injectables, éventuellement implanter un cathéter IP pour les injections d&#39;entretien. </li><li> Maintenir l&#39;animal hydraté avec une solution saline ou une solution de Ringer-lactate tout au long de l&#39;expérience (environ 3 ml / kg / h), par exemple en utilisant un cocktail anesthésiant diluée de manière appropriée pour des injections d&#39;entretien (tableau 1). </li><li> Placer l&#39;animal dans un dispositif de maintien de tête stéréotaxique ou autre. Assurez-vous que le crâne est bien fixé. Cela est essentiel pour le maintien des enregistrements de cellules individuelles stables. </li><li> Appliquer une pommade pour les yeux opthalamic pour prévenir la sécheresse. Maintenir la température du corps à 36,5 à 37 ° C. </li><li> Effectuer une vidange cisternal pour la stabilité d&#39;enregistrement supplémentaire. En utilisant une lame de scalpel, enlever les tissus de la face postérieure du crâne pour exposer la grande citerne. Faire une petite entaille dans la dure-mère pour drainer le liquide céphalo-rachidien. Utilisez seulement la pointe de la lame, et éviter tout contact avec la tige de cervelet ou du cerveau. </li><li> Utilisation du scalpel, effectuer une petite craniotomie (~ 2 mm 2) sur la zone corticale d&#39;intérêt (par auditifcortex, environ -2,3 mm postérieure du bregma, 4,5 mm latéral de la ligne médiane). </li><li> Retirer la dure-mère et couvrir le cortex exposé avec une couche d&#39;agarose chaud de 0,5 à 1 mm d&#39;épaisseur (1,5% d&#39;agarose dans une solution saline, 0,9% de NaCl; appliquer à ~ 37 ° C). Gardez le agarose humide tout au long de l&#39;expérience en appliquant périodiquement quelques gouttes de solution saline. </li></ol> 2. Enregistrement Set-up <ol><li> Préparer une électrode de tungstène (7-14 MQ, 127 um de diamètre, 12 ° pointe effilée, avec revêtement époxy). Superglue l&#39;électrode à un tube capillaire en verre et ajouter une gaine thermorétractable de chaleur pour une prise (Figure 1). </li><li> Monter l&#39;électrode sur un micromanipulateur motorisé (ou hydraulique), mis à voyager perpendiculaire à la surface corticale. Faites glisser un terrain de fil sous la peau, contre le crâne. Eviter le contact avec les muscles sur le côté de la tête et l&#39;arrière du cou, car ils peuvent générer des artefacts électromyographiques. </li><li> Amplifier le signal électrique en utilisant un ampli extracellulaireli fi cateur approprié pour l&#39;enregistrement unitaire, de préférence équipé d&#39;un mode de contrôle d&#39;impédance. Surveiller l&#39;activité de dopage en cours (passe-bande 300 - 5000 Hz) avec deux oscilloscopes et un ensemble de haut-parleurs. Ici, les données enregistrées sont numérisées en continu à un débit de 10 kHz d&#39;échantillonnage; les pointes sont extraits hors ligne. </li><li> Avancer l&#39;électrode à travers l&#39;agarose jusqu&#39;à la pointe atteint la surface du cortex. Observez cette étape à travers un microscope. &quot;Zéro&quot; de cette position sur le micromanipulateur. </li><li> Pour mieux se rapprocher de la profondeur de l&#39;enregistrement, vérifiez visuellement que la pointe de l&#39;électrode sort de la surface corticale à une profondeur de zéro lors du retrait de l&#39;électrode à la fin d&#39;une pénétration. REMARQUE: Un décalage entre la lecture de manipulateur et la position de l&#39;électrode observé indique qu&#39;il a dérivé par rapport au tissu. Cela peut être causé par l&#39;instabilité du cerveau ou de l&#39;animal, ou la dérive de manipulateur. <ol><li> Comme une vérification supplémentaire pour assurer quecette valeur de consigne de zéro correspond à la surface du cortex, surveiller les potentiels de champ évoqués stimulation tout en avançant à travers les quelques premières centaines de micromètres de tissu. Lors de la surveillance des potentiels de champ, abaisser le plus bas passe-bande de coupure du filtre de l&#39;amplificateur extracellulaire de 10 Hz. Assurez-vous que la polarité du potentiel de champ local inverse autour d&#39;une profondeur d&#39;environ 100 um, à proximité de la couche I / II frontière 13. Ceci est un point de référence fiable pour cortex auditif, mais il peut être différent pour d&#39;autres aires corticales. </li></ol></li><li> Monter une fibre optique couplée à une source de lumière bleue (figure 2) sur un micromanipulateur manuel. Utilisation du microscope, positionner l&#39;extrémité de la fibre au plus près de la surface de l&#39;agarose en tant que possible. Centrer le faisceau où l&#39;électrode va entrer dans le tissu. </li><li> Réguler la sortie de la source de lumière avec une unité de commande capable de fournir un TTL (logique transistor-transistor), le train d&#39;impulsions de largeur et duratio spécifién-, par exemple, un Arduino (figure 3), une carte informatique d&#39;E / S (comme illustré), ou un générateur d&#39;impulsions disponibles dans le commerce. <ol><li> Surveiller le signal sur les deux oscilloscopes. </li></ol></li><li> Mesurer la puissance totale de la lumière (mW) à l&#39;extrémité de la fibre optique à l&#39;aide d&#39;un wattmètre. Utiliser cette valeur pour le calcul de l&#39;éclairement énergétique (mW / mm 2) en divisant par la surface de section transversale du coeur de la fibre. Commencer avec une valeur d&#39;intensité dans la gamme de 10 à 15 mW / mm 2 et d&#39;ajuster vers le bas, si nécessaire, jusqu&#39;à ce que les artefacts sont éliminés. </li><li> Vérifiez objets légers avec l&#39;électrode dans l&#39;agarose, prêt à entrer dans le tissu. Lancer la recherche train d&#39;impulsions (par exemple, 30 impulsions lumineuses ms, 500 ms d&#39;intervalle interstimulus (ISI)). <ol><li> Éliminer les artefacts lumineux transitoires (figure 4) par le repositionnement de la fibre optique par rapport à l&#39;électrode de changer l&#39;angle de la lumière incidente. Si artefacts persistent, essayez de diminuer la puissance lumineuse. En ee de rares cas que les objets ne peuvent pas être complètement éliminés (seulement réduit), prolonger la durée de l&#39;impulsion de la recherche. Cela peut aider à identifier les véritables pointes neuronales. </li></ol></li></ol> 3. Droit PINP-in &#39; <ol><li> Avancer l&#39;électrode lentement à travers le cerveau à une vitesse d&#39;environ 1 um / sec. Utilisez un oscilloscope pour surveiller l&#39;activité en cours. D&#39;autre part, déclencher les impulsions laser. </li><li> Écoutez le «hachage» faible de pointes légères évoqués sur le moniteur audio, ce qui indique que l&#39;électrode est proche d&#39;une cellule ChR2 + et peut souvent être perçue bien avant que le signal électrique est apparent sur l&#39;oscilloscope. Ralentir le rythme de l&#39;avance. NOTE: Si la cellule se trouve directement dans la trajectoire de l&#39;électrode, le voyant d&#39;activité évoqués se grandir (et fort). Comme l&#39;électrode se rapproche d&#39;un seul interneurones, le hachage résoudre en petits mais bien définis pointes de taille uniforme et la forme. </li><li> Dès que lipointes GHT évoqués sont assez grands pour déclencher de façon individuelle sur l&#39;oscilloscope, commencer à le faire. Réglez l&#39;échelle sur l&#39;axe horizontal à observer la forme exacte de la forme d&#39;onde de pic. </li><li> Arrêtez-vous et attendez que le tissu à régler. Résistez à la tentation de déplacer l&#39;électrode plus près de la cellule. Cette étape est essentielle pour un enregistrement stable. Si après plusieurs minutes, le rapport signal-sur-bruit n&#39;a pas amélioré - ce qui est, le tissu est réglé, mais il n&#39;a pas apporté la cellule tout près de la pointe de l&#39;électrode - avance de 5 um et plus encore attendre. <ol><li> Répétez ce processus jusqu&#39;à ce que le pic ou le creux de potentiel d&#39;action peuvent être capturés de façon fiable avec un seuil de tension bien placé au-dessus du bruit de fond (par exemple, +/- 300 mV ou plus). REMARQUE: Il ya peu de risque de faux positifs ou ambiguïté quand PINPing interneurones inhibiteurs. La plupart des cellules peuvent facilement suivre un train d&#39;impulsions d&#39;une durée de 30 ms et inter-pulintervalle de 500 ms ou plus rapide, avec un temps de latence fiable premier pic de 2-5 ms (Figure 5) soi. La majorité des cellules photovoltaïques +, en particulier, peut soutenir la cuisson pour un plein 1 sec (Figure 6). Parce que ce sont les neurones inhibiteurs, disynaptique activation (indirect) est pas une préoccupation majeure. </li></ol></li><li> Pendant l&#39;enregistrement, surveiller l&#39;activité en cours sur le premier oscilloscope. Gardez un œil sur la taille et la forme des pointes en utilisant la deuxième oscilloscope. Notez la qualité de leur son sur le haut-parleur. <ol><li> Écoutez attentivement les changements brusques, qui indiquent que la cellule est soit tire trop proche de l&#39;électrode (où elle risque d&#39;être empalé ou endommagé), ou est à la dérive. Si les pointes deviennent grands et déformée, revenir en arrière; si elles poussent plus petit, avancer l&#39;électrode. Déplacez-vous lentement en 2 étapes um. </li><li> Confirmer la qualité de la post-hoc de l&#39;enregistrement par superposition de toutes les formes d&#39;onde de pic, alignés à pic ou un creux. Variez les thres de tensiontenir. Dans un large éventail de valeurs de seuil, il devrait jamais être une forme de pic cohérent de hauteur uniforme (figure 5a). </li></ol></li><li> Si à tout moment le signal est contaminée avec des pointes d&#39;un neurone voisin, avancer. Avancer lentement, sur le peu de chance, l&#39;électrode va passer hors de portée de la cellule voisine, mais rester à portée du neurone cible. (Cela est généralement sans succès.) </li><li> Déplacer l&#39;électrode à travers toute la profondeur du cortex (900 + pm) à chaque pénétration. Si aucun neurones sensibles à la lumière sont rencontrés après de nombreuses pénétrations ou, au contraire, les enregistrements sont régulièrement contaminés avec des pointes à partir de cellules ChR2- voisins, essayez une nouvelle électrode. NOTE: Même à l&#39;intérieur d&#39;un seul lot, l&#39;impédance et pointe géométrie des électrodes individuelles peut varier considérablement. Ces deux facteurs contribuent à la &quot;rayon écoute&quot; efficace de l&#39;électrode, qui doit être suffisamment grande pour détecter des pics de lumière évoqués while recherche, mais suffisamment restreint pour permettre l&#39;enregistrement d&#39;un seul interneurones dans l&#39;isolement. </li><li> Tester l&#39;impédance d&#39;électrodes comme vous le souhaitez. Pour éviter d&#39;endommager le tissu, le faire avec la pointe de l&#39;électrode dans la partie supérieure de la couche d&#39;agarose, bien en dehors du cerveau. Dans la gamme de 7-14 MQ, l&#39;impédance exact est pas un indicateur sûr de la performance, ou rendement, pour cette application. Cela dit, il est une approximation raisonnable de rayon écoute: électrodes à faible impédance ramasser plusieurs unités; de plus grande impédance, moins. </li><li> A la fin de l&#39;expérience, euthanasier l&#39;animal au moyen institutionnel approuvées, comme surdose d&#39;un anesthésique ou la dislocation cervicale sous un plan d&#39;anesthésie profonde. </li></ol>

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	<li>Lima, S. Q., Hromadka, T., Znamenskiy, P., Zador, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PINP:+a+new+method+of+tagging+neuronal+populations+for+identification+during+in+vivo+electrophysiological+recording.">PINP: a new method of tagging neuronal populations for identification during in vivo electrophysiological recording.</a> PLoS One. 4, (2009).</li><li>Moore, A. K., Wehr, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parvalbumin-expressing+inhibitory+interneurons+in+auditory+cortex+are+well-tuned+for+frequency.">Parvalbumin-expressing inhibitory interneurons in auditory cortex are well-tuned for frequency.</a> J Neurosci. 33, 13713-13723 (2013).</li><li>Merchant, H., de Lafuente, V., Pena-Ortega, F., Larriva-Sahd, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+impact+of+interneuronal+inhibition+in+the+cerebral+cortex+of+behaving+animals.">Functional impact of interneuronal inhibition in the cerebral cortex of behaving animals.</a> Prog Neurobiol. 99, 163-178 (2012).</li><li>Markram, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interneurons+of+the+neocortical+inhibitory+system.">Interneurons of the neocortical inhibitory system.</a> Nat Rev Neurosci. 5, 793-807 (2004).</li><li>Atallah, B. 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The authors have no competing financial interests.

Bien PINP est conceptuellement simple, il peut être difficile dans la pratique. Un facteur déterminant de la réussite est le choix de l&#39;électrode. Le rayon d&#39;écoute électrique est le paramètre critique. Il doit être suffisamment grand pour détecter les pics de lumière évoquée lorsque la pointe est encore loin d&#39;une cellule ChR2 +, de sorte que l&#39;on peut régler la vitesse d&#39;avance en conséquence. Dans le même temps, elle doit être limitée suffisamment bonne pour permettre l&#39;isolem...

Characterizing the myriad cell types that comprise the mammalian brain has been a central, but long-elusive goal of neurophysiology. For instance, the properties and function of different inhibitory cell types in the cerebral cortex are topics of great interest but are still relatively unknown. This is in part because conventional blind in vivo recording techniques are limited in their ability to distinguish between different cell types. Extracellular spike width can be used to separate putative parvalbumin-positive inhibitory neurons from excitatory pyramidal cells, but this method is subject to both type I and type II errors2,3. Alternatively, recorded neurons can be filled, recovered, and stained to later confirm their morphological and molecular identity, but this is a pain-staking and time-consuming process. Recently, genetically identified populations of inhibitory interneurons have become accessible by means of calcium imaging or visually guided patch recordings. In these approaches, viral or transgenic expression of a calcium reporter (such as GCaMP) or fluorescent protein (such as GFP) allows identification and characterization of cell types defined by promoter expression. These approaches use 2-photon microscopy, which requires expensive equipment, and are also limited to superficial cortical layers due to the light scattering properties of brain tissue. 
Recently, Lima and colleagues1 developed a novel application of optogenetics to target electrophysiological recordings to genetically identified neuronal types in vivo, termed &#8220;PINP&#8221; &#8211; or Photostimulation-assisted Identification of Neuronal Populations. 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. Unlike many other optogenetic applications, the goal is not to manipulate circuit function but simply to identify neurons belonging to a genetically-defined class, which can then be characterized during normal brain function. The technique can be implemented with standard extracellular recording equipment and can therefore serve as an accessible and inexpensive alternative to calcium imaging or visually-guided patching. Here we describe an approach to PINPing specific cell types in the anesthetized auditory cortex, with the expectation that the more general points can be usefully applied in other preparations and brain regions.
In cortex, PINP holds particular promise for investigating the in vivo response properties of inhibitory interneurons. GABAergic interneurons comprise a small, heterogeneous subset of cortical neurons4. Different subtypes, marked by the expression of particular molecular markers, have recently been shown to perform different computational roles in cortical circuits5-9. As genetic tools improve it may eventually be possible to distinguish morphologically- and physiologically-separable types that fall within these broad classes. We here share our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. This strategy was developed specifically for targeting parvalbumin-positive (PV+) cells, but we have found that it works for other interneuron types as well, such as somatostatin-expressing (SOM+) and calretinin-expressing (CR+) interneurons. Although PINPing is conceptually straightforward, it can be surprisingly unyielding in practice. We learned a number of tips and tricks through trial-and-error that may be useful to others attempting the method.

<table border="0" cellpadding="0" cellspacing="0" width="1482">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Name of Material/Equipment</td>
			<td style="width:171px;">Company</td>
			<td style="width:341px;">Product/Stock Number</td>
			<td style="width:708px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ChR2-EYFP Line</td>
			<td style="width:171px;">Jackson Colonies</td>
			<td style="width:341px;">12569</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pvalb-iCre (PV) Line</td>
			<td style="width:171px;">Jackson Colonies</td>
			<td style="width:341px;">8069</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sst-iCre (SOM) Line</td>
			<td style="width:171px;">Jackson Colonies</td>
			<td style="width:341px;">13044</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cr-iCre (CR) Line</td>
			<td style="width:171px;">Jackson Colonies</td>
			<td>10774</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Agarose</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:341px;">A9793</td>
			<td>Type III-A, High EEO</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Micro Point (dural hook)</td>
			<td style="width:171px;">FST</td>
			<td style="width:341px;">10066-15</td>
			<td style="width:708px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Surgical Scissors</td>
			<td style="width:171px;">FST</td>
			<td>14084-09</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Scalpel</td>
			<td style="width:171px;">FST</td>
			<td>10003-12 (handle), 10011-00 (blades)</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Puralube Ophthalmic Ointment</td>
			<td style="width:171px;">Foster &#38; Smith</td>
			<td style="width:341px;">9N-76855</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Homeothermic Blanket</td>
			<td style="width:171px;">Harvard Apparatus</td>
			<td style="width:341px;"><a name="RANGE!C11">507220F</a></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tungsten Microelectrodes</td>
			<td style="width:171px;">A-M Systems</td>
			<td>577200</td>
			<td>12 M&#937; AC resistance, 127 &#956;m diameter, 12&#176; tapered tip, epoxy-coated</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Capillary Glass Tubing</td>
			<td style="width:171px;">Warner Instruments</td>
			<td style="width:341px;">G150TF-3</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Heat Shrink Tubing</td>
			<td style="width:171px;">DigiKey</td>
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a guide to in vivo single-unit recording from optogenetically identified cortical inhibitory interneurons

Un défi majeur dans la neurophysiologie a été de caractériser les propriétés de réponse et la fonction de nombreux types de cellules inhibiteur dans le cortex cérébral. Nous partageons ici notre stratégie pour obtenir stables, bien isolées enregistrements unitaires de interneurones inhibiteurs identifiés dans le cortex de souris anesthésiée à l&#39;aide d&#39;une méthode développée par Lima et ses collègues 1. Les enregistrements sont effectués dans des souris exprimant channelrhodopsin-2 (ChR2) dans les sous-populations neuronales spécifiques. Les membres de la population sont identifiés par leur réponse à un bref éclair de lumière bleue. Cette technique - appelée «PINP&quot;, ou identification Photostimulation assistée de neurones populations - peuvent être mises en œuvre avec l&#39;équipement d&#39;enregistrement extracellulaire standard. Il peut servir comme une alternative peu coûteuse et accessible à l&#39;imagerie de calcium ou patch visuellement guidé, dans le but de cibler des enregistrements extracellulaires de cellules génétiquement déterminées. Havant que nous fournissons un ensemble de lignes directrices pour l&#39;optimisation de la méthode dans la pratique quotidienne. Nous avons affiné notre stratégie spécifiquement pour cibler des cellules de parvalbumin positif (PV +), mais nous avons trouvé que cela fonctionne pour d&#39;autres types de interneurones ainsi, comme (CR +) interneurones Calretinin exprimant la somatostatine exprimant (SOM +) et.

Nous ici partageons notre stratégie pour obtenir des enregistrements unitaires de interneurones inhibiteurs génétiquement petites dans le cortex de souris anesthésiée, en utilisant une méthode optogenetic développé par Lima et al. Tableau 1 détaille le cocktail anesthésie suggéré, la kétamine-médétomidine-acépromazine (1. &quot; KMA &quot;). La figure 1 illustre une micro-électrode de tungstène, préparée pour l&#39;enregistrement. La figur...

Watch this Scientific Journal Video about A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons at JoVE.com

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 ...

a-guide-to-vivo-single-unit-recording-from-optogenetically-identified

Research

JoVE Journal

Neuroscience

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

19.0K vues.  University of Oregon. 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. 

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

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 ...

Recherche

Neurosciences

Recording Behavioral Responses to Reflection in Crayfish

Social behavior depends on sensory input from the visual, mechanical and olfactory systems. One important issue concerns the relative roles of each sensory modality in guiding behavior. The role of visual inputs has been examined by isolating visual stimuli from mechanical and chemosensory stimuli. In some studies (Bruski &amp; Dunham, 1987: Delgado-Morales et al., 2004) visual inputs have been removed with blindfolds or low light intensity, and effects of remaining sensory modalities have been elucidated. An alternative approach is to study the effects of visual inputs in the absence of any appropriate mechanical and chemosensory cues. This approach aims to identify the exclusive role of visual inputs. 
We have used two methods to provide visual stimuli to crayfish without providing chemical and mechanical cues. In one method, crayfish are videotaped in an aquarium where half of the walls are covered in mirrors to provide a reflective environment, and the other half are covered in a non-reflective (matte finish) plastic. This gives the crayfish a choice between reflective and non-reflective environments. The reflective environment provides visual cues in the form of reflected images of the crayfish as it moves throughout half of the tank; these visual cues are missing from the non-reflective half of the tank. An alternative method is to videotape the behavior of crayfish in an aquarium separated by a smaller chamber at each end, with a crayfish in one small chamber providing visual cues and an inert object in the opposite small chamber providing visual input from a non-moving, non-crayfish source. 
Our published results indicate that responses of crayfish to the reflective environment depend on socialization and dominance rank. Socialized crayfish spent more time in the reflective environment and exhibited certain behaviors more frequently there than in the non-reflective environment; isolated crayfish showed no such differences. Crayfish that were housed in same-sex pairs developed a social rank of either dominant or subordinate. Responses to reflection differed between dominant and subordinate crayfish (May &amp; Mercier, 2006; May &amp; Mercier, 2007). Dominant crayfish spent more time on the reflective side, entered reflective corners more frequently and spent more time in reflective corners compared to the non-reflective side. Subordinate crayfish walked in reverse more often on the reflective side than on the non-reflective side. Preliminary data suggest similar effects from visual cues provided by a crayfish in a small adjoining chamber (May et al., 2008).

We have developed two methods for studying effects of visual cues on behavior in the absence of tactile and chemical cues. One method involves videotaping responses of crayfish to reflective walls in an aquarium; the other examines effects of visual inputs provided by a live crayfish behind a transparent partition.

Enregistrement des réponses comportementales à la réflexion chez les écrevisses

Social behavior depends on sensory input from the visual, mechanical and olfactory systems.  One important issue concerns the relative roles of each ...

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 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 ...

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 ...

Subtype-selective Electroporation of Cortical Interneurons

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression of genes of interest to specific neuronal subtypes has proven remarkably challenging due to the relative scarcity of specific promoter elements. GABAergic interneurons constitute a neuronal population with extensive genetic and morphological diversity. Indeed, more than 11 different subtypes of GABAergic interneurons have been characterized in the mouse cortex1. Here we present an adapted protocol for selective targeting of GABAergic populations. We achieved subtype selective targeting of GABAergic interneurons by using the enhancer element of the homeobox transcription factors Dlx5 and Dlx6, homologues of the Drosophila distal-less (Dll) gene2,3, to drive the expression of specific genes through in utero electroporation.

This procedure shows how to target interneurons in the developing mouse forebrain by means of in utero electroporation. This technique was particularly efficient to achieve selective gene expression in interneuron subtypes destined to the superficial layers of the cortex.

Sous-sélective électroporation corticaux Interneurones

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression ...

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

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

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

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

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

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

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing system was developed that allows for the injection of a drug while recording electrophysiological signals from the affected neuronal population. This method provides a simple and economical alternative to commercial solutions. A glass pipette was modified by combining it with a hypodermic needle and a silver filament. The injectrode is attached to commercial microsyringe pump for drug delivery. This results in a technique that provides real-time pharmacodynamics feedback through multi-unit extracellular signals originating from the site of drug delivery. As a proof of concept, we recorded neuronal activity from the superior colliculus elicited by flashes of light in rats, concomitantly with delivery of drugs through the injectrode. The injectrode recording capacity permits the functional characterization of the injection site favoring precise control over the localization of drug delivery. Application of this method also extends far beyond what is demonstrated here, as the choice of chemical substance loaded into the injectrode is vast, including tracing markers for anatomic experiments.

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 ...