Nous tenons à remercier Britta Kaltwasser pour son excellente assistance technique et Mahesh Kumar Teli à organiser la préparation tournage vidéo.

1. Animaux <ol><li> Des expériences ont été réalisées selon les directives du NIH pour le soin et l&#39;utilisation des animaux de laboratoire et approuvé par les autorités locales. Pour toutes les expériences, C57BL6 / J souris de type sauvage, apolipoprotéine - / - (ApoE KO) et SV129 souris (12-16 semaines, 26-30 g de poids corporel, 5-6 animaux par groupe expérimental) ont été utilisés. </li></ol> 2. Coloration des vaisseaux cérébraux avec du latex coloré <ol><li> Préparer un mélange de 25 ul encre de carbone noir (Herlitz, Allemagne) avec 0,5 ml de composé de latex (Pébéo, France) dans un rapport 1:20 dans un tube PE et faites chauffer le mélange à 37 ° C dans un bain-marie. Recueillir le mélange dans une seringue 2 ml munie d&#39;une aiguille de 28-30G avant que l&#39;animal anesthésié. </li><li> Dissolvez 50 mg de poudre de chlorhydrate papavarine dans 1 ml de solution saline normale stérile. Recueillir la solution dans une seringue à insuline. Casser l&#39;aiguille d&#39;une autre seringue à insuline stérile. Placer cette aiguille à l&#39;extrémité d&#39;un c 20m PE10 tube. Maintenant fixer ce tube PE10 sur l&#39;aiguille de la seringue contenant la solution de chlorhydrate d&#39;insuline papavarine à être injecté dans la veine fémorale pour assurer un remplissage convenable et vasodilatation des vaisseaux. </li><li> Préparer deux autres seringues à insuline contenant chacun 1 ml de solution saline. Seringues à insuline permet une libération lente du fluide injecté à un endroit précis. Toutefois, en raison de la viscosité élevée de latex, de remplacement des seringues de 1 ml avec aiguilles amovibles plus larges devraient être utilisés. </li><li> Prendre toutes les seringues et les instruments stérilisés dissection proches de la phase d&#39;exploitation. Étendre une feuille de drapier chirurgicale autour de la phase d&#39;exploitation. La phase d&#39;exploitation devrait être suffisamment éclairée et d&#39;un microscope opératoire peut être utilisé si nécessaire. </li><li> Maintenant, mesurer le poids du corps d&#39;une souris C57BL6 / J puis induire une anesthésie avec l&#39;isoflurane 5% vaporisé dans 70% de N 2 O et 30% d&#39;O 2. Continuer anesthésie avec de l&#39;isoflurane 1% vaporisé. Après POSITIONNEMENT bong de la souris sur le dos et les branches de fixation, d&#39;évaluer la profondeur de l&#39;anesthésie. Ensuite, couper la peau au-dessus de la veine fémorale gauche et localiser la veine. Insérez l&#39;extrémité pointue de l&#39;aiguille placée sur le tube PE10 et injecter lentement une solution de chlorhydrate papavarine à un taux de 100 pi par min (50 poids corporel mg / kg). </li><li> Après l&#39;injection, couper la cavité abdominale et percer la membrane. Couper les côtes pour exposer le cœur sans endommager ou ecchymoses il. Fixez le aorte thoracique descendante avec une pince hémostatique. Faites une coupe nette sur l&#39;oreillette droite pour permettre au sang veineux s&#39;écouler. Injecter 2 ml de solution saline dans le ventricule gauche, suivie par l&#39;injection du latex coloré manuellement avec une légère pression sur 18-20 sec. Utiliser les seringues pré-remplies l&#39;une après l&#39;autre. Éviter d&#39;injecter des bulles. </li><li> Après l&#39;injection de latex, de laisser l&#39;animal pendant 5 min. Décapiter l&#39;animal et retirez avec précaution l&#39;ensemble du cerveau. Prenez bien soin de ne pas blesser l&#39;architecture des navires et cortex tout en enlevant les os du crâne et des méninges. Placez le cerveau dans une boîte de culture cellulaire de 60 mm contenant 6-8 ml de PFA à 4% pour prendre une photo. Placez une règle de mesure sous la parabole transparente. Prenez des photos avec un grossissement 25x de la dorsale et la face ventrale du cerveau avec une caméra fixée dans le microscope opératoire. Les photos doivent être prises à un angle de 90 ° entre la boîte de Pétri et l&#39;objectif du microscope afin de quantifier la distance d&#39;origine. </li><li> Répétez la procédure dans un autre animaux 4-5. </li></ol> 3. Coloration des vaisseaux cérébraux avec mélange d&#39;encres noir de carbone <ol><li> Pour comparer les résultats de latex à base de coloration vasculaire cérébral avec notre protocole, préparer un mélange de 100 pi de Herlitz Stempelfarbe d&#39;encre (CB1) avec 900 pi de Pelican Scribtol encre Schwarz (CB2) dans un tube de 1,5 ml EP. Préparer deux tubes PE pour obtenir un volume total de 2 ml de mélange de CB1 et CB2 dans un rapport de 1:9. Recueillir le mélange dans deux seringues à insuline séparées (1ml chacune). Reboucher les seringues et les placer dans un bain d&#39;eau à chauffer à 37 ° C. Recueillir 2 ml de solution saline dans un autre seringues à insuline deux et placez-les dans un bain d&#39;eau aussi bien. </li><li> Anesthésier l&#39;animal et répétez la même procédure que celle décrite précédemment pour l&#39;injection de chlorhydrate de papavarine. Suite à l&#39;injection de 2 ml salaine dans le ventricule gauche, injecter un mélange de 2 ml d&#39;encres noir de carbone à la place du latex coloré. Effectuer les injections à la main avec une légère pression sur 18-20 sec par ml. Fixez le aorte thoracique descendante avant les injections. </li><li> Sacrifier l&#39;animal, retirez le cerveau et prendre des photos. </li><li> Pour préservation à long terme du cerveau, mettre le cerveau en PFA à 4% durant la nuit suivie d&#39;une incubation avec du saccharose avec une concentration progressivement croissante jusqu&#39;à ce que le cerveau flottant submerge totalement (5% puis 15% puis 30%). Cerveaux des animaux perfusés avec du latex coloré peut aussi être conservé de la même manière. </li><li> Effectuez l&#39;procédure dans un autre animaux 4-5. Pour comparer la différence de l&#39;anatomie vasculaire cérébral dû à bagage génétique différent ou souches, répéter le protocole en nombre égal de ApoE KO et SV129 souris, respectivement. </li><li> Pour étudier l&#39;anatomie vasculaire dans des conditions ischémiques, induire une ischémie cérébrale focale transitoire pendant 45 min ou 90 min selon un protocole standard de l&#39;occlusion intraluminale de la MCA sous anesthésie profonde (une description détaillée de l&#39;opération dépasse le cadre de cet article, le lecteur est renvoyé à Doeppner et al., 2010). A la fin de la période de reperfusion prévu (1 jour ou 5 jours), effectuer la coloration vasculaire avec CB1 CB2 + uniquement les encres et tacher le cerveau entier avec une solution à 2% TTC à 37 ° C pendant 5-10 min pour identifier le volume de l&#39;infarctus . TTC est réduit en formazan coloré en rouge par les enzymes mitochondriales (en particulier le succinate déshydrogénase). Après coloration TTC, les taches des tissus métaboliquement actifs profonde en rouge lorsque le tissu infarci reste unstainie et apparaît en blanc en raison de dysfonctionnements enzyme mitochondriale et dénaturé. Recueillir des images de la même manière que décrit ci-dessus. </li></ol> 4. Etude des Territoires vasculaires cérébraux <ol><li> Pour analyser l&#39;anatomie des vaisseaux cérébraux, des images de la surface dorsale du cerveau, soit colorées avec du latex coloré ou CB1 CB2 + encres peuvent être analysées avec le logiciel ImageJ (un logiciel Java domaine public utilisé pour le traitement d&#39;images et l&#39;analyse). Brains perfusés avec du latex coloré peut présenter des degrés variables de coloration navire situé sur la face dorsale, tandis que CB1 CB2 + perfusion tacher tous les navires sur les deux faces ventrales et dorsales. </li><li> Ouvrir un fichier image avec le logiciel ImageJ. Identifier tous les points anastomotiques entre l&#39;ACA et le MCA. Un point d&#39;anastomose est défini comme le lieu où le diamètre de la cuve est plus étroit ou la moitié de la distance entre les points les plus proches de ramification de l&#39;ACA et les branches MCA, respectivement 2. <sup&gt; Tracez une ligne imaginaire (ligne anastomotique) en marquant les points anastomotiques avec l&#39;aide d&#39;outil de dessin en ligne segmentée et le plugin &quot;Dotted Line&quot;. </li><li> Définition de l&#39;échelle en mm. Mesurez la distance entre la ligne anastomotique et la ligne médiane de 4 mm caudale du pôle frontal du cerveau en utilisant l&#39;outil de dessin en ligne droite et «mesure» de l&#39;outil. Faites de même à 6 mm caudalement du pôle frontal. Analyser les images 3-4 par animal et calculer la valeur moyenne. Comparer les valeurs entre les différents groupes d&#39;animaux pour identifier la variation de l&#39;anatomie vasculaire cérébral. En C57BL6 / J animaux, la ligne anastomotique entre l&#39;ACA et le MCA peut être retracée plus proche de la ligne médiane de 4 mm à 6 mm de caudale du pôle frontal. Souris KO ApoE présentera pas de différence significative à cet égard. Au contraire, dans SV129 souris la ligne anastomotique se situera mm plus loin et parallèle à la ligne médiane à la fois à 4 mm et 6 caudale du pôle frontal. </li><li> Pour analyser l&#39;anatomie vasculaire in des conditions ischémiques, effectuer les mêmes calculs mentionnés ci-dessus. Identifier la frontière infarctus que la marge de tissu de couleur rouge et blanc représentant métaboliquement active et le tissu nécrotique du tissu non coloré, respectivement. Mesurer la distance de la bordure de la ligne médiane du myocarde à 4 mm et 6 mm à partir du pôle caudal frontal du cerveau. Récupérer la valeur moyenne à partir d&#39;images 3-4 par animal. Comparer les résultats entre les périodes d&#39;ischémie reperfusion et différente. </li></ol>

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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsal+cerebral+arterial+collaterals+of+the+rat.">Dorsal cerebral arterial collaterals of the rat.</a> The Anatomical Record. 203, 397-404 (1982).</li><li>Coyle, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsal+cerebral+collaterals+of+stroke-prone+spontaneously+hypertensive+rats+(SHRSP)+and+Wistar+Kyoto+rats+(WKY).">Dorsal cerebral collaterals of stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar Kyoto rats (WKY).</a> The Anatomical Record. 218, 40-44 (1987).</li><li>Hasan, M. R., Herz, J., 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=Visualization+of+macroscopic+cerebral+vessel+anatomy&#8212;A+new+and+reliable+technique+in+mice.">Visualization of macroscopic cerebral vessel anatomy&#8212;A new and reliable technique in mice.</a> Journal of Neuroscience Methods. 204, 249-253 (2012).</li><li>Doeppner, T. R., Nagel, F., 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=TAT-Hsp70-mediated+neuroprotection+and+increased+survival+of+neuronal+precursor+cells+after+focal+cerebral+ischemia+in+mice.">TAT-Hsp70-mediated neuroprotection and increased survival of neuronal precursor cells after focal cerebral ischemia in mice.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 29, 1187-1196 (2009).</li><li>Todo, K., Kitagawa, K., 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=Granulocyte-macrophage+colony-stimulating+factor+enhances+leptomeningeal+collateral+growth+induced+by+common+carotid+artery+occlusion.">Granulocyte-macrophage colony-stimulating factor enhances leptomeningeal collateral growth induced by common carotid artery occlusion.</a> Stroke. 39 (6), 1875-1882 (2008).</li><li>Sugiyama, Y., Yagita, Y., 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=Granulocyte+colony-stimulating+factor+enhances+arteriogenesis+and+ameliorates+cerebral+damage+in+a+mouse+model+of+ischemic+stroke.">Granulocyte colony-stimulating factor enhances arteriogenesis and ameliorates cerebral damage in a mouse model of ischemic stroke.</a> Stroke. 42 (3), 770-775 (2011).</li><li>Busch, H. 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Neurobiology of Diseases. 6, 101-108 (1999).</li><li>Wang, Y., Kilic, E., 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=VEGF+overexpression+induces+post-ischaemic+neuroprotection,+but+facilitates+haemodynamic+steal+phenomena.">VEGF overexpression induces post-ischaemic neuroprotection, but facilitates haemodynamic steal phenomena.</a> Brain. 128 (1), 52-63 (2005).</li><li>ElAli, A., Doeppner, T. R., 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=Increased+blood-brain+barrier+permeability+and+brain+edema+after+focal+cerebral+ischemia+induced+by+hyperlipidemia:+Role+of+lipid+peroxidation+and+calpain+1/2,+matrix+metalloproteinase-2/9,+and+RhoA+overoxidation.">Increased blood-brain barrier permeability and brain edema after focal cerebral ischemia induced by hyperlipidemia: Role of lipid peroxidation and calpain 1/2, matrix metalloproteinase-2/9, and RhoA overoxidation.</a> Stroke. 42, (2011).</li><li>Coyle, P., Jokelainen, P. 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Aucun conflit d&#39;intérêt déclaré.

La perfusion de CB1 CB2 + par injection manuelle peut être effectuée avec succès par l&#39;absence de formation intensive car il ne comporte pas de dispositif spécifique pour impliquer une certaine pression 2,3. L&#39;hétérogénéité des résultats de perfusion dans notre protocole est également négligeable. 1 seul animal sur 16 animaux non ischémiques et 3 des 20 animaux ischémiques ont montré perfusion incomplète. Dans ces cas, l&#39;incorporation de bulles pendant la perfusion saline conduisan...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Nom de la société </td><td> Catalog No. </td></tr><tr><td> Scribtol Schwarz (CB2) </td><td> Pelican, Allemagne </td><td> 221 135 </td></tr><tr><td> Stempelfarbe (CB1) </td><td> Herlitz PBS AG, Allemagne </td><td> 10417202 </td></tr><tr><td> Gedeo Latex </td><td> Pébéo, France </td><td> 13042B </td></tr></tbody></table>

intravascular perfusion of carbon black ink allows reliable visualization of cerebral vessels

La structure anatomique des vaisseaux cérébraux est un facteur déterminant pour l&#39;hémodynamie du cerveau ainsi que la gravité des blessures à la suite lésions ischémiques. Le système vasculaire cérébral répond dynamiquement aux différents états physiopathologiques et elle présente des différences considérables entre les souches et dans des conditions de manipulations génétiques. Essentiellement, une technique fiable pour la coloration vaisseaux intracrâniens est indispensable pour étudier la pathogenèse de l&#39;AVC ischémique. Jusqu&#39;à tout récemment, un ensemble de techniques différentes ont été utilisées pour visualiser la vascularisation cérébrale, y compris l&#39;injection de résine de faible viscosité, araldite F, de la gélatine mélangée avec divers colorants 1 (c.-à-rouge carmin, encre de Chine) ou en latex avec 2 ou sans 3 du noir de carbone. Perfusion de latex composé blanc à travers l&#39;aorte ascendante a été d&#39;abord rapporté par Coyle et Jokelainen 3. Maeda et al. 2 ont modifié le protocole en ajoutant carbon encre noire pour le composé de latex pour un meilleur contraste de visualisation des vaisseaux après une perfusion saline du cerveau. Cependant, la perfusion inefficace et un mauvais remplissage des navires sont souvent vécu en raison de la viscosité élevée du composé de latex 4. Par conséquent, nous avons décrit une technique simple et rentable en utilisant un mélange de deux encres de carbone commercialement disponibles noir (CB1 et CB2) pour visualiser la vascularisation cérébrale de façon reproductible 5. Nous avons montré que la perfusion avec CB1 CB2 + dans les résultats de la souris, la coloration des vaisseaux cérébraux significativement plus petits à une densité plus élevée par rapport à la perfusion latex 5. Ici, nous décrivons notre protocole d&#39;identifier les points anastomotiques entre la partie antérieure (ACA) et les artères cérébrales moyennes (MCA) pour étudier les variations des navires dans les souris de différentes origines génétiques. Finalement, nous démontrons la faisabilité de notre technique dans un modèle transitoire ischémie cérébrale focale chez la souris en combinant CB1 +Coloration navire CB2 médiée par coloration TTC à des degrés divers de lésions ischémiques.

Le protocole décrit ici permet de surmonter les limitations techniques de visualisation classique à base de latex de la vascularisation cérébrale de rongeurs. La figure 1A montre que la perfusion suivante du latex coloré, seuls les grands navires sur la face ventrale sont tachés, laissant toute la face dorsale non colorées. Le résultat est également très variable. Un seul animal sur six spectacles de coloration partielle de l&#39;ACA et le MCA visible sur la face dorsale du cerveau (données n...

Watch this Scientific Journal Video about Intravascular Perfusion of Carbon Black Ink Allows Reliable Visualization of Cerebral Vessels at JoVE.com

Intravascular Perfusion of Carbon Black Ink Allows Reliable Visualization of Cerebral Vessels

Analyse de l&#39;anatomie vasculaire cérébral des rongeurs joue un rôle important dans la recherche sur l&#39;AVC expérimental. Dans ce contexte, la perfusion intravasculaire avec du latex coloré a été considéré comme un outil standard pour plusieurs années. Cependant, cette technique implique des limitations techniques distinctes, qui nuisent à sa reproductibilité. Nous décrivons ici une méthode simple pour visualiser les vaisseaux cérébraux de façon reproductible. Injection d&#39;un mélange de deux encres noires de carbone disponibles dans le commerce par le biais des résultats du myocarde du ventricule gauche dans le remplissage adéquat des vaisseaux cérébraux avec visualisation à contraste élevé. Nous avons appliqué avec succès cette technique pour identifier les points anastomotiques entre les territoires vasculaires cérébraux de souris avec différents fonds génétiques. Nous avons finalement témoigner que ce nouveau procédé et simple pour la coloration navire peut être combiné avec du chlorure de triphényltétrazolium (TTC) coloration - un outil largement utilisé pour observer et analyser volumes d&#39;infarctus chez la souris.

La perfusion intravasculaire de carbone d&#39;encre noire permet une visualisation fiable des vaisseaux cérébraux

The anatomical structure of cerebral vessels is a key determinant for brain hemodynamics as well as the severity of injury following ischemic insults. The ...

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Neuroscience

15.3K vues.  University of Duisburg-Essen Medical School. Analyse de l'anatomie vasculaire cérébral des rongeurs joue un rôle important dans la recherche sur l'AVC expérimental. Dans ce contexte, la perfusion intravasculaire avec du latex coloré a été considéré comme un outil standard pour plusieurs années. Cependant, cette technique implique des limitations techniques distinctes, qui nuisent à sa reproductibilité. Nous décrivons ici une méthode simple pour visualiser les vaisseaux cérébraux de façon rep...

Vidéo: Intravascular Perfusion of Carbon Black Ink Allows Reliable Visualization of Cerebral Vessels

Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable clinical state; in the development of animal models of focal ischemia, however, achieving reproducibility of experimentally induced infarct volume is essential. The rat is a widely used animal model for stroke due to its relatively low animal husbandry costs and to the similarity of its cranial circulation to that of humans2,3. In humans, the middle cerebral artery (MCA) is most commonly affected in stroke syndromes and multiple methods of MCA occlusion (MCAO) have been described to mimic this clinical syndrome in animal models. Because recanalization commonly occurs following an acute stroke in the human, reperfusion after a period of occlusion has been included in many of these models. In this video, we demonstrate the transient endovascular suture MCAO model in the spontaneously hypertensive rat (SHR). A filament with a silicon tip coating is placed intraluminally at the MCA origin for 60 minutes, followed by reperfusion. Note that the optimal occlusion period may vary in other rat strains, such as Wistar or Sprague-Dawley. Several behavioral indicators of stroke in the rat are shown. Focal ischemia is confirmed using T2-weighted magnetic resonance images and by staining brain sections with 2,3,5-triphenyltetrazolium chloride (TTC) 24 hours after MCAO.

Surgical induction of ischemic brain damage in the rat is a widely used model for stroke research. Here we demonstrate the induction of focal cerebral ischemia by occlusion of the middle cerebral artery. Visualization of the resulting infarct by histological staining and magnetic resonance imaging is also shown.

Focale modèle d&#39;ischémie cérébrale par occlusion suture endovasculaire de l&#39;artère cérébrale moyenne chez le rat

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable ...

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Neurosciences

Automated Interactive Video Playback for Studies of Animal Communication

Video playback is a widely-used technique for the controlled manipulation and presentation of visual signals in animal communication. In particular, parameter-based computer animation offers the opportunity to independently manipulate any number of behavioral, morphological, or spectral characteristics in the context of realistic, moving images of animals on screen. A major limitation of conventional playback, however, is that the visual stimulus lacks the ability to interact with the live animal. Borrowing from video-game technology, we have created an automated, interactive system for video playback that controls animations in response to real-time signals from a video tracking system. We demonstrated this method by conducting mate-choice trials on female swordtail fish, Xiphophorus birchmanni. Females were given a simultaneous choice between a courting male conspecific and a courting male heterospecific (X. malinche) on opposite sides of an aquarium. The virtual male stimulus was programmed to track the horizontal position of the female, as courting males do in the wild. Mate-choice trials on wild-caught X. birchmanni females were used to validate the prototype's ability to effectively generate a realistic visual stimulus.

Video playback is a widely used technique in animal behavior. We created and evaluated a program that applies rules-based, interactive playback of 3-D computer animations in response to real-time, automated data on subject behavior.

Lecture automatique Interactive Video pour les études de la communication animale

Video playback is a widely-used technique for the controlled manipulation and presentation of visual signals in animal communication. In particular, ...

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 ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

Visualisation et la manipulation génétique des dendrites et des épines dans le cortex cérébral et l&#39;hippocampe de souris à l&#39;aide<em&gt; In utero</em&gt; Électroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Deux enregistrements électrophysiologiques des synapses-réponses évoquées astrogliales et neuronales aiguës dans des coupes d&#39;hippocampe

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Electrophysiology on Isolated Brainstem-spinal Cord Preparations from Newborn Rodents Allows Neural Respiratory Network Output Recording

While it is well known that the central respiratory drive is located in the brainstem, several aspects of its basic function, development, and response to stimuli remain to be fully understood. To overcome the difficulty of accessing the brainstem in the whole animal, isolation of the brainstem and part of the spinal cord is performed. This preparation is maintained in artificial cerebro-spinal fluid where gases, concentrations, and temperature are controlled and monitored. The output signal from the respiratory network is recorded by a suction electrode placed on the fourth ventral root. In this manner, stimuli can be directly applied onto the brainstem, and the effect can be recorded directly. The signal recorded is linked to the inspiratory signal sent to the diaphragm via the phrenic nerve, and can be described as bursts (around 8 bursts per minute). Analysis of these bursts (frequency, amplitude, length, and area under the curve) allows precise characterization of the stimulus effect on the respiratory network. The main limitation of this method is the viability of the preparation beyond the early post-natal stages. Thus, this method greatly focuses on the study of the whole network without the peripheral inputs in the newborn rat.

The central respiratory drive is located in the brainstem. Spontaneous respiratory motor output from an isolated brainstem-spinal cord is recorded by placing an electrode on the fourth ventral root. This experimental approach is valuable for pharmacological investigations or the assessment of respiratory challenges and genetic manipulations on rhythmic motor behavior.

Électrophysiologie sur isolés Cord Préparatifs du tronc cérébral-spinal de la naissance Rongeurs Permet Neural respiratoire Réseau d&#39;enregistrement de sortie

While it is well known that the central respiratory drive is located in the brainstem, several aspects of its basic function, development, and response to ...

Purification of Mouse Brain Vessels

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and immune cells between the brain and the blood. Moreover, the huge neuronal metabolic demand requires a moment-to-moment regulation of blood flow. Notably, abnormalities of these regulations are etiological hallmarks of most brain pathologies; including glioblastoma, stroke, edema, epilepsy, degenerative diseases (ex: Parkinson&#8217;s disease, Alzheimer&#8217;s disease), brain tumors, as well as inflammatory conditions such as multiple sclerosis, meningitis and sepsis-induced brain dysfunctions. Thus, understanding the signaling events modulating the cerebrovascular physiology is a major challenge. Much insight into the cellular and molecular properties of the various cell types that compose the cerebrovascular system can be gained from primary culture or cell sorting from freshly dissociated brain tissue. However, properties such as cell polarity, morphology and intercellular relationships are not maintained in such preparations. The protocol that we describe here is designed to purify brain vessel fragments, whilst maintaining structural integrity. We show that isolated vessels consist of endothelial cells sealed by tight junctions that are surrounded by a continuous basal lamina. Pericytes, smooth muscle cells as well as the perivascular astrocyte endfeet membranes remain attached to the endothelial layer. Finally, we describe how to perform immunostaining experiments on purified brain vessels.

We describe a protocol allowing the purification of the mouse brain's vascular compartment. Isolated brain vessels include endothelial cells linked by tight junctions and surrounded by a continuous basal lamina, pericytes, vascular smooth muscle cells, as well as perivascular astroglial membranes.

Purification de souris vaisseaux cérébraux

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and ...

Isolation and Cannulation of Cerebral Parenchymal Arterioles

Intracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance blood vessels branching out from pial arteries and arterioles and diving into the brain parenchyma. Individual PA perfuse a discrete cylindrical territory of the parenchyma and the neurons contained within. These arterioles are a central player in the regulation of cerebral blood flow both globally (cerebrovascular autoregulation) and locally (functional hyperemia). PAs are part of the neurovascular unit, a structure that matches regional blood flow to metabolic activity within the brain and also includes neurons, interneurons, and astrocytes. Perfusion through PAs is directly linked to the activity of neurons in that particular territory and increases in neuronal metabolism lead to an augmentation in local perfusion caused by dilation of the feed PA. Regulation of PAs differs from that of better-characterized pial arteries. Pressure-induced vasoconstriction is greater in PAs and vasodilatory mechanisms vary. In addition, PAs do not receive extrinsic innervation from perivascular nerves &#8212; innervation is intrinsic and indirect in nature through contact with astrocytic endfeet. Thus, data regarding contractile regulation accumulated by studies using pial arteries does not directly translate to understanding PA function. Further, it remains undetermined how pathological states, such as hypertension and diabetes, affect PA structure and reactivity. This knowledge gap is in part a consequence of the technical difficulties pertaining to PA isolation and cannulation. In this manuscript we present a protocol for isolation and cannulation of rodent PAs. Further, we show examples of experiments that can be performed with these arterioles, including agonist-induced constriction and myogenic reactivity. Although the focus of this manuscript is on PA cannulation and pressure myography, isolated PAs can also be used for biochemical, biophysical, molecular, and imaging studies.

This manuscript describes a simple and reproducible protocol for isolation of intracerebral arterioles (a group of blood vessels encompassing parenchymal arterioles, penetrating arterioles and pre-capillary arterioles) from mice, to be used in pressure myography, immunofluorescence, biochemistry, and molecular studies.

Isolement et Cannulation de parenchyme cérébral artérioles

Intracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance ...

Intracerebroventricular and Intravascular Injection of Viral Particles and Fluorescent Microbeads into the Neonatal Brain

In the study on the pathogenesis of viral encephalitis, the infection method is critical. The first of the two main infectious routes to the brain is the hematogenous route, which involves infection of the endothelial cells and pericytes of the brain. The second is the intracerebroventricular (ICV) route. Once within the central nervous system (CNS), viruses may spread to the subarachnoid space, meninges, and choroid plexus via the cerebrospinal fluid. In experimental models, the earliest stages of CNS viral distribution are not well characterized, and it is unclear whether only certain cells are initially infected. Here, we have analyzed the distribution of cytomegalovirus (CMV) particles during the acute phase of infection, termed primary viremia, following ICV or intravascular (IV) injection into the neonatal mouse brain. In the ICV injection model, 5 &#181;l of murine CMV (MCMV) or fluorescent microbeads were injected into the lateral ventricle at the midpoint between the ear and eye using a 10-&#181;l syringe with a 27 G needle. In the IV injection model, a 1-ml syringe with a 35 G needle was used. A transilluminator was used to visualize the superficial temporal (facial) vein of the neonatal mouse. We infused 50 &#181;l of MCMV or fluorescent microbeads into the superficial temporal vein. Brains were harvested at different time points post-injection. MCMV genomes were detected using the in situ hybridization method. Fluorescent microbeads or green fluorescent protein expressing recombinant MCMV particles were observed by fluorescent microscopy. These techniques can be applied to many other pathogens to investigate the pathogenesis of encephalitis.

Here, we describe a simple method of intracerebroventricular and intravascular injection of viral particles or fluorescent microbeads into the neonatal mouse brain. The localization pattern of the virus and nanoparticles could be detected by microscopic evaluation or by in situ hybridization.

Intracérébroventriculaire et intravasculaire Injection de particules virales et fluorescentes microbilles dans le cerveau néonatal

In the study on the pathogenesis of viral encephalitis, the infection method is critical. The first of the two main infectious routes to the brain is the ...

A Rat Model of Mild Intrauterine Hypoperfusion with Microcoil Stenosis

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies of this phenomenon have been performed in either models with severe intrauterine ischemia or models with gradient degree of intrauterine hypoperfusion. No study has been performed in a model on uniform mild intrauterine hypoperfusion (MIUH). Two models have been used for studies of MIUH: a model based on suture ligation of either side of the arterial arcade formed with the uterine and ovarian arteries, and a transient model based on clipping the bilateral ovarian arteries and aorta having patency. Those two rodent models of MIUH have some limitations, e.g., not all fetuses are subjected to MIUH, depending on their position in the uterine horn. In our MIUH model, all fetuses are subjected to a comparable level of intrauterine hypoperfusion. MIUH was achieved by mild stenosis of all four arteries feeding the uterus, i.e., the bilateral uterine and ovarian arteries.
Arterial stenosis was induced by metal microcoils wrapped around the feeding arteries. Producing arterial stenosis with microcoils allowed us to control, optimize, and reproduce decreased blood flow with very little inter-animal variability and a low mortality rate, thus enabling accurate evaluation. When microcoils with an inner diameter of 0.24 mm were used, the blood flow in both the placenta and fetus was mildly decreased (approximately 30% from the pre-stenosis level in the placenta). The offspring of our MIUH model clearly demonstrates long-lasting alterations in neurological, neuroanatomical and behavioral test results.

Mild intrauterine hypoperfusion was produced by artery stenosis with metal microcoils wrapped around the uterine and ovarian arteries in rats at embryonic day 17. This procedure produced prenatal hypoperfusion and intrauterine growth restriction.

Un modèle de Rat de doux Hypoperfusion intra-utérine avec sténose de le Microcoil

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies ...