AK est soutenu par la Fondation Oppenheim et HR est soutenu par la Fondation Velux. Nous remercions I. Krumbein pour Buder technique et K. de soutien histologique, et J. Goldschmidt (Institut Leibniz de neurobiologie, Magdeburg, Allemagne) pour des conseils techniques sur TIMM coloration.

Toutes les interventions sur les animaux sont effectuées en conformité avec la Convention européenne pour la protection des animaux et l&#39;utilisation des animaux de laboratoire et de la Déclaration ARVO pour l&#39;utilisation d&#39;animaux dans ophtalmique et Vision Research. Toutes les expériences sont approuvés par le comité d&#39;éthique local. La procédure de ON blessures chez les souris est décrit ailleurs 9. Une. Injection intravitréenne de manganèse <ol><li> Effectuer le Mn 2 + injection 24 heures avant le MR scanner à l&#39;aide d&#39;un assistant. Anesthésier les animaux par injection intrapéritonéale d&#39;une solution d&#39;hydrate de chloral à 5% (420 à 450 mg / kg de poids corporel dans du PBS stérile). Pour une anesthésie topique supplémentaire, appliquer une goutte de liquide conjuncain (0,4% de chlorhydrate de oxybuprocaïne) de la cornée avant ponction œil. Pour injecter 15 nmol Mn 2 + par oeil préparer une mM MnCl solution 7,5 2, par exemple, en diluant 1 L de 1 M MnCI2 solution stock dans 132 LH 2 &lt;/ Sub&gt; O. Charge 5 ul de la solution finale dans une seringue de Hamilton de 5 ul relié à un petit moyeu amovible aiguille 34 G (aiguille RN). </li><li> Lors du démarrage de l&#39;œil droit, placez la souris du côté gauche sous un microscope binoculaire et ouvrez-le délicatement et fixer l&#39;œil droit entre le pouce et l&#39;index de votre main gauche. Prenez la seringue avec votre main droite et s&#39;emparer de l&#39;aiguille près de la pointe. Pour ponction atraumatique de l&#39;ampoule d&#39;oeil, insérer délicatement l&#39;aiguille dans le corps vitré à la circonférence de inféro-temporal environ 1 mm distale du limbe, épargnant ainsi les navires scléral. </li><li> Ensuite, l&#39;assistant applique lentement le volume total de 2 pi tout en contrôlant l&#39;échelle de la seringue Hamilton. Au cours de cette procédure, surveiller le placement optimal de l&#39;aiguille sous le microscope et d&#39;éviter la perforation de la lentille ou de déversement du liquide. Gardez l&#39;aiguille insérée statiquement pour un supplément de 30 secondes, puis retirer lentement pour minimiser liquidedes fuites provenant du site d&#39;injection. </li><li> Tout au long de la procédure, une attention particulière doit être prise pour éviter la pression de l&#39;œil. De même, éviter les tentatives difficiles ou nombreuses à percer l&#39;ampoule d&#39;oeil. Depuis Mn 2 + absorption dans les CGR et le transport le long de la ON est déjà saturé à 15 nmol MnCI2, ce qui minimise les variations de signal par des volumes d&#39;injection légèrement imprécises. Pour le signal amélioration bilatérale de la projection visuelle, répétez la procédure d&#39;injection pour l&#39;oeil gauche. </li><li> Appliquer ofloxacine contenant (3 mg / ml) collyre et pommade contenant du panthénol-fois après la procédure de prévenir les infections oculaires et le séchage de l&#39;œil. Remettre les souris dans leurs cages, dans des conditions normales de logement jusqu&#39;à ce que le début de l&#39;examen IRM. </li></ol> 2. Préparation des animaux pour l&#39;IRM <ol><li> Anesthésier les souris par administration d&#39;un mélange à 2% / 98% d&#39;isoflurane gaz / oxygène. Montez la souris sur un support de la souris dans une position presque horizontale sans torsion. Insert-la dans la bobine de MR, qui est ensuite ajustée à l&#39;intérieur de l&#39;appareil d&#39;IRM. Surveillance de la respiration et du rythme cardiaque par des systèmes appropriés. Pour plus de détails techniques, voir Herrmann et al 20. </li><li> Au cours de l&#39;IRM, l&#39;anesthésie par insufflation d&#39;alimentation en continu d&#39;un mélange gazeux initialement 1,5% / 98,5% d&#39;isoflurane / oxygène par l&#39;intermédiaire d&#39;un évaporateur relié au support de la tête de la souris par un tube intégré. Lors de l&#39;analyse, ajuster la profondeur de l&#39;anesthésie en fonction des paramètres vitaux enregistrés (c. viser un taux de respiration stable d&#39;environ 40 respirations par minute). L&#39;utilisation d&#39;un dispositif de chauffage à maintenir la température de surface du corps stable entre 35 et 37 ° C, telle que mesurée par un capteur thermique placé à l&#39;emplacement de l&#39;abdomen de la souris. Pour plus de détails techniques, voir Herrmann et al 17. </li><li> Après le scan, libérer la souris de son support et fournir de l&#39;oxygène pur à accélérer la reprise de l&#39;anesthésie. En outre, maintenir la température corporelle stable en utilisantune source de chauffage de la lumière rouge. </li></ol> 3. Protocole IRM <ol><li> Le protocole est validé pour une Tesla scanner 3 équipé d&#39;une bobine dédié, SNR efficace petit animal (bobine Litz polarisée linéairement) avec un champ de vue effectif de 35 mm × 38 mm de diamètre. Actionner la bobine en mode d&#39;émission-réception. </li><li> Avec l&#39;animal dans sa position finale, ajuster la mise au point et la partie de la bobine à l&#39;aide d&#39;un analyseur de fréquence. Ajuster manuellement la tension de référence de l&#39;émetteur et les courants cales afin d&#39;optimiser l&#39;image homogénéité et la qualité. </li><li> Acquérir T 1 pondérés images 2D EST avec une résolution de 0,5 mm x 0,5 mm x 2 mm en vue sagittale et transversale de la planification. Utilisation des examens IRM de planification, d&#39;acquérir les images MEMR en direction de mesure coronale, tourné pour être parallèle à la tête de l&#39;animal avec codage de phase dans le sens gauche-droite. Pour réduire le temps d&#39;acquisition, utiliser un champ rectangulaire de vue adaptée à laDimensions de la tête réels. Employer une séquence FLASH 3D gâté (VIBE 3D) en utilisant les paramètres suivants: la matrice de base 256, champ de vision 54 mm x 50.65 mm x 14.08 mm, utilisant un champ rectangulaire de 93,8% de vue en direction de codage de phase, et 128 tranches de 0,11 mm l&#39;épaisseur de la tranche à la résolution de la tranche fixée à 61%. </li><li> Activer l&#39;interpolation dans le plan pour créer des images finales avec 512 × 480 × 128, offrant une résolution effective de 0,21 mm x 0,21 mm x 0,18 mm (0,1 mm x 0,1 mm x 0,09 mm interpolés), temps d&#39;écho T E = 6,51 ms, temps de répétition T R = 16 ms, bande passante = 160 Hz / px, angle de bascule = 22 °. Appliquer deux moyennes et trois répétitions pour obtenir un temps d&#39;acquisition total (T A) d&#39;environ 30 min. </li></ol> 4. Analyse des données IRM <ol><li> Analyser les données en utilisant le FastView syngo logiciel. Pour l&#39;amélioration quantitative de signal, sélectionnez régions définies ointérêt de f en 2D planaires enregistrements IRM et déterminer l&#39;intensité du signal (SI) de la structure renforcée (SI MEMRI), fond de tissu (SI backgr), et l&#39;écart type du bruit (SD N). Si nécessaire, utiliser un cerveau de souris atlas pour faciliter l&#39;orientation de neuro-anatomique pour LGN et les structures de SC. Calculer le rapport contraste sur bruit (CNR) selon la formule suivante: </li><li> CNR = (SI MEMRI - SI backgr) / SD N </li><li> Quantifier trois images consécutives pour le calcul CNR moyenne pour chaque échantillon. Chez les animaux injectés bilatéralement, analyser chaque hémisphère indépendamment. </li><li> Pour la représentation des images horizontales, frontal et sagittal, calculer reconstructions multiplanaires de l&#39;ensemble original de données IRM 3D. Ces images traitées ne sont pas recommandés pour l&#39;analyse quantitative. Pour créer des reconstructions 3D animés (projections d&#39;intensité maximale, PMI) de la rétinopathie-Tectale projection, utiliser un module logiciel de post-traitement angiographie. </li></ol> 5. Mn 2 + Autometallography (TIMM coloration) <ol><li> Pour TIMM coloration de Mn 2 structures cérébrales + tracées suivant MEMRI, injecter une dose de 15-150 nmol Mn 2 + ivit 24 heures avant l&#39;imagerie. </li><li> Après numérisation du MR, perfuser les animaux avec 30 ml glacée 0,325% de Na 2 S dans du PBS (pH 7,4). Disséquer la rétine et des échantillons de gel dans la section congelés. </li><li> Couper, sections équatoriales séquentielles de 15 um d&#39;épaisseur sur une cryotome. </li><li> Effectuer TIMM coloration 21 en l&#39;absence de fixateur et de cryoprotection selon Angenstein 22 et al. </li></ol> 6. Analyse statistique Effectuer des analyses statistiques en utilisant le test t de Student pour les comparaisons simples, suivie par la poste ANOVA hoc. Les données sont présentées comme moyenne ± erreur-type. Des numéros individuels N sontséparément pour chaque expérience. Résultats atteignant P ≤ 0,05 sont considérées comme statistiquement significatives (P ≤ 0,05, *, P ≤ 0,01, **, P ≤ 0,001, ***).

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Les auteurs déclarent qu&#39;ils n&#39;ont aucun intérêt financier concurrents.

MEMRI du système visuel s&#39;étend techniques classiques neurobiologiques pour évaluer la fonctionnalité dans des conditions pathologiques et naïfs. En plus de fournir un aperçu unique de l&#39;intégrité d&#39;un isolé CNS faisceau de fibres, MEMRI peut être facilement complétée par des tests de comportement, par exemple, l&#39;optométrie et des tâches à base d&#39;eau visuellement, pour enquêter sur les conséquences immédiates d&#39;un paradigme donné pour la perception visuelle. Il relie ?...

Sur la base de sa structure neuro-anatomique favorable du système visuel rongeur offre des possibilités uniques pour évaluer les composés pharmacologiques et leur capacité à servir de médiateur neuroprotection 1 ou effets pro-régénératives 2,3. En outre, il permet des études sur les caractéristiques fonctionnelles et neuro-anatomique de mutants de souris, comme l&#39;a récemment illustré pour les souris dépourvues de la protéine d&#39;échafaudage présynaptique Basson 4. En outre, un large éventail d&#39;outils complémentaires offre supplémentaire comportant des cellules rétiniennes ganglionnaires (GRC) et le nombre d&#39;axones RGC ainsi que l&#39;activité RGC, par exemple, par électrorétinographie et des tests de comportement, et la détermination des réarrangements corticales par imagerie optique des signaux intrinsèques. Les derniers développements techniques de microscopie laser permettent la visualisation in situ de RGC régénération de tissu profond imagerie de fluorescence dans des spécimens entiers de montage de nerf optique (ON) et le cerveau. Dans ce histologapproche ical, tétrahydrofuranne base compensation des tissus en combinaison avec la lumière microscopie à fluorescence de la feuille permet la résolution des fibres simples qui ré-entrer dans le ON déafférenté et des voies optiques 5. Bien que de telles techniques pourrait être supérieure à la résolution et la détermination de profils de croissance, ils ne permettent pas d&#39;analyses répétitives et longitudinaux des événements de croissance individuels, qui sont particulièrement souhaités pour évaluer le processus de régénération à long terme. Contraste l&#39;IRM a été utilisée pour la visualisation invasive de la projection rétino-tectale chez la souris et le rat 6,7. Ceci peut être réalisé par dépôt intra-oculaire directe des ions paramagnétiques (par exemple, Mn 2 +) à des cellules de la rétine. Comme un analogue de calcium, Mn 2 + est incorporé dans RGC soma via des canaux calciques voltage-dépendants et transporté activement le long du cytosquelette axonal de l&#39;ON intacte et des voies optiques. Bien qu&#39;il s&#39;accumule dans les noyaux du cerveaude la projection visuelle, c&#39;est à dire le corps genouillé latéral (CGL) et colliculus supérieur (SC), propagation transsynaptique dans le cortex visuel primaire semble négligeable 8,9, mais il peut se produire 10,11. Sous séquençage MR, paramagnétique Mn 2 + augmente M. contraste principalement en raccourcissant T 1 spin-réseau temps de relaxation 12. Cette Mn 2 + amélioré IRM (MEMRI) a été appliquée avec succès dans diverses études neuro-anatomiques et fonctionnelles des rats, y compris l&#39;évaluation de la régénération axonale et la dégénérescence après ON blessures 13,14, la cartographie anatomique précise de la projection 15 rétino-tectale , ainsi que la détermination des caractéristiques de transport axonal après le traitement pharmacologique 16. Améliorations récentes dans les doses, la toxicité, et la cinétique des protocoles IRM neuronales Mn 2 + absorption et le transport, ainsi que l&#39;amélioration ont étendu son application à des études sur transgénique9 souris en utilisant 3 Tesla, couramment utilisés dans la pratique clinique 17. Ici, nous présentons un protocole approprié pour MEMRI longitudinal imagerie in vivo de la projection rétino-tectale souris et illustrons son applicabilité en évaluant Mn 2 + amélioration du signal dépend des conditions de neurodégénérescence naïfs et divers. Notre protocole met l&#39;accent spécifique sur l&#39;acquisition de données MR dans un champ magnétique modéré 3 T qui est généralement plus accessibles que les scanners pour animaux dédiés. Chez les souris naïves, nous illustrons comment intensité de signal spécifique à l&#39;appareil peut être sensiblement et de façon reproductible devenir augmenté après intravitréenne (ivit) Mn 2 + application. Quantitativement, Mn 2 + propagation le long de la projection visuelle se produit indépendamment du processus normal de vieillissement (mesurée entre les souris de 3 et 26 mois) et l&#39;augmentation est réfractaire à une stimulation visuelle et de l&#39;adaptation à l&#39;obscurité. En revanche, Mn <sjusqu&#39;à&gt; 2 + enrichissement dans les centres thalamiques et du mésencéphale est diminuée suivant aiguë ON écrasement blessures 18 ainsi que dans les souris knock-out NFKB1 (p50 KO) souffre de la mort spontanée RGC apoptotique et sur ​​la dégénérescence 19. Ainsi, l&#39;expansion à l&#39;analyse histologique conventionnelle, analyse MEMRI longitudinal de chaque animal permet le profilage de la cinétique uniques de processus neurodégénératifs. Cela devrait s&#39;avérer utile pour des études sur la neuroprotection et la régénération axonale associés aux interventions pharmacologiques ou génétiques.

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			<td height="21" style="height:21px;width:363px;">Manganese (II) chloride solution 1M</td>
			<td style="width:269px;">Sigma Aldrich, Taufkirchen, Germany</td>
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			<td>Dr. Mann Pharma, Berlin, Germany</td>
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			<td>0.4% oxybuprocaine hydrochloride</td>
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			<td>Dr. Mann Pharma, Berlin, Germany</td>
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			<td style="width:269px;">Sigma Aldrich, Taufkirchen, Germany</td>
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			<td>420-450 mg/kg body weight</td>
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			<td>Actavis, Munich, Germany</td>
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			<td style="width:269px;">Hamilton Company, Reno, NV, USA</td>
			<td style="width:119px;">207434/00</td>
			<td>removable needle RN, 34 gauge, lenght 38.1 mm, point style 4</td>
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			<td style="width:269px;">Zeiss, Oberkochen, Germany</td>
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			<td height="21" style="height:21px;">3T MRI scanner Magnetom TIM Trio</td>
			<td colspan="2">Siemens Medical Solutions, Erlangen, Germany</td>
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			<td>Doty Scientific Inc., Columbia, SC, USA</td>
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			<td height="42" style="height:42px;width:363px;">Frozen section medium NEG-50</td>
			<td style="width:269px;">Thermo Fisher Scientific, Schwerte, Germany</td>
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			<td>tissue embedding for cryo-sections</td>
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			<td>Merck, Darmstadt, Germany</td>
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			<td height="21" style="height:21px;width:363px;">Sodium sulfide nonahydrate (Na2S &#215; 9 H2O)</td>
			<td>Sigma Aldrich, Taufkirchen, Germany</td>
			<td style="width:119px;">208043</td>
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			<td height="21" style="height:21px;width:363px;">gum arabic</td>
			<td style="width:269px;">Roth, Arlesheim, Switzerland</td>
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			<td rowspan="5">for TIMM staining</td>
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			<td style="width:269px;">Roth, Arlesheim, Switzerland</td>
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			<td style="width:269px;">Merck, Darmstadt, Germany</td>
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in vivo imaging of optic nerve fiber integrity by contrast-enhanced mri in mice

Le système visuel de rongeur englobe les cellules ganglionnaires de la rétine et de leurs axones qui forment le nerf optique pour entrer centres thalamiques et du mésencéphale et projections post-synaptiques dans le cortex visuel. Sur la base de sa structure anatomique distinct et l&#39;accessibilité pratique, il est devenu la structure privilégiée pour les études sur la survie neuronale, la régénération axonale et la plasticité synaptique. Les récents progrès dans l&#39;IRM ont permis la visualisation in vivo de la partie rétino-tectale de cette projection à l&#39;aide de manganèse médiation amélioration du contraste (MEMRI). Ici, nous présentons un protocole MEMRI pour illustration de la projection visuelle chez la souris, que les résolutions de (200 um) 3 peuvent être obtenus en utilisant communes à 3 Tesla. Nous montrons comment l&#39;injection intravitréenne d&#39;une dose unique de 15 nmol MnCI2 conduit à une amélioration saturé de la projection intact dans les 24 heures. A l&#39;exception de la rétine, des changements dans l&#39;intensité du signal sont indédent de la stimulation visuelle coïncidé ou vieillissement physiologique. Nous appliquons en outre cette technique pour suivre longitudinalement dégénérescence axonale suite à une lésion du nerf optique aiguë, un paradigme qui Mn 2 + transport complètement arrestations sur le site de la lésion. Inversement, Mn 2 + transport actif est quantitativement proportionnelle à la viabilité, le nombre et l&#39;activité électrique des fibres axonales. Pour cette analyse, nous illustrons Mn 2 + cinétique de transport le long du trajet optique dans un modèle de souris transgénique (NF-kB p50 KO) l&#39;affichage de l&#39;atrophie spontanée sensorielle, notamment visuelle, des projections. Chez ces souris, MEMRI indique réduite mais non retardé Mn 2 + transport par rapport aux souris de type sauvage, révélant ainsi des signes de dégradations structurales et / ou fonctionnelles des mutations par NF-kB. En résumé, MEMRI comble bien des essais in vivo et post mortem histologie pour la characterizatisur l&#39;intégrité et l&#39;activité des fibres nerveuses. Il est très utile pour les études longitudinales sur la dégénérescence et la régénération axonale, et les enquêtes de souris mutantes pour phénotypes authentiques ou inductibles.

La capacité de cette technique d&#39;imagerie afin d&#39;évaluer avec précision la vitalité et la fonctionnalité de la projection visuelle repose sur une application précise de Mn 2 + posologie non toxique pour le corps vitré et son absorption par les CGR. Cette hypothèse majeure est testée à la figure 1, où la couche spécifique Mn 2 + absorption est démontrée par autometallography (TIMM coloration) 21. Sections de la rétine ont été analysées à 24 heur...

Watch this Scientific Journal Video about In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice at JoVE.com

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

Cette vidéo illustre un procédé, en utilisant un T scanner clinique 3, pour l&#39;imagerie RM de renforcement du contraste de la projection visuelle de la souris naïve et pour des études répétitives et longitudinaux in vivo de la dégénérescence du nerf optique associés à une lésion du nerf optique à l&#39;écrasement aiguë et de la dégénérescence du nerf optique chronique dans souris knock-out (KO p50).

Dans l&#39;imagerie in vivo de Optic Nerve Fiber intégrité par IRM de contraste amélioré chez la souris

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and ...

in-vivo-imaging-optic-nerve-fiber-integrity-contrast-enhanced-mri

Research

JoVE Journal

Neuroscience

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

13.2K vues.  Jena University Hospital.  Cette vidéo illustre un procédé, en utilisant un T scanner clinique 3, pour l'imagerie RM de renforcement du contraste de la projection visuelle de la souris naïve et pour des études répétitives et longitudinaux in vivo de la dégénérescence du nerf optique associés à une lésion du nerf optique à l'écrasement aiguë et de la dégénérescence du nerf optique chronique dans souris knock-out (KO p50). 

Vidéo: In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

Methods for Experimental Manipulations after Optic Nerve Transection in the Mammalian CNS

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals through the vitreous fluid ensures that they act upon the entire RGC population. Viral vectors, plasmids or short interfering RNAs (siRNAs) can also be delivered to the vitreous chamber in order to infect or transfect retinal cells 5-12. The high tropism of Adeno-Associated Virus (AAV) vectors is beneficial to target RGCs, with an infection rate approaching 90% of cells near the injection site 6, 7, 13-15. Moreover, RGCs can be selectively transfected by applying siRNAs, plasmids, or viral vectors to the cut end of the optic nerve 16-19 or injecting vectors into their target the superior colliculus 10. This allows researchers to study apoptotic mechanisms in the injured neuronal population without confounding effects on other bystander neurons or surrounding glia. RGC apoptosis has a characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a window for experimental manipulations directed against pathways involved in apoptosis. Manipulations that directly target RGCs from the transected optic nerve stump are performed at the time of axotomy, immediately after cutting the nerve. In contrast, when substances are delivered via an intraocular route, they can be injected prior to surgery or within the first 3 days after surgery, preceding the initiation of apoptosis in axotomized RGCs. In the present article, we demonstrate several methods for experimental manipulations after optic nerve transection.

Optic Nerve transection is a widely used model of adult CNS injury. This model is ideal for performing a number of experimental manipulations that target the retina globally or directly target the injured neuronal population of retinal ganglion cells.

Méthodes de manipulations expérimentales après section du nerf optique dans le SNC des mammifères

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be ...

Recherche

Neurosciences

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

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

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

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

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

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

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Utilisation de neuro-imagerie fonctionnelle par ultrasons rupture de la barrière hémato-encéphalique et le manganèse-IRM

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has been heavily studied. While many imaging studies treat the hippocampus as a single unitary neuroanatomical structure, it is, in fact, composed of several subfields that have a complex three-dimensional geometry. As such, it is known that these subfields perform specialized functions and are differentially affected through the course of different disease states. Magnetic resonance (MR) imaging can be used as a powerful tool to interrogate the morphology of the hippocampus and its subfields. Many groups use advanced imaging software and hardware (&#62;3T) to image the subfields; however this type of technology may not be readily available in most research and clinical imaging centers. To address this need, this manuscript provides a detailed step-by-step protocol for segmenting the full anterior-posterior length of the hippocampus and its subfields: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus (DG), strata radiatum/lacunosum/moleculare (SR/SL/SM), and subiculum. This protocol has been applied to five subjects (3F, 2M; age 29-57, avg. 37). Protocol reliability is assessed by resegmenting either the right or left hippocampus of each subject and computing the overlap using the Dice&#39;s kappa metric. Mean Dice&#39;s kappa (range) across the five subjects are: whole hippocampus, 0.91 (0.90-0.92); CA1, 0.78 (0.77-0.79); CA2/CA3, 0.64 (0.56-0.73); CA4/dentate gyrus, 0.83 (0.81-0.85); strata radiatum/lacunosum/moleculare, 0.71 (0.68-0.73); and subiculum 0.75 (0.72-0.78). The segmentation protocol presented here provides other laboratories with a reliable method to study the hippocampus and hippocampal subfields in vivo using commonly available MR tools.

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The goal of this manuscript is to study the hippocampus and hippocampal subfields using MRI. The manuscript describes a protocol for segmenting the hippocampus and five hippocampal substructures: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus, strata radiatum/lacunosum/moleculare, and subiculum.

Haute résolution<em&gt; In Vivo</em&gt; Protocole de segmentation manuelle pour les sous-zones de l&#39;hippocampe humain Utilisation 3T imagerie par résonance magnétique

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has ...

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

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

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

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

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

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

Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to monitor the disease course in the follow-up period. Changes in the VEP parameters closely correlate with pathological damage in the optic nerve. This protocol provides a detailed description about the rodent model of optic nerve microinjection, in which a partial demyelination lesion is produced in the optic nerve. VEP recording techniques are also discussed. Using skull implanted electrodes, we are able to acquire reproducible intra-session and between-session VEP traces. VEPs can be recorded on individual animals over a period of time to assess the functional changes in the optic nerve longitudinally. The optic nerve demyelination model, in conjunction with the VEP recording protocol, provides a tool to investigate the disease processes associated with demyelination and remyelination, and can potentially be employed to evaluate the effects of new remyelinating drugs or neuroprotective therapies.

Focal demyelination is induced in the optic nerve using lysolecithin microinjection. Visual evoked potentials are recorded via skull electrodes implanted over the visual cortex to examine the signal conduction along the visual pathway in vivo. This protocol details the surgical procedures underlying electrode implantation and optic nerve microinjection.

Enregistrement Potentiel évoqué visuel dans un modèle de rat of Experimental Optic Nerve démyélinisation

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to ...

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of structural MRI in vivo is ultimately limited by physiological noise, including pulsation, respiration and head movement. Although imaging hardware continues to improve, it is still difficult to resolve structures on the millimeter scale. For example, the primary visual sensory pathways synapse at the lateral geniculate nucleus (LGN), a visual relay and control nucleus in the thalamus that normally is organized into six interleaved monocular layers. Neuroimaging studies have not been able to reliably distinguish these layers due their small size that are less than 1 mm thick.
The resolving limit of structural MRI, in a postmortem brain was tested using multiple images averaged over a long duration (~24 h). The purpose was to test whether it was possible to resolve the individual layers of the LGN in the absence of physiological noise. A proton density (PD)1 weighted pulse sequence was used with varying resolution and other parameters to determine the minimum number of images necessary to be registered and averaged to reliably distinguish the LGN and other subcortical regions. The results were also compared to images acquired in living human brains. In vivo subjects were scanned in order to determine the additional effects of physiological noise on the minimum number of PD scans needed to differentiate subcortical structures, useful in clinical applications.

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

Here we present a protocol to determine the minimum number images that needed to be registered and averaged to resolve subcortical structures and test whether the individual layers of the LGN could be resolved in the absence of physiological noise.

Haute résolution structurelle Imagerie par résonance magnétique de la sous-cortex humain<I&gt; In Vivo</I&gt; Et Postmortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

In Vivo Transfert de gènes à des cellules de Schwann dans le nerf sciatique Rongeur par électroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging de neurones corticaux en souris néonatales

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.