We would like to thank Erica Lorenzetti for technical assistance on the injections and Irene Costantini for making figure 1. The research leading to these results has received funding from LASERLABEUROPE (Grant 284464, European Commission&#8217;s Seventh Framework Programme). This research project has also been supported by the Italian Ministry for Education, University and Research in the framework of the Flagship Project NANOMAX and by Italian Ministry of Health in the framework of the &#8220;Stem Cells Call for Proposals.&#8221; This work is part of the research activities of the European Flagship Human Brain Project and has been carried out in the framework of the International Center of Computational Neurophotonics foundation supported by &#8220;Ente Cassa di Risparmio di Firenze&#8221;.

1. Étiquetage axonale <ol><li> Fibres d&#39;escalade peut être marqué soit en injectant des colorants organiques conjugués à des dextrans de haut poids moléculaire ou des plasmides / virus qui induisent l&#39;expression de protéines fluorescentes de 26 à 29. Dans ce protocole, le colorant organique Alexa Fluor Dextran 488 est injecté dans l&#39;olive inférieure à étiqueter fibres grimpantes et les visualiser dans le cortex cérébelleux (figure 1). Toutes les procédures décrites ici ont été approuvés par le ministère italien de la Santé. </li><li> Préparer le tube capillaire en verre en le tirant sur un étirage de micropipettes. Couper la pointe du tube capillaire en verre avec des ciseaux jusqu&#39;à ce que le diamètre extérieur est d&#39;environ 30 um. </li><li> Charger le capillaire avec 1-2 ul du colorant de dextrane conjugué. </li><li> Avant de commencer, stériliser tous les instruments chirurgicaux dans un autoclave. Au cours de la procédure, de réduire les risques de contamination à l&#39;aide d&#39;un stérilisateur à billes de verre. </li><li> Anesthésier la souris (9-12semaines) par injection intraperitoneale de kétamine (90 mg / kg) et de xylazine (9 mg / kg). Assurez-vous que l&#39;animal est entièrement sous sédation en utilisant la queue et / ou pincées d&#39;orteil. Utilisez vétérinaire pommade sur les yeux pour prévenir la sécheresse tandis que sous anesthésie. </li><li> Raser les poils au-dessus du cou avec une lame de rasoir ou la crème dépilatoire. Badigeonner le site chirurgical deux fois avec de la bétadine, en alternance avec de l&#39;éthanol 70%. </li><li> Placez la souris sur un support stéréotaxique. Gardez l&#39;animal au chaud avec un coussin chauffant. </li><li> Poussez doucement les earbars sur l&#39;os à côté des oreilles de sorte que la tête est maintenue fermement. Tourner la tête pour former un angle de 140 ° avec le corps. L&#39;orientation souhaitée peut être obtenue en modifiant légèrement la hauteur de la tige de support de nez sur le support stéréotaxique. </li><li> Appliquer quelques gouttes de lidocaïne sur la peau entre les oreilles. Utiliser une pince ou une lame de rasoir pour effectuer une incision verticale d&#39;environ 1 cm sur la peau au-dessous de l&#39;occiput. </li><li> Séparez délicatement le tissu sous-cutané et les muscles par pince émoussées sous le microscope de dissection. Abaissez le faisceau musculaire horizontale et tenir en dehors des deux muscles verticaux fascicules afin d&#39;exposer la dure sur le foramen magnum. </li><li> Utilisation de la plus grande prudence, disséquer la durée avec une aiguille de seringue ou une pince très pointus pour exposer le tronc cérébral. Une petite quantité de liquide céphalo-rachidien peut se répandre lorsque la durée a été réduite. </li><li> Sur le micromanipulateur stéréotaxique, faire tourner le support du tube capillaire à 45 ° de la verticale. Placer le tube capillaire sur la ligne médiane, à mi-chemin entre le bord caudal du cortex cérébelleux et la première vertèbre cervicale. Insérer la pipette à une profondeur de 2 mm de la surface. </li><li> Délivrer un volume 0,5-1,5 pi de colorant sur 10-15 min. Un système de distribution de micro-injection permet de délivrer de la pression contrôlée du liquide dans le capillaire (pression d&#39;impulsion = 20 psi; durée d&#39;impulsion = 3-4 ms, la fréquence des impulsions de livraison = 12 Hz). </li><li> Laissez le capillaire en place pendant 15 min, puis slowly (en 2-3 min) retirez-le. Délicatement réaligner les muscles et suturer la peau au-dessus. Gardez les muscles bien hydratés par l&#39;application de certaines saline toute la procédure. </li><li> Pour éviter la déshydratation, injecter sous-cutanée de 0,5 ml de NaCl à 0,9%. Garder l&#39;animal dans une cage chauffée jusqu&#39;à récupération complète de l&#39;anesthésie. </li><li> Sous-cutanée administrer Carprofen (5 mg / kg) par jour pendant 2-3 jours après la chirurgie. </li></ol> 2. Fenêtre optique sur le cervelet Cortex NOTE: Le colorant est transporté dans 2 semaines du noyau olivaire inférieur à la cortex cérébelleux aux bornes du SCF (voir la figure 1). Les SADC peu marquées peuvent être visualisés sous la fenêtre crânienne permanent qui peut être effectué comme suit: <ol><li> Stériliser tous les instruments chirurgicaux. </li><li> Anesthésier la souris comme décrit ci-dessus. Utilisez pincées d&#39;orteil pour vérifier l&#39;animal est entièrement sous sédation. </li><li> Raser les cheveux au-dessus de la tête, essuyez le skavec des balayages alternés de 70% d&#39;alcool et de la bétadine et appliquer une pommade oculaire. Placez la souris dans un cadre stéréotaxique et positionner les barres d&#39;oreilles afin de maintenir fermement la tête. </li><li> Sous-cutanée administrer la dexaméthasone (0,2 mg / kg) et Carprofen (5 mg / kg) pour éviter le gonflement du cerveau et les inflammations possibles à la fenêtre le site crânienne. </li><li> Appliquez une goutte de la lidocaïne sur la peau au-dessus du crâne et couper un volet, d&#39;entre les oreilles jusqu&#39;au-dessus des yeux. Appliquer la solution de lidocaïne à nouveau sur le périoste avant de gratter avec un scalpel. </li><li> Utilisez la fraise de dentiste à une mince région semi-circulaire de crâne au-dessus du cervelet. La fenêtre est habituellement placé central et vers l&#39;arrière de suture, de sorte que la profondeur de crâne est à peu près constante tout au long du pourtour de la demi-cercle. Positionnement de la fenêtre trop près de la suture lambda et forage répétitif sur cette région très épais peut entraîner une surchauffe du cerveau et de la formation d&#39;oedèmes. </li><li> Appliquer hémostatiqueéponge sur le crâne en cas de saignement. </li><li> Couper une lamelle en deux parties avec l&#39;aide d&#39;une lame de rasoir et une pince à épiler. Vérifiez que la lamelle est légèrement plus grand que l&#39;île de l&#39;os et a une forme similaire avant de retirer le volet de l&#39;os avec la pince à épiler; sinon, remodeler la lamelle ou en choisir un autre. Ne touchez pas la dure-mère avec la pince à épiler en tirant de l&#39;os. Poser doucement sur la lamelle sur la durée. NOTE: L&#39;adhérence de la totalité de la surface de la vitre sur la dure-mère est prédisait de longues fenêtres crâniens durables. </li><li> Retirer les bulles d&#39;air sous le verre en le rinçant à plusieurs reprises avec une solution saline et séchage avec des bâtons de coton. Cela aidera également à éliminer des restes de sang qui pourraient être déversés sur des petits capillaires après le positionnement de la vitre. Sceller la fenêtre avec un mélange de la colle acrylique et le ciment dentaire 30. </li><li> Gardez l&#39;animal sur un coussin chauffant dans une chambre de récupération jusqu&#39;à ce que complètement récupéré de l&#39;anesthésie. Ne pas réutilisertourner la souris dans la cage de la maison jusqu&#39;à ce que complètement rétabli; ne les laissez pas sans surveillance jusqu&#39;à ce qu&#39;ils repris conscience suffisante pour maintenir décubitus sternal. </li><li> Sous-cutanée administrer Carprofen (5 mg / kg) par jour pendant 2-3 jours après la chirurgie. </li></ol> 3. In Vivo multi-photons laser axotomie <ol><li> Attendez 2-7 jours de la chirurgie avant d&#39;effectuer la première session d&#39;imagerie. Le construit sur mesure à deux photons microscope utilisé ici est pourvue d&#39;un Ti: source laser saphir (120 fs impulsions en largeur, le taux de répétition de 90 MHz), qui est d&#39;abord numérisé par une paire de miroirs galvanométriques et ensuite focalisé sur l&#39;échantillon par une immersion dans l&#39;eau 20x objectif (NA 0,95, WD 2 mm). Une étape piézoélectrique en boucle fermée exécute les déplacements axiaux jusqu&#39;à 400 um de l&#39;objectif. tubes photomultiplicateurs collectent le signal de fluorescence. </li><li> Anesthésier l&#39;animal, puis placez la tête dans le support stéréotaxique de sorte que la fenêtre crânienne fixe parallèle à l&#39;objectivationve plan focal. </li><li> Explorez avec TPF imagerie du cortex sous la fenêtre crânienne. Choisissez l&#39;axone d&#39;être endommagés parmi les plus brillants. </li><li> Acquérir un Z-stack (généralement le champ de vision est de 100 x 100 um 2, à 512 x 512 pixels, 2 pm axe z étape) pour obtenir une 3D reconstructions de l&#39;axone ciblée. Dans le volume imagé, choisir le site de la lésion sur une branche axonale qui est la plupart du temps parallèle au plan focal. Cette orientation permet de marquer rapidement des changements morphologiques, qui sont prédictifs d&#39;une dissection de succès. </li><li> Irradier avec une énergie forte dose au point sélectionné sur une portion distale d&#39;un CF. Cela se fait par le logiciel Labview: augmenter la puissance du laser 5-10x plus de la puissance utilisée pour l&#39;imagerie (≈ 300 mW, mesurée après l&#39;objectif), définir la position des miroirs galvanométriques pour faire un scan en ligne (~ 4 - 10 pm) sur le site de la lésion et ouvrir l&#39;obturateur de laser pour 400 à 600 msec. Orienter le balayage de ligneperpendiculaire au plan CF à sectionner complètement l&#39;axone. Irradier taches lumineuses (par exemple., Varicosités) permet généralement de dissection de l&#39;axone. La longueur d&#39;onde de laser axotomie est le même que celui utilisé pour l&#39;imagerie (920 nm). </li><li> Habituellement, la fluorescence de l&#39;axone dans la zone irradiée diminue passagèrement en raison de photoblanchiment. L&#39;acquisition d&#39;une pile de la même région, quelques minutes après l&#39;irradiation pour assurer l&#39;ablation a été bien effectuée. Si la région a commencé le gonflement et l&#39;axone formé des structures en forme de perles, le neurone a été disséqué avec succès. Dans ce cas, en quelques heures (de quelques dizaines de minutes à 24 heures) de la partie distale de l&#39;axone va dégénérer et disparaître complètement. </li><li> Malheureusement, la dose d&#39;énergie nécessaire pour produire une axotomie complète n&#39;est pas toujours prévisible. Si le seul résultat de l&#39;irradiation est photoblanchiment, la fluorescence devrait être récupéré rapidement en raison de la diffusion de colorant, et l&#39;irradiation doit être répétée à nouveau à efficacement disséquer l&#39;axone. En outre, dans le cas des signes de gonflement n&#39;apparaissent pas dans les 5 - 20 min, ou le gonflement est suivie par la récupération (. Analogue à ce qui a été décrit pour dendrotomy laser, voir Allegra Mascaro et al 23), essayez à nouveau avec de nouveaux scans de plus en plus la le temps de séjour et / ou la puissance du laser. </li><li> Surveillance de la rénovation éventuelle de l&#39;axone dans les jours qui suivent la axotomy laser avec deux photons imagerie in vivo (figures 2 et 3). Le réseau vasculaire est utilisé comme modèle de référence pour récupérer l&#39;axone disséqué dans les jours suivants. </li><li> Sous-cutanée administrer Carprofen (5 mg / kg) chaque séance d&#39;imagerie pour toute la durée de l&#39;expérience. </li></ol>

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We have nothing to disclose.

Ce protocole montre comment étiqueter les neurones de l&#39;olive inférieure avec un colorant fluorescent. Par la suite, le procédé pour réaliser une fenêtre crânienne sur le cortex cérébelleux est décrite. Cette technique permet un accès optique à la partie terminale de neurones olivocerebellar, les fibres d&#39;escalade. Malheureusement, le résultat à la fois de l&#39;étiquetage et de la chirurgie de craniotomie est assez faible, même dans les mains d&#39;opérateurs qualifiés (généralement 1 sur 3 ...

Transection axonale résultant d&#39;une lésion mécanique, agression toxique ou les maladies neurodégénératives est généralement suivie par la dégénérescence de la partie distale de l&#39;axone qui est séparée du corps de la cellule de 10 à 13. À quelques exceptions près 2,7,14,15, les axones sectionnés dans le SNC des animaux adultes sont généralement incapables d&#39;activer un programme de régénération 16. On sait peu sur la dynamique en temps réel des événements dégénératives au niveau cellulaire et subcellulaire. Le développement de nouvelles stratégies pour limiter les dommages neuronaux et favoriser la repousse neuronale nécessite, dans un premier temps, de clarifier le mécanisme par lequel les cellules neuronales singulièrement blessés dégénèrent et se régénèrent. Cette étude est la plus directement visée par le suivi de la dynamique d&#39;un seul neurone in vivo. Bien que les techniques à un photon de fluorescence imagerie sont limitées par la diffusion intense de la lumière visible, excitation à deux photons atteint couches corticales profondes live souris avec subcellulaire résolution 3,4,17. Profitant de souris transgéniques dans lesquelles les protéines fluorescentes sont exprimés sélectivement dans les sous-populations de neurones 18-20, TPF microscopie a été appliqué à l&#39;exploration de la plasticité synaptique et l&#39;allongement axonal pendant le développement in vivo 21,22. L a capacité des neurones endommagés singulièrement à repousser après une blessure peut être étudiée par couplage dans le suivi in vivo par imagerie à deux photons avec un modèle de lésion spécifiquement ciblées sur l&#39;axone d&#39;intérêt. Multi-photon absorption d&#39;impulsions femtosecondes a été utilisée pour perturber dendrites individuelles ou même des épines simples 5,23. En outre, cette blessure paradigme permet de couper des branches axonales simples sans perturber la dendrite contact 6. Dans le cadre de disséquer les fonctionnalités qui permettent population neuronale spécifique pour régénérer leurs axones fois endommagés, fibres grimpantes du cervelet (FC) sont un modèle si utilence ils conservent des propriétés plastiques remarquables après une blessure, même chez les animaux adultes 24,25. Récemment, l&#39;imagerie à long terme des FC a montré que ces axones sont capables de repousse dans les jours qui suivent axotomy laser 6. Ce protocole décrit la façon d&#39;étiqueter les neurones olivocerebellar et leur allongement des axones à travers antérograde traçage. Une fois que les neurones d&#39;intérêt sont marqués par fluorescence, ils peuvent être contrôlés de manière répétée à des instants arbitraires pendant des semaines ou des mois de moins d&#39;une fenêtre crânienne. La procédure de disséquer branches axonales simples par axotomie laser in vivo sera ensuite illustrée. Les techniques présentées ici fournissent de nouveaux aperçus dans le mécanisme de remodelage des axones in vivo et peuvent aider le développement de stratégies thérapeutiques pour limiter la dégénérescence neuronale et favoriser la repousse axonale.

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	<tbody>
		<tr>
			<td>Lab standard stereotaxic, rat and mouse</td>
			<td>Stoelting&#160;</td>
			<td>51670</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass with filament</td>
			<td>Sutter Instrument Inc</td>
			<td>BF100-50-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Germinator 500 (Glass bead sterilizer)</td>
			<td>Roboz</td>
			<td></td>
			<td></td>
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			<td>Microinjection dispense system</td>
			<td>Picospritzer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Small diameter round cover glass, #1 thickness, 3 mm, 100 pack (CS-3R)</td>
			<td>Warner Instruments&#160;</td>
			<td>64-0720</td>
			<td></td>
		</tr>
		<tr>
			<td>Ti:Sapphire laser, 120 fs width pulses,&#160; 90 MHz repetition rate</td>
			<td>Coherent</td>
			<td>Chameleon&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Spongostan, haemostatic sponge&#160;</td>
			<td>Ferrosan</td>
			<td>MS0005</td>
			<td></td>
		</tr>
		<tr>
			<td>Galvanometric mirrors&#160;</td>
			<td>GSI Lumonics</td>
			<td>VM500+</td>
			<td></td>
		</tr>
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			<td>Objective&#160;</td>
			<td>&#160;Olympus</td>
			<td>XLUMPLFLN 20XW</td>
			<td></td>
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		<tr>
			<td>Piezoelectric stage&#160;</td>
			<td>Physik Instrumente</td>
			<td>P-721</td>
			<td></td>
		</tr>
		<tr>
			<td>Photomultiplier modules&#160;</td>
			<td>Hamamatsu Photonics</td>
			<td>H7710-13</td>
			<td></td>
		</tr>
		<tr>
			<td>LabVIEW System Design Software</td>
			<td>National Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Voren, 1 mg/ml (dexamethasone-21- isonicotinate)</td>
			<td>Boheringer Ingelheim</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rymadil (carprofen)&#160;</td>
			<td>Pfizer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lidocaine clorohydrate 2%</td>
			<td>ATI</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa488 dextran</td>
			<td>Life Technologies</td>
			<td>D22910</td>
			<td></td>
		</tr>
	</tbody>
</table>

laser nanosurgery of cerebellar axons in vivo

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex5-9.
This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury.

Ce protocole décrit comment effectuer l&#39;étiquetage axonale, l&#39;imagerie in vivo et axotomy laser sur les neurones simples. Le calendrier de l&#39;expérience est illustré à la figure 1. Un exemple de FC marquées avec Alexa Fluor 488 Dextran et visualisées sous la fenêtre crânienne in vivo par microscopie à deux photons est rapporté sur la figure 2. 6,27 Comme indiqué précédemment, les branches ascendantes pré...

Watch this Scientific Journal Video about Laser Nanosurgery of Cerebellar Axons In Vivo at JoVE.com

Laser Nanosurgery of Cerebellar Axons In Vivo

Imagerie à deux photons, couplé à nanodissection laser, sont des outils utiles pour étudier les processus dégénératifs et régénératifs dans le système nerveux central avec une résolution subcellulaire. Ce protocole indique comment étiqueter, image, et disséquer fibres grimpantes simples dans le cortex cérébelleux in vivo.

Laser nanochirurgie de cervelet axones<em&gt; In Vivo</em

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to ...

laser-nanosurgery-of-cerebellar-axons-in-vivo

Research

JoVE Journal

Neuroscience

Laser Nanosurgery of Cerebellar Axons In Vivo

8.1K vues.  University of Florence.  Imagerie à deux photons, couplé à nanodissection laser, sont des outils utiles pour étudier les processus dégénératifs et régénératifs dans le système nerveux central avec une résolution subcellulaire. Ce protocole indique comment étiqueter, image, et disséquer fibres grimpantes simples dans le cortex cérébelleux in vivo. 

Vidéo: Laser Nanosurgery of Cerebellar Axons In Vivo

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different neurological diseases. Defects in this essential process can have detrimental effects on neuronal functioning and development. We have developed a dissection protocol that is designed to expose the Drosophila larval segmental nerves to view axonal transport in real time. We have adapted this protocol for live imaging from the one published by Hurd and Saxton (1996) used for immunolocalizatin of larval segmental nerves. Careful dissection and proper buffer conditions are critical for maximizing the lifespan of the dissected larvae. When properly done, dissected larvae have shown robust vesicle transport for 2-3 hours under physiological conditions. We use the UAS-GAL4 method 1 to express GFP-tagged APP or synaptotagmin vesicles within a single axon or many axons in larval segmental nerves by using different neuronal GAL4 drivers. Other fluorescently tagged markers, for example mitochrondria (MitoTracker) or lysosomes (LysoTracker), can be also applied to the larvae before viewing. GFP-vesicle movement and particle movement can be viewed simultaneously using separate wavelengths.

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

This protocol discusses the live dissection of Drosophila larvae for the purpose of imaging the movement of GFP tagged axonal vesicles on microtubule tracks.

Visualisation in vivo des vésicules synaptiques Dans Drosophila larves axones segmentaire

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

Recherche

Neurosciences

In vivo Laser Axotomy in C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron is damaged by injury or disease, it may regenerate. Cell-intrinsic and extrinsic factors influence the ability of a neuron to regenerate and restore function. Recently, the nematode C. elegans has emerged as an excellent model organism to identify genes and signaling pathways that influence the regeneration of neurons1-6. The main way to initiate neuronal regeneration in C. elegans is laser-mediated cutting, or axotomy. During axotomy, a fluorescently-labeled neuronal process is severed using high-energy pulses. Initially, neuronal regeneration in C. elegans was examined using an amplified femtosecond laser5. However, subsequent regeneration studies have shown that a conventional pulsed laser can be used to accurately sever neurons in vivo and elicit a similar regenerative response1,3,7.
We present a protocol for performing in vivo laser axotomy in the worm using a MicroPoint pulsed laser, a turnkey system that is readily available and that has been widely used for targeted cell ablation. We describe aligning the laser, mounting the worms, cutting specific neurons, and assessing subsequent regeneration. The system provides the ability to cut large numbers of neurons in multiple worms during one experiment. Thus, laser axotomy as described herein is an efficient system for initiating and analyzing the process of regeneration.

In vivo Laser Axotomy in C. elegans

A protocol to cut neurons in C. elegans with a MicroPoint pulsed laser is presented. We describe setting up the system, immobilizing worms, and severing labeled neurons. Advantages include a relatively low-cost system and the ability to sever neuronal processes or ablate cells in vivo.

In vivo laser axotomie en C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron ...

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 ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

Manipulation génétique des neurones du cervelet granules<em&gt; In Vitro</em&gt; Et<em&gt; In Vivo</em&gt; À étudier neuronale Morphologie et migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then radial migration in the molecular layer and the Purkinje cell layer to reach the internal granular layer of the cerebellar cortex. Default in migratory processes induces either cell death or misplacement of the neurons, leading to deficits in diverse cerebellar functions. Centripetal granule cell migration involves several mechanisms, such as chemotaxis and extracellular matrix degradation, to guide the cells towards their final position, but the factors that regulate cell migration in each cortical layer are only partially known. In our method, acute cerebellar slices are prepared from P10 rats, granule cells are labeled with a fluorescent cytoplasmic marker and tissues are cultured on membrane inserts from 4 to 10 hr before starting real-time monitoring of cell migration by confocal macroscopy at 37 &#176;C in the presence of CO2. During their migration in the different cortical layers of the cerebellum, granule cells can be exposed to neuropeptide agonists or antagonists, protease inhibitors, blockers of intracellular effectors or even toxic substances such as alcohol or methylmercury to investigate their possible role in the regulation of neuronal migration.

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal cerebellum development, immature granule cells originating from the germinal zone exhibit distinct modalities of migration to reach their final destination and to establish neuronal networks. This protocol describes the preparation of cerebellar slices and the confocal macroscopic approach used to investigate the factors that regulate neuronal migration.

Ex Vivo Imaging postnatal du cervelet granules migration cellulaire utilisant confocale Macroscopie

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then ...

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 ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

Modélisation de la mort neuronale et la dégénérescence de neurones granules primaires de cervelet de souris

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this protocol, we aim to guide the reader not simply through the technical considerations of generating laser-induced lesions to trigger neovascular processes, but rather focus on the powerful information that can be obtained from multimodal longitudinal in vivo imaging throughout the follow-up period.
The laser-induced mouse CNV model was generated by a diode laser administration. Multimodal in vivo imaging techniques were used to monitor CNV induction, progression and regression. First, spectral domain optical coherence tomography (SD-OCT) was performed immediately after the lasering to verify a break of Bruch&#39;s membrane. Subsequent in vivo imaging using fluorescein angiography (FA) confirmed successful damage of Bruch&#39;s membrane from serial images acquired at the choroidal level. Longitudinal follow-up of CNV proliferation and regression on days 5, 10, and 14 after the lasering was performed using both SD-OCT and FA. Simple and reliable grading of leaky CNV leasions from FA images is&#160;presented. Automated segmentation for measurement of total retinal thickness, combined with manual caliber application for measurement of retinal thickness at CNV sites, allow unbiased evaluation of the presence of edema. Finally, histological verification of CNV is performed using isolectin GS-IB4 staining on choroidal flatmounts. The staining is thresholded, and the isolectin-positive area is calculated with ImageJ.
This protocol is especially useful in therapeutics studies requiring high-throughput-like screening of CNV pathology as it allows fast, multimodal, and reliable classification of CNV pathology and retinal edema. In addition, high resolution SD-OCT enables the recording of other pathological hallmarks, such as the accumulation of subretinal or intraretinal fluid. However, this method does not provide a possibility to automate CNV volume analysis from SD-OCT images, which has to be performed manually.

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Here, we present the usefulness of longitudinal in vivo imaging in the follow-up of morphological changes of laser-induced choroidal neovascularization in mice.

In Vivo Imagerie multimodale et analyse du modèle de néovascularisation choroïdienne souris induite par Laser

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this ...

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

In a mammalian nervous system, myelin provides an electrical insulation by enwrapping the axon fibers in a multilayered spiral. Inspired by its highly-organized subcellular architecture, we recently developed a new imaging modality, named spectral reflectometry (SpeRe), which enables unprecedented label-free nanoscale imaging of the live myelinated axons in situ. The underlying principle is to obtain nanostructural information by analyzing the reflectance spectrum of the multilayered subcellular structure. In this article, we describe a detailed step-by-step protocol for performing a basic SpeRe imaging of the nervous tissues using a commercial confocal microscopic system, equipped with a white-light laser and a tunable filter. We cover the procedures of sample preparation, acquisition of spectral data, and image processing for obtaining nanostructural information.

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

Here, we present a step-by-step protocol for imaging myelinated axons in a fixed brain slice using a label-free nanoscale imaging technique based on spectral reflectometry.

Microscopie spectrale réflectométrique sur les axones myélinisés In Situ

In a mammalian nervous system, myelin provides an electrical insulation by enwrapping the axon fibers in a multilayered spiral. Inspired by its ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from parallel fibers (PF) to Purkinje cells (PC) is considered the basis for motor learning, and deficiencies of both LTD and motor learning are observed in various gene-manipulated animals. Common motor learning sets, such as adaptation of the optokinetic reflex (OKR), the vestibular-ocular reflex (VOR), and rotarod test were used for evaluation of motor learning ability. However, results obtained from the GluA2-carboxy terminus modified knock-in mice demonstrated normal adaptation of the VOR and the OKR, despite lacking PF-LTD. In that report, induction of LTD was only attempted using one type of stimulation protocol at room temperature. Thus, conditions to induce cerebellar LTD were explored in the same knock-in mutants using various protocols at near physiological temperature. Finally, we found stimulation protocols, by which LTD could be induced in these gene-manipulated mice. In this study, a set of protocols are proposed to evaluate LTD-induction, which will more accurately allow examination of the causal relationship between LTD and motor learning. In conclusion, experimental conditions are crucial when evaluating LTD in gene-manipulated mice.

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are shown.

Évaluation de l'induction à long terme de dépression dans les tranches de cérévelateur adultes

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...