Name: laser nanosurgery of cerebellar axons in vivo
Uploaded: 2014-07-28T13:00:00
Description: Watch this Scientific Journal Video about Laser Nanosurgery of Cerebellar Axons In Vivo at JoVE.com

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 décrit...

<ol>
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L., 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=In+vivo+single+branch+axotomy+induces+GAP-43-dependent+sprouting+and+synaptic+remodeling+in+cerebellar+cortex.">In vivo single branch axotomy induces GAP-43-dependent sprouting and synaptic remodeling in cerebellar cortex.</a> Proc Natl Acad Sci USA. 110, 10824-10829 (2013).</li><li>Ylera, B., 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=Chronically+CNS-injured+adult+sensory+neurons+gain+regenerative+competence+upon+a+lesion+of+their+peripheral+axon.">Chronically CNS-injured adult sensory neurons gain regenerative competence upon a lesion of their peripheral axon.</a> Curr Biol. 19, 930-936 (2009).</li><li>Canty, A. 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=In-vivo+single+neuron+axotomy+triggers+axon+regeneration+to+restore+synaptic+density+in+specific+cortical+circuits.">In-vivo single neuron axotomy triggers axon regeneration to restore synaptic density in specific cortical circuits.</a> Nat Commun. 4, 2038 (2013).</li><li>Canty, A. 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=Synaptic+elimination+and+protection+after+minimal+injury+depend+on+cell+type+and+their+prelesion+structural+dynamics+in+the+adult+cerebral+cortex.">Synaptic elimination and protection after minimal injury depend on cell type and their prelesion structural dynamics in the adult cerebral cortex.</a> J Neurosci. 33, 10374-10383 (2013).</li><li>Waller, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experiments+on+the+Section+of+the+Glossopharyngeal+and+Hypoglossal+Nerves+of+the+Frog,+and+Observations+of+the+Alterations+Produced+Thereby+in+the+Structure+of+Their+Primitive+Fibres.">Experiments on the Section of the Glossopharyngeal and Hypoglossal Nerves of the Frog, and Observations of the Alterations Produced Thereby in the Structure of Their Primitive Fibres.</a> Philosophical Transactions of the Royal Society of London. 140, 423-429 .</li><li>Hilliard, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+degeneration+and+regeneration+a+mechanistic+tug+of+war.">Axonal degeneration and regeneration a mechanistic tug of war.</a> J Neurochem. 108, 23-32 (2009).</li><li>Kerschensteiner, M., Schwab, M. E., Lichtman, J. W., Misgeld, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+axonal+degeneration+and+regeneration+in+the+injured+spinal+cord.">In vivo imaging of axonal degeneration and regeneration in the injured spinal cord.</a> Nat Med. 11, 572-577 (2005).</li><li>Luo, L., O'Leary, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axon+retraction+and+degeneration+in+development+and+disease.">Axon retraction and degeneration in development and disease.</a> Annu Rev Neurosci. 28, 127-156 (2005).</li><li>Chauvet, N., Prieto, M., Alonso, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tanycytes+present+in+the+adult+rat+mediobasal+hypothalamus+support+the+regeneration+of+monoaminergic+axons.">Tanycytes present in the adult rat mediobasal hypothalamus support the regeneration of monoaminergic axons.</a> Exp Neurol. 151, 1-13 (1998).</li><li>Morrison, E. E., Costanzo, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+olfactory+sensory+neurons+and+reconnection+in+the+aging+hamster+central+nervous+system.">Regeneration of olfactory sensory neurons and reconnection in the aging hamster central nervous system.</a> Neurosci Lett. 198, 213-217 (1995).</li><li>Cafferty, W. B., McGee, A. W., Strittmatter, S. 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Neuroscience. 10, 647-658 (2009).</li><li>De Paola, V., 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=Cell+type-specific+structural+plasticity+of+axonal+branches+and+boutons+in+the+adult+neocortex.">Cell type-specific structural plasticity of axonal branches and boutons in the adult neocortex.</a> Neuron. 49, 861-875 (2006).</li><li>Allegra Mascaro, A., Sacconi, L., Pavone, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-photon+nanosurgery+in+live+brain.">Multi-photon nanosurgery in live brain.</a> Frontiers in neuroenergetics. 2, 21 (2010).</li><li>Rossi, F., van der Want, J., Wiklund, L., Strata, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reinnervation+of+cerebellar+Purkinje+cells+by+climbing+fibres+surviving+a+subtotal+lesion+of+the+inferior+olive+in+the+adult+rat.+II.+Synaptic+organization+on+reinnervated+Purkinje+cells.">Reinnervation of cerebellar Purkinje cells by climbing fibres surviving a subtotal lesion of the inferior olive in the adult rat. 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Synaptic organization on reinnervated Purkinje cells.</a> The Journal of comparative neurology. 308, 536-554 (1991).</li><li>Carulli, D., Buffo, A., Strata, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reparative+mechanisms+in+the+cerebellar+cortex.">Reparative mechanisms in the cerebellar cortex.</a> Progress in neurobiology. 72, 373-398 (2004).</li><li>Grasselli, G., Mandolesi, G., Strata, P., Cesare, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+sprouting+and+axonal+atrophy+in+cerebellar+climbing+fibres+following+in+vivo+silencing+of+the+growth-associated+protein+GAP-43.">Impaired sprouting and axonal atrophy in cerebellar climbing fibres following in vivo silencing of the growth-associated protein GAP-43.</a> PLoS One. 6, e20791 (2011).</li><li>Nishiyama, H., Fukaya, M., Watanabe, M., Linden, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+motility+and+its+modulation+by+activity+are+branch-type+specific+in+the+intact+adult+cerebellum.">Axonal motility and its modulation by activity are branch-type specific in the intact adult cerebellum.</a> Neuron. 56, 472-487 (2007).</li><li>Shinoda, Y., Sugihara, I., Wu, H. S., Sugiuchi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+entire+trajectory+of+single+climbing+and+mossy+fibers+in+the+cerebellar+nuclei+and+cortex.">The entire trajectory of single climbing and mossy fibers in the cerebellar nuclei and cortex.</a> Prog Brain Res. 124, 173-186 (2000).</li><li>Strata, P., Tempia, F., Zagrebelsky, M., Rossi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reciprocal+trophic+interactions+between+climbing+fibres+and+Purkinje+cells+in+the+rat+cerebellum.">Reciprocal trophic interactions between climbing fibres and Purkinje cells in the rat cerebellum.</a> Prog Brain Res. 114, 263-282 (1997).</li><li>Mostany, R., Portera-Cailliau, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+craniotomy+surgery+procedure+for+chronic+brain+imaging.">A craniotomy surgery procedure for chronic brain imaging.</a> J Vis Exp. (12), (2008).</li></ol>

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.

<table border="0" cellpadding="0" cellspacing="0">
	<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>
		</tr>
		<tr>
			<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>
		<tr>
			<td>Objective&#160;</td>
			<td>&#160;Olympus</td>
			<td>XLUMPLFLN 20XW</td>
			<td></td>
		</tr>
		<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 ...

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Laser Nanosurgery of Cerebellar Axons In Vivo

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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

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

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

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

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

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 ...