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.. Axonale Mærkning <ol><li> Klatring fibre kan mærkes ved at injicere enten organiske farvestoffer konjugeret højmolekylære dextraner eller plasmid / vira, der inducerer ekspression af fluorescerende proteiner 26-29. I denne protokol, er den organiske farvestof Alexa Fluor Dextran 488 sprøjtes ind i ringere oliven til at mærke klatring fibre og visualisere dem i den cerebellare cortex (Figur 1). Alle her beskrevne procedurer er blevet godkendt af det italienske sundhedsministerium. ...

<ol>
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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. 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Denne protokol viser, hvordan at mærke neuroner i ringere oliven med et fluorescerende farvestof. Efterfølgende metode til at udføre en kraniel vindue på cerebellar cortex beskrevet. Denne teknik giver optisk adgang til den terminale del af olivocerebellar neuroner, klatring fibre. Desværre, resultatet af både mærkning og craniotomy kirurgi er ganske lav, selv i hænderne på dygtige operatører (normalt 1 ud af 3 mus er mærket, og 1 ud af 3 kranie vinduer forbliver klar efter 1-2 uger). <p class="jove_conte...

Axonal transection følge af mekanisk skade, giftige fornærmelse eller neurodegenerative sygdomme er normalt efterfulgt af degeneration af den distale del af Axon, der er løsrevet fra cellen kroppen 10-13. Med nogle få undtagelser 2,7,14,15, afhuggede axoner i CNS af voksne dyr er som regel i stand til at aktivere en genvækst program 16. Lidt er kendt om real-time dynamik degenerative begivenheder på celleniveau og subcelleniveauet. Udviklingen af ​​nye strategier til begrænsning af neuronal skade og fremme neuronal genvækst kræver som et første skridt, der præciserer den mekanisme, som ualmindeligt sårede neuronale celler degenerere og regenerere. Denne undersøgelse er mest direkte behandlet ved at overvåge dynamikken i en enkelt neuron in vivo. Mens den ene-foton fluorescens billeddannelse teknikker er begrænset af intens spredning af synligt lys, to-foton excitation når dybe kortikale lag i live mus med subcellulær opløsning 3,4,17. Drage fordel af transgene mus, hvor fluorescerende proteiner er selektivt udtrykt i subpopulationer af neuroner 18-20 har TPF mikroskopi blevet anvendt til udforskning af synaptisk plasticitet og axonforlængende under udviklingen in vivo 21,22. T han evne ualmindeligt beskadigede neuroner til regrow efter skade kan undersøges ved kobling in vivo overvågning af to-foton billeddannelse med en model af skade specifikt rettet til Axon af interesse. Multi-foton absorption af femtosekund pulser er blevet brugt til at forstyrre enkelt dendritter eller endda enkelte pigge 5,23. Desuden er denne skade paradigme tillader skæring enkelt axonale grene uden at forstyrre kontakte dendritceller 6. Inden for rammerne af dissekere de funktioner, der giver specifik neuronal befolkning til at regenerere deres axoner engang beskadigede, cerebellare klatring fibre (CFS) er en nyttig model since de bevarer bemærkelsesværdige plastiske egenskaber efter skaden, selv i voksne dyr 24,25. For nylig, langsigtet billeddannelse CFS viste, at disse axoner er i stand til fornybare i de dage, der følger laser axotomi 6. Denne protokol beskriver, hvordan man mærke olivocerebellar neuroner og deres axonforlængende gennem anterograd sporing. Når neuroner af interesse fluorescens-mærkede, kan de blive overvåget gentagne gange på vilkårlige tidspunkter for uger eller måneder under et kranie vindue. Proceduren for at dissekere en enkelt axonale grene ved hjælp af laser axotomi in vivo vil derefter blive vist. De teknikker, der præsenteres her giver ny indsigt i den mekanisme af axonal remodeling in vivo, og kan bidrage til udviklingen af terapeutiske strategier til at begrænse neuronal degeneration og fremme axonal genvækst.

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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>
		</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.

Denne protokol beskrevet, hvordan du udfører axon mærkning, in vivo billeddannelse og laser axotomi på enkelte neuroner. Tidslinje for eksperimentet er vist i figur 1. Et eksempel på CFS mærket med Alexa Fluor 488 Dextran og visualiseret under kranie vindue ved in vivo to-foton mikroskopi er rapporteret i figur 2.. Som tidligere rapporteret 6,27, de opstigende grene vise en høj stabilitet i hele observationsperioden på fle...

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

Laser Nanosurgery of Cerebellar Axons In Vivo

To-foton billeddannelse, koblet til laser nanodissection, er nyttige værktøjer til at studere degenerative og regenerative processer i centralnervesystemet med subcellulær opløsning. Denne protokol viser, hvordan til at mærke, billede, og dissekere enkelt klatring fibre i den cerebellare cortex in vivo.

Laser nanosurgery af cerebellare Axoner<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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Neuroscience

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 Multimodal billedbehandling og analyse af mus Laser-induceret 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 ...

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

Spectral Reflectometric mikroskopi på myelinerede axoner 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

Vurdering af langvarig depression induktion i voksne cerebellar skiver

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