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. Labeling axonal <ol><li> Fibras de escalada podem ser marcados quer por injecção de corantes orgânicos conjugados com dextranos de elevado peso molecular ou de plasmídeo / vírus que induzem a expressão de proteínas fluorescentes 26-29. Neste protocolo, o corante orgânico Alexa Fluor Dextran 488 é injetado na oliva inferior a rotular fibras escalada e visualizá-los no córtex cerebelar (Figura 1). Todos os procedimentos aqui descritos foram aprovados pelo Ministério da Saúde i...

<ol>
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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. 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(12), (2008).</li></ol>

Este protocolo apresenta a forma de rótulo neurónios do oliva inferior com um corante fluorescente. Subsequentemente, o método para executar uma janela craniana no córtex cerebelar é descrito. Esta técnica permite o acesso óptico para a porção terminal de neurónios olivocerebellar, as fibras de escalada. Infelizmente, o resultado de ambos rotulagem e cirurgia craniotomia é bastante baixa, mesmo nas mãos de operadores qualificados (geralmente 1 de 3 camundongos é rotulado, e 1 em cada 3 janelas cranianas per...

Transecção axonal resultante de uma lesão mecânica, lesão tóxica ou doenças neurodegenerativas é geralmente seguido por degeneração da parte distal do axónio que é destacada do corpo celular 10-13. Com poucas exceções 2,7,14,15, os axônios cortados no SNC de animais adultos são geralmente incapazes de ativar um programa de rebrota 16. Pouco se sabe sobre a dinâmica em tempo real de eventos degenerativas no nível celular e subcelular. O desenvolvimento de novas estratégias para limitar a lesão neuronal e na promoção recrescimento neuronal requer, como um primeiro passo, que clarifica o mecanismo pelo qual as células neuronais lesionados singularmente degenerar e regenerar. Este estudo é mais diretamente dirigida pelo monitoramento da dinâmica de um único neurônio in vivo. Embora as técnicas de um fóton de fluorescência de imagem são limitados por intensa dispersão de luz visível, de dois fótons de excitação atinge camadas corticais profundas na live ratos com resolução 3,4,17 subcelular. Tirando partido de ratinhos transgénicos nos quais as proteínas fluorescentes são expressas selectivamente em subpopulações de neurónios 18-20, microscopia TPF foi aplicado para a exploração de plasticidade sináptica e elongação axonal durante o desenvolvimento in vivo 21,22. Que t capacidade de neurónios danificados a singularmente regredir após a lesão pode ser investigado pelo acoplamento no monitoramento vivo pela imagem de dois fótons com um modelo de lesão especificamente orientadas para o axônio de interesse. Absorção multi-fotão de pulsos femtosecond foi utilizado para destruir a dendritos individuais ou mesmo espinhas individuais 5,23. Além disso, este paradigma ferimento permite cortar ramos axonais individuais sem interromper o dendrito contato 6. Dentro do contexto da dissecando as características que permitem a população neuronal específica para regenerar os seus axónios uma vez danificados, fibras de escalada de cerebelo (SFC) é um modelo útil Since eles mantêm notáveis ​​propriedades plásticas após a lesão, mesmo em animais adultos 24,25. Recentemente, a imagem latente a longo prazo de FC mostraram que estes são capazes de axónios renováveis ​​nos dias que se seguem axotomia de laser 6. Este protocolo descreve como rotular neurônios olivocerebellar e seu alongamento axonal através de rastreamento anterógrado. Uma vez que os neurónios de interesse são marcadas por fluorescência, que pode ser monitorado repetidamente nos pontos de tempo arbitrárias durante semanas ou meses ao abrigo de uma janela craniana. O procedimento para dissecar ramos axonais individuais por axotomia do laser in vivo serão então ilustrado. As técnicas apresentadas aqui fornecer novos insights sobre o mecanismo de remodelação axonal in vivo e pode ajudar o desenvolvimento de estratégias terapêuticas para limitar a degeneração neuronal e promover o crescimento axonal.

<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.

Este protocolo descrito como realizar rotulagem axonal, imagem in vivo e axotomia laser sobre os neurônios individuais. A linha do tempo da experiência é mostrado na Figura 1. Um exemplo de FCs marcadas com Alexa Fluor 488 Dextrano e visualizadas sob a janela craniana in vivo por microscopia de dois fotões é apresentado na Figura 2. Como relatado anteriormente 6,27, os ramos ascendentes exibir uma elevada estabilidade ao lon...

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

Laser Nanosurgery of Cerebellar Axons In Vivo

Imagem de dois fótons, acoplado a nanodissection laser, são ferramentas úteis para estudar processos degenerativos e regenerativos no sistema nervoso central, com resolução subcelular. Este protocolo mostra como rotular, imagem e dissecar fibras escalada individuais no córtex cerebelar in vivo.

Laser Nanocirurgia de cerebelo axônios<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 ...

Research

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Neuroscience

Laser Nanosurgery of Cerebellar Axons In Vivo

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Simultâneo<em&gt; Ex vivo</em&gt; Teste Funcional de Dois retinas por<em&gt; In vivo</em&gt; Sistema Eletrorretinograma

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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

Na Vivo Imagiologia multimodal e análise do modelo de neovascularização coroide de induzida por Laser de Mouse

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

Microscopia de reflectométrica espectral em dendritos 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

Avaliação da indução de depressão em longo prazo em fatias Cerebelares adultas

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