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. Axonal Labeling <ol><li> Kletterfasern können durch Injektion, entweder organische Farbstoffe mit hohem Molekulargewicht Dextran oder ein Plasmid / Viren, die die Expression von fluoreszierenden Proteinen induzieren 26-29 konjugiert bezeichnet werden. In diesem Protokoll wird der organische Farbstoff Dextran Alexa Fluor 488 in den unteren Olive injiziert Kletter Fasern kennzeichnen und zu visualisieren in der Kleinhirnrinde (Abbildung 1). Alle hier beschriebenen Verfahren von der italie...

<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. 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Dieses Protokoll zeigt, wie Neuronen der unteren Olive mit einem Fluoreszenzfarbstoff zu markieren. Anschließend wird das Verfahren zur Herstellung eines Schädel Fenster auf der Kleinhirnrinde zuführen beschrieben. Diese Technik stellt einen optischen Zugang zu dem Anschlussabschnitt der olivocerebellar Neuronen, die Kletterfasern. Leider ist das Ergebnis sowohl der Kennzeichnung und Schädelchirurgie auch in den Händen von Facharbeitern recht niedrig (in der Regel 1 von 3 Mäusen markiert ist, und 1 von 3 Schädelf...

Axonalen Durchtrennung von mechanischen Verletzungen toxischen Insult oder neurodegenerativen Erkrankungen resultiert, wird gewöhnlich durch die Degeneration des distalen Teils des Axons, die von dem Zellkörper 10-13 gelöst wird, gefolgt. Mit wenigen Ausnahmen 2,7,14,15 sind getrennt Axone im ZNS von erwachsenen Tieren in der Regel nicht in der Lage, ein Nachwachsen Programm 16 zu aktivieren. Wenig ist über die Echtzeit-Dynamik von degenerativen Ereignisse auf zellulärer und subzellulärer Ebene bekannt. Die Entwicklung neuer Strategien zur Begrenzung neuronalen Schäden und Förderung der neuronalen Nachwachsen erfordert, als einen ersten Schritt, die Klärung der Mechanismus, mit dem einzigartig verletzten Nervenzellen degenerieren und zu regenerieren. Diese Studie wird am direktesten durch die Überwachung der Dynamik eines einzelnen Neurons in vivo gerichtet. Beim Ein-Photonen-Fluoreszenz-Imaging-Techniken durch intensive Streuung des sichtbaren Lichts beschränkt, tief kortikalen Schichten in li erreicht Zweiphotonenanregungve Mäuse mit subzellulärer Auflösung 3,4,17. Unter Ausnutzung von transgenen Mäusen, bei denen fluoreszierende Proteine ​​werden selektiv in Subpopulationen von Neuronen 18-20 ausgedrückt hat TPF-Mikroskopie zur Erforschung der synaptischen Plastizität und axonalen Dehnung während der Entwicklung in vivo 21,22 angewandt. T er Fähigkeit der geschädigten Neuronen einzeln zu nachwachsen nach Verletzung kann durch Kopplung in-vivo-Überwachung von Zwei-Photonen-Bildgebung mit einem Modell der Schädigung spezifisch an das Axon von Interesse gezielt untersucht werden. Multi-Photonen-Absorption von Femtosekundenpulsen verwendet wurde, um einzelne Dendriten oder auch Einzel Stacheln 5,23 stören. Darüber hinaus ermöglicht diese Verletzung Paradigma Schneiden Einzel axonalen Verzweigungen, ohne dass der Kontakt mit Dendriten-6. Im Rahmen der sezieren die Funktionen, die spezifischen neuronalen Population zu ermöglichen, ihre Axone zu regenerieren einmal beschädigt, Kleinhirnkletterfasern (CF) sind ein nützliches Modell since sie bemerkenswerte Kunststoffeigenschaften nach der Verletzung auch bei erwachsenen Tieren 24,25 zu behalten. Kürzlich, langfristige Darstellung von CF zeigten, dass diese Axone sind in der Lage, die in den nachwachsenden Laser-Axotomie 6 Tagen folgen. Dieses Protokoll beschreibt, wie olivocerebellar Neuronen und ihre axonalen Dehnung durch anterograde Tracing zu beschriften. Sobald die Neuronen von Interesse fluoreszierend markiert sind, können sie wiederholt an beliebigen Zeitpunkten für Wochen oder Monate nach einer Schädelfenster überwacht werden. Das Verfahren, um einzelne axonalen Verzweigungen durch Laser-Axotomie in vivo sezieren wird dann dargestellt werden. Die hier vorgestellten Techniken bieten neue Einblicke in den Mechanismus der axonalen Umbau in vivo und können die Entwicklung von therapeutischen Strategien helfen, neuronale Degeneration zu begrenzen und zu fördern axonale Nachwachsen.

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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.

Dieses Protokoll beschrieben, wie axonalen Etikettierung, in-vivo-Imaging und Laser-Axotomie an einzelnen Nervenzellen durchzuführen. Die Zeitachse des Experiments ist in Fig. 1 gezeigt. Ein Beispiel für CF mit Alexa Fluor 488 Dextran markiert und unter der Schädelfenster in vivo Zweiphotonenmikroskopie sichtbar ist in Abbildung 2 dargestellt. Wie bereits berichtet 6,27, zeigen die aufsteigenden Äste eine hohe Stabilität in ...

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

Laser Nanosurgery of Cerebellar Axons In Vivo

Zwei-Photonen-Imaging, Laser Nanodissektions gekoppelt ist, sind nützliche Tools, degenerativen und regenerativen Prozessen im zentralen Nervensystem mit subzellulärer Auflösung zu studieren. Dieses Protokoll zeigt, wie beschriften, Bild-und sezieren einzigen Kletterfasern in der Kleinhirnrinde in vivo.

Laser-Nanochirurgie der Kleinhirn-Axone<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

JoVE Journal

Neuroscience

Laser Nanosurgery of Cerebellar Axons In Vivo

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

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Genetische Manipulation von Kleinhirnkörnerzellen<em&gt; In-vitro-</em&gt; Und<em&gt; In Vivo</em&gt;, Um neuronale Morphologie und Migration Studieren

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

Gleichzeitig<em&gt; Ex vivo</em&gt; Funktionsprüfung von zwei Netzhäute von<em&gt; In vivo</em&gt; Elektroretinogramm-System

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

Modellierung von neuronalen Tod und Degeneration in Maus primären zerebelläre Granulat Neuronen

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 Multimodale Bildgebung und Analyse der laserinduzierten choroidale Neovaskularisation Mausmodell

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

Spektrale Reflectometric Mikroskopie auf Markhaltige Axone 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

Bewertung der Langzeit-Depressionsinduktion bei erwachsenen Cerebellarscheiben

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