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. Etiquetado Axonal <ol><li> Escalada fibras pueden ser etiquetados mediante la inyección de cualquiera de los tintes orgánicos conjugados con dextranos de alto peso molecular o de plásmido / virus que inducen la expresión de proteínas fluorescentes 26-29. En este protocolo, el colorante orgánico Alexa Fluor 488 dextrano se inyecta en la oliva inferior para etiquetar fibras trepadoras y visualizarlos en la corteza cerebelosa (Figura 1). Todos los procedimientos descritos aquí han sido aprobados por el Ministerio de Sanidad italiano. </li><li> Preparar el tubo capilar de vidrio tirando de él en un extractor de micropipeta. Recorte la punta del tubo capilar de vidrio con tijeras hasta que el diámetro externo es de aproximadamente 30 micras. </li><li> Cargue el capilar con 1-2 l de la teñir-dextrano conjugado. </li><li> Antes de comenzar, esterilizar todos los instrumentos quirúrgicos en un autoclave. Durante el procedimiento, reducir los riesgos de contaminación por el uso de un esterilizador de cuentas de vidrio. </li><li> Anestesiar el ratón (9-12semanas) mediante inyección intraperitoneal de ketamina (90 mg / kg) y xilazina (9 mg / kg). Asegúrese de que el animal está completamente sedado utilizando cola y / o pellizcos del dedo del pie. Use ungüento veterinario en los ojos para evitar la sequedad, mientras que bajo anestesia. </li><li> Afeitar el vello por encima del cuello, ya sea con una hoja de afeitar o crema depilatoria. Limpie el sitio de la cirugía dos veces con betadine, alternado con etanol al 70%. </li><li> Coloque el ratón sobre un soporte estereotáxica. Mantenga al animal caliente con una almohadilla térmica. </li><li> Empuje suavemente las earbars en el hueso al lado de los oídos para que la cabeza se mantiene con firmeza. Girar la cabeza para formar un ángulo de 140 ° con el cuerpo. La orientación deseada se puede conseguir cambiando suavemente la altura de la varilla de soporte de la nariz en el soporte estereotáctica. </li><li> Aplique unas gotas de lidocaína en la piel entre las orejas. El uso de fórceps o una hoja de afeitar para llevar a cabo una incisión vertical de aproximadamente 1 cm en la piel por debajo de la protuberancia occipital. </li><li> Separar suavemente el tejido subcutáneo y los músculos por pinza romas bajo el microscopio de disección. Empuje hacia abajo el haz muscular horizontal y mantenga separados los dos fascículos musculares verticales a fin de exponer la duramadre por encima del foramen magnum. </li><li> Usando extremo cuidado, diseccionar la duramadre con una aguja de jeringa o pinzas muy afiladas para exponer el tronco cerebral. Una pequeña cantidad de fluido cerebro-espinal puede derramarse cuando la dura ha sido cortado. </li><li> En el micromanipulador estereotáctica, girar el soporte del capilar 45 ° de la vertical. Coloque el capilar en la línea media, en el punto medio entre el extremo caudal de la corteza cerebelar y la primera vértebra cervical. Inserte la pipeta a una profundidad de 2 mm desde la superficie. </li><li> Entregar un volumen 0,5-1,5 l de tinte durante 10-15 min. Un sistema de dispensación de microinyección permite la entrega controlada de la presión del líquido en el capilar (presión de pulso = 20 psi; duración de pulso = 3-4 mseg; frecuencia de pulso de entrega = 12 Hz). </li><li> Deje el capilar en su lugar durante 15 min, a continuación, slowly (en 2-3 min) quitarla. Delicadamente realinear los músculos y suturar la piel por encima. Mantenga los músculos bien hidratados aplicando alguna solución salina a través de todo el procedimiento. </li><li> Para evitar la deshidratación, se inyecta por vía subcutánea 0,5 ml de NaCl al 0,9%. Mantenga al animal en una jaula calienta hasta la recuperación total de la anestesia. </li><li> Por vía subcutánea administrar Carprofeno (5 mg / kg) al día durante 2-3 días después de la cirugía. </li></ol> 2. Ventana óptica en la corteza cerebelosa NOTA: El colorante es transportado en 2 semanas desde el núcleo olivar inferior hasta la corteza cerebelosa a los terminales de SFC (ver Figura 1). Los FCs escasamente marcados pueden ser visualizados debajo de la ventana craneal permanente que se puede realizar de la siguiente manera: <ol><li> Esterilice todos los instrumentos quirúrgicos. </li><li> Se anestesia el ratón como se describe anteriormente. Utilice pellizcos del dedo del pie para comprobar que el animal está completamente sedado. </li><li> Afeitar el cabello por encima de la cabeza, limpie el skcon alternancia de golpes de 70% de alcohol y betadine y aplicar pomada para los ojos. Coloque el ratón en un marco estereotáxico y coloque las barras de oído con el fin de sujetar firmemente la cabeza. </li><li> Por vía subcutánea administrar dexametasona (0,2 mg / kg) y carprofeno (5 mg / kg) para prevenir la inflamación del cerebro y posibles inflamaciones en el lugar de la ventana craneal. </li><li> Aplique una gota de lidocaína en la piel sobre el cráneo y cortar una solapa, de entre los oídos para arriba de los ojos. Aplicar una solución de lidocaína de nuevo en el periostio antes de raspar con un escalpelo. </li><li> Utilice el taladro dental para diluir una región semicircular del cráneo por encima del cerebelo. La ventana se coloca generalmente central y hacia la sutura de vuelta, de modo que la profundidad del cráneo es casi constante en todo el perímetro del semicírculo. Colocar la ventana demasiado cerca de la sutura lambda y de perforación repetitivo sobre esta región muy grueso puede dar lugar a un sobrecalentamiento del cerebro y la formación de edemas. </li><li> Aplicar hemostáticoesponja sobre el cráneo en caso de sangrado. </li><li> Corte un cubreobjetos en dos mitades con la ayuda de una cuchilla de afeitar y pinzas. Compruebe que el cubreobjetos es ligeramente más grande que la isla de los huesos y tiene una forma similar antes de quitar la tapa de la médula con las pinzas; si no, cambiar la forma del cubreobjetos o elegir otro tema. No toque la duramadre con las pinzas mientras se dibuja en el hueso. Coloque con cuidado por el cubreobjetos sobre la dura. NOTA: El cumplimiento de la totalidad de la superficie del vidrio en la duramadre está prediciendo de largas ventanas craneales duraderos. </li><li> Eliminar las burbujas de aire bajo el cristal de un enjuague varias veces con solución salina y secado con bastoncillos de algodón. Esto también ayudará a quitar un poco de sangre residual que pueda derramarse hacia fuera de pequeños capilares después de colocar el cristal. Sellar la ventana con una mezcla de pegamento acrílico y cemento dental 30. </li><li> Mantenga al animal en una almohadilla térmica en una cámara de recuperación hasta que se recupere completamente de la anestesia. No vuelva aconvertir ratones en la jaula hasta que se recupere plenamente; no los deje sin supervisión hasta que recobró el conocimiento suficiente para mantener decúbito esternal. </li><li> Por vía subcutánea administrar Carprofeno (5 mg / kg) al día durante 2-3 días después de la cirugía. </li></ol> 3. En Vivo multi-fotón Láser Axotomía <ol><li> Espere 2-7 días de la cirugía antes de realizar la primera sesión de imágenes. La costumbre construido microscopio de dos fotones utilizado aquí se proporciona con un Ti: Sapphire fuente láser (120 fs pulsos de ancho, frecuencia de repetición de 90 MHz), que primero es escaneado por un par de galvanómetros de espejo y luego se centró sobre la muestra por una inmersión en agua 20X objetivo (NA 0,95, WD 2 mm). Una etapa piezoeléctrico de bucle cerrado realiza desplazamientos axiales de hasta 400 m del objetivo. Tubos fotomultiplicadores recogen la señal de fluorescencia. </li><li> Anestesiar al animal, entonces la posición de la cabeza en el soporte estereotáctico para que la ventana craneal establece paralelo a la objetivaciónve plano focal. </li><li> Explore con TPF imágenes de la corteza bajo la ventana craneal. Elija el axón a dañarse entre los más brillantes. </li><li> Adquirir una pila Z (normalmente el campo de visión es de 100 x 100 m 2, en 512 x 512 píxeles, 2 micras paso del eje z) para obtener un 3D reconstrucciones del axón apuntado. Dentro del volumen reflejado, elegir el lugar de la lesión axonal en una rama que se encuentra principalmente en paralelo al plano focal. Esta orientación permite marcar rápidamente los cambios morfológicos, que son predictivos de una disección de éxito. </li><li> Irradiar con una alta dosis de energía el punto seleccionado en una porción distal de un CF. Esto se realiza a través del software Labview: aumentar la potencia del láser 5-10x más que el poder utiliza para la imagen (≈ 300 mW, medida después de la lente del objetivo), ajuste la posición de los espejos galvanométricos para hacer una exploración de línea (~ 4 - 10 micras) en el sitio de la lesión y abrir el obturador de láser para: 400 - 600 mseg. Orientar la exploración de líneaperpendicular al plano de CF a seccionar completamente el axón. La irradiación de los puntos más brillantes (por ejemplo., Varices) generalmente ayuda a la disección del axón. La longitud de onda para la axotomía láser es el mismo utilizado para la formación de imágenes (920 nm). </li><li> Por lo general, la fluorescencia del axón en el área irradiada disminuye transitoriamente debido a photobleaching. Adquirir una pila de la misma región pocos minutos después de la irradiación para garantizar la ablación ha sido bien realizado. Si la región se inició la hinchazón y el axón formó estructuras en forma de cuentas, la neurona se ha diseccionado con éxito. En este caso, en unas pocas horas (desde pocas decenas de minutos a 24 horas) la porción distal del axón se degenerar y desaparecer por completo. </li><li> Desafortunadamente la dosis de energía necesaria para producir una axotomía completa no siempre es predecible. Si el único resultado de la irradiación se photobleaching, la fluorescencia debe ser recuperado rápidamente debido a la difusión del colorante, y la irradiación debe repetirse de nuevo para efectivaly diseccionar el axón. También, en caso de signos de inflamación no aparecen dentro de 5 a 20 minutos, o la hinchazón va seguido de recuperación (. Forma análoga a lo que se ha descrito para dendrotomy láser, ver Allegra Mascaró et al 23), intente de nuevo con nuevas exploraciones aumentar el tiempo de permanencia y / o la potencia del láser. </li><li> Monitorear la eventual remodelación del axón en los días que siguen a la axotomía láser con dos fotones de imágenes in vivo (Figuras 2 y 3). El patrón vascular se utiliza como patrón de referencia para recuperar el axón diseccionado en los siguientes días. </li><li> Por vía subcutánea administrar carprofeno (5 mg / kg) de cada sesión de formación de imágenes para toda la duración del experimento. </li></ol>

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We have nothing to disclose.

Este protocolo muestra cómo etiquetar las neuronas de la oliva inferior con un colorante fluorescente. Posteriormente, el método para llevar a cabo una ventana craneal en la corteza cerebelosa se describe. Esta técnica proporciona acceso óptico a la parte terminal de las neuronas olivocerebellar, las fibras de escalada. Por desgracia, el resultado de tanto el etiquetado y la cirugía craneotomía es bastante bajo, incluso en manos de operadores calificados (por lo general 1 de cada 3 ratones se etiqueta, y 1 de cada...

Transección axonal que resulta de una lesión mecánica, agresión tóxica o las enfermedades neurodegenerativas es generalmente seguido por la degeneración de la parte distal del axón que se separa del cuerpo celular 10-13. Con unas pocas excepciones 2,7,14,15, axones dañados en el SNC de animales adultos son generalmente incapaces de activar un programa de recrecimiento 16. Poco se sabe acerca de la dinámica en tiempo real de eventos degenerativos a nivel celular y subcelular. El desarrollo de nuevas estrategias para limitar el daño neuronal y promover el nuevo crecimiento neuronal requiere, como primer paso, aclarar el mecanismo por el cual las células neuronales singularmente lesionados degeneran y se regeneran. Este estudio se dirige más directamente mediante el control de la dinámica de una sola neurona in vivo. Mientras que las técnicas de imagen de fluorescencia de un fotón están limitados por una intensa dispersión de la luz visible, la excitación de dos fotones alcanza profundas capas corticales en lihe ratones con resolución subcelular 3,4,17. Tomando ventaja de los ratones transgénicos en los que las proteínas fluorescentes se expresan selectivamente en subpoblaciones de neuronas 18-20, microscopía de TPF ha sido aplicada a la exploración de la plasticidad sináptica y la elongación axonal durante el desarrollo in vivo 21,22. L a la capacidad de las neuronas dañadas singularmente a volver a crecer después de la lesión puede ser investigado por el acoplamiento en el seguimiento in vivo de dos fotones de imágenes con un modelo de lesión dirigida específicamente al axón de interés. Absorción multi-fotón de pulsos de femtosegundos se ha utilizado para interrumpir dendritas individuales o incluso espinas individuales 5,23. Por otra parte, este paradigma lesión permite cortar ramas axonales individuales sin interrumpir la dendrita de contactos 6. En el contexto de la disección de las características que permiten a la población neuronal específica de regenerar sus axones vez dañadas, las fibras trepadoras del cerebelo (SFC) son un modelo útil sina vez que retienen propiedades plásticas notables después de la lesión, incluso en los animales adultos 24,25. Recientemente, proyección de imagen a largo plazo de las FC mostró que estos axones son capaces de recrecimiento en los días que siguen a la axotomía láser 6. Este protocolo describe cómo etiquetar las neuronas olivocerebellar y su elongación axonal a través de rastreo anterógrada. Una vez que las neuronas de interés están marcados con fluorescencia, que se pueden monitorizar varias veces en los puntos de tiempo arbitrarios para semanas o meses en virtud de una ventana craneal. El procedimiento para diseccionar ramas axonales individuales por axotomía láser in vivo A continuación, se ilustra. Las técnicas presentadas aquí proporcionan nuevos conocimientos sobre los mecanismos de remodelación axonal in vivo y pueden ayudar al desarrollo de estrategias terapéuticas para limitar la degeneración neuronal y promover la regeneración axonal.

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		<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>
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			<td>Germinator 500 (Glass bead sterilizer)</td>
			<td>Roboz</td>
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			<td>Microinjection dispense system</td>
			<td>Picospritzer</td>
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			<td></td>
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		<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>
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			<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 describe cómo llevar a cabo el etiquetado axonal, imágenes in vivo y axotomía láser en las neuronas individuales. La línea de tiempo del experimento se muestra en la Figura 1. Un ejemplo de SFC marcada con Alexa Fluor 488 de dextrano y visualizados debajo de la ventana craneal por microscopía in vivo de dos fotones se informó en la Figura 2. Como se informó anteriormente 6,27, las ramas ascendentes muestran...

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

Laser Nanosurgery of Cerebellar Axons In Vivo

Dos fotones de imágenes, acoplado a nanodissection láser, son herramientas útiles para el estudio de los procesos degenerativos y regenerativos en el sistema nervioso central con resolución subcelular. Este protocolo muestra cómo etiquetar, imagen y diseccionar las fibras individuales de escalada en la corteza cerebelosa in vivo.

Laser Nanocirugía del cerebelo Los axones<em&gt; En 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 vistas.  University of Florence.  Dos fotones de imágenes, acoplado a nanodissection láser, son herramientas útiles para el estudio de los procesos degenerativos y regenerativos en el sistema nervioso central con resolución subcelular. Este protocolo muestra cómo etiquetar, imagen y diseccionar las fibras individuales de escalada en la corteza cerebelosa in vivo. 

Video: 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.

Visualización en vivo de las vesículas sinápticas En Drosophila segmentaria axones larvas

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

Investigación

Neurociencias

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

La transfección de células de ratón del ganglio de la retina por In vivo La electroporación

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

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.

En vivo láser axotomía 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.

Neuroimagen Funcional Utilizando ultrasonidos hematoencefálica perturbación de la barrera y la RM-manganeso mayor

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.

La manipulación genética de las neuronas cerebelosa gránulo<em&gt; In Vitro</em&gt; Y<em&gt; En Vivo</em&gt; Estudiar Neuronal Morfología y Migración

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 de Postnatal cerebelosa gránulo migración celular utilizando confocal Macroscopía

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.

Simultáneo<em&gt; Ex vivo</em&gt; Pruebas funcionales de dos retinas por<em&gt; In vivo</em&gt; Sistema Electrorretinograma

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.

Modelado de la muerte Neuronal y la degeneración en las neuronas del gránulo cerebeloso primario de ratón

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.

En Vivo La proyección de imagen multimodal y análisis del modelo de neovascularización coroidea inducida por láser 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

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.