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 italiano. </li><li> Prepare o tubo capilar de vidro, puxando-o em um extrator micropipeta. Corte a ponta do tubo capilar de vidro com uma tesoura até o diâmetro externo é de cerca de 30 mm. </li><li> Carregar o capilar com 1-2 mL do corante de dextrano conjugado. </li><li> Antes de começar, esterilizar todos os instrumentos cirúrgicos em uma autoclave. Durante o procedimento, a reduzir os riscos de contaminação, usando um esterilizador de contas de vidro. </li><li> Anestesiar o rato (9-12semanas) por injecção intraperitoneal de cetamina (90 mg / kg) e xilazina (9 mg / kg). Certifique-se de que o animal está totalmente sedado com cauda e / ou beliscões dedo do pé. Use veterinário pomada nos olhos para evitar a secura sob anestesia. </li><li> Raspar os cabelos acima do pescoço ou com uma lâmina de barbear ou creme de depilação. Limpe o local cirúrgico, duas vezes com betadine, alternadas com etanol a 70%. </li><li> Posicione o mouse sobre um suporte estereotáxico. Mantenha o animal aquecido com uma almofada de aquecimento. </li><li> Suavemente empurrar os earbars no osso ao lado das orelhas, de modo que a cabeça é mantida firmemente. Rotação da cabeça de modo a formar um ângulo de 140 ° com o corpo. A orientação desejada pode ser obtida, alterando ligeiramente a altura da haste de suporte de nariz no suporte estereotáxico. </li><li> Aplique algumas gotas de lidocaína na pele entre as orelhas. Use uma pinça ou uma lâmina de barbear para efectuar uma incisão vertical de aproximadamente 1 cm na pele abaixo do occipital. </li><li> Delicadamente separar o tecido subcutâneo e os músculos por pinça sem cortes sob o microscópio de dissecação. Empurre para baixo o feixe muscular horizontal e mantenha para além dos dois músculos fascículos verticais, a fim de expor a dura sobre o forame magno. </li><li> Usando extrema cautela, dissecar a dura com uma agulha de seringa ou uma pinça muito afiada para expor o tronco cerebral. Uma pequena quantidade de fluido cérebro-espinhal pode derramar quando a dura foi cortada. </li><li> No micromanipulador estereotáxica, rodar o titular do capilar a 45 ° em relação à vertical. Colocar o capilar na linha média, no ponto médio entre a extremidade caudal do córtex cerebelar e da primeira vértebra cervical. Inserir a pipeta, a uma profundidade de 2 mm a partir da superfície. </li><li> Entregar um volume de 0,5-1,5 mL de corante ao longo de 10-15 min. Um sistema de distribuição de microinjecção permite o fornecimento de pressão controlada do líquido no tubo capilar (pressão de pulso = 20 psi; duração do impulso = 3-4 mseg; frequência de pulso entrega = 12 Hz). </li><li> Deixar o capilar no local durante 15 minutos, em seguida, slowly (em 2-3 min) removê-lo. Delicadamente realinhar os músculos e suturar a pele acima. Mantenha os músculos bem hidratados, aplicando alguns salina durante todo o procedimento. </li><li> Para evitar a desidratação, injectar por via subcutânea 0,5 ml de NaCl a 0,9%. Mantenha o animal em uma gaiola aquecida até a recuperação completa da anestesia. </li><li> Subcutânea administrar Carprofen (5 mg / kg) por dia por 2-3 dias após a cirurgia. </li></ol> 2. Janela óptica no Córtex Cerebelar NOTA: O corante é transportado em 2 semanas a partir do núcleo olivar inferior até o córtex cerebelar aos terminais CFs (ver Figura 1). Os FCs escassamente marcadas podem ser visualizadas sob a janela craniana permanente que pode ser realizada como se segue: <ol><li> Esterilizar todos os instrumentos cirúrgicos. </li><li> Anestesiar o rato, tal como descrito acima. Use pitadas de dedo para verificar o animal está completamente sedada. </li><li> Raspar o cabelo acima da cabeça, limpe o skcom alternância de furtos de 70% de álcool e betadine e aplicar pomada. Posicione o mouse em um quadro estereotáxico e posicionar as barras de ouvido, a fim de segurar com firmeza a cabeça. </li><li> Subcutaneamente administrar dexametasona (0,2 mg / kg) e carprofeno (5 mg / kg) para prevenir a expansão do cérebro e possíveis inflamações no local da janela craniana. </li><li> Aplicar uma gota de lidocaína na pele acima do crânio e cortar uma aba, de entre as orelhas para acima dos olhos. Aplicar solução de lidocaína novamente para o periósteo antes de raspá-lo com um bisturi. </li><li> Use a broca dental para diluir uma região semicircular do crânio acima do cerebelo. A janela é geralmente colocado central e em direcção à volta da sutura, de modo que a profundidade do crânio é quase constante em todo o perímetro do semicírculo. Posicionando a janela muito perto da sutura lambda e perfuração repetitivo sobre esta região muito grosso pode resultar em superaquecimento do cérebro e da formação de edemas. </li><li> Aplicar hemostáticoesponja no crânio no caso de hemorragia. </li><li> Cortar uma lamela em duas metades, com a ajuda de uma lâmina de barbear e uma pinça. Verifique se a lamela é ligeiramente maior do que a ilha de osso e tem uma forma semelhante, antes de remover a tampa do osso com a pinça; se não, reformular a lamela ou escolha outro. Não toque na dura-máter com a pinça enquanto desenha fora do osso. Cuidadosamente estabelecer a lamela sobre a dura-máter. NOTA: A adesão de toda a superfície do vidro sobre a dura-máter está predizendo de janelas longas cranianos duradouros. </li><li> Remova as bolhas de ar sob o vidro de lavagem repetidas vezes com solução salina e secagem com cotonetes. Isso também irá ajudar a remover um pouco de sangue residual que possa ser derramado para fora de pequenos capilares depois de posicionar o vidro. Selar a janela com uma mistura de cola de acrílico e cimento dental 30. </li><li> Mantenha o animal em uma almofada de aquecimento em uma câmara de recuperação até que esteja totalmente recuperado da anestesia. Não retransformar os ratos na gaiola até que esteja totalmente recuperado; não deixá-los abandonados, até que recuperou a consciência suficiente para manter decúbito esternal. </li><li> Subcutânea administrar Carprofen (5 mg / kg) por dia por 2-3 dias após a cirurgia. </li></ol> 3. In Vivo Multi-fóton Laser axotomia <ol><li> Espere 2-7 dias da cirurgia antes de realizar a primeira sessão de imagem. O costume construído microscópio de dois fótons usada aqui é fornecido com um Ti: fonte de laser Sapphire (120 fs pulsos de largura, taxa de repetição de 90 MHz), o que é verificado pela primeira vez por um par de espelhos galvanométricos e depois se concentrou na amostra por uma imersão em água 20X objetivo (NA 0,95, WD 2 mm). A fase piezoelétrico de circuito fechado realiza deslocamentos axiais de até 400 mM do objetivo. Tubos fotomultiplicadores recolher o sinal de fluorescência. </li><li> Anestesiar o animal, em seguida, posicione a cabeça no suporte estereotáxica para que a janela craniana estabelece paralelos ao objective plano focal. </li><li> Explore com TPF imagens do córtex sob a janela craniana. Escolha o axônio de ser danificado entre os mais brilhantes. </li><li> Adquirir uma pilha-Z (em geral o campo de visão é de 100 x 100 mm 2, de 512 x 512 pixels, 2 um passo eixo z) para obter um 3D-reconstruções do axónio alvo. Dentro do volume fotografada, escolher o local da lesão axonal num ramo que se encontra principalmente em paralelo ao plano focal. Esta orientação permite marcar rapidamente alterações morfológicas, que são preditivos de uma dissecção de sucesso. </li><li> Irradiar com uma dose elevada de energia no ponto seleccionado sobre uma parte distal de um CF. Isto é feito através do software Labview: aumentar a potência do laser 5-10x mais do que a energia usada para geração de imagens (≈ 300 mW, medida após a lente objetiva), defina a posição dos espelhos galvanométricos para fazer uma varredura de linha (~ 4 - 10 mm) no local da lesão e abrir o obturador laser para 400-600 ms. Orientar a verificação das linhasperpendicular ao plano de CF para transecção completamente o axónio. Irradiando pontos brilhantes (por exemplo., Varizes) geralmente ajuda a dissecar o axônio. O comprimento de onda do laser para axotomia é o mesmo utilizado para a imagem latente (920 nm). </li><li> Normalmente, a fluorescência do axónio na área irradiada transiente diminui devido à fotodegradação. Adquirir uma pilha dos mesmos região poucos minutos após a irradiação para assegurar a ablação tem sido bem realizada. Se a região começou a inchar e do axônio formaram estruturas de contas-like, o neurônio foi dissecado com sucesso. Neste caso, em poucas horas (de algumas dezenas de minutos a 24 horas), a porção distal do axónio degenera e desaparecer completamente. </li><li> Infelizmente, a dose de energia necessária para produzir uma axotomia total, nem sempre é previsível. Se o único resultado da irradiação é a fotodegradação, a fluorescência deveria ser rapidamente recuperados, devido à difusão do corante, e a irradiação deve ser repetido mais uma vez a eficácialy dissecar o axônio. Além disso, no caso de sinais de inchaço não aparecem dentro de 5 - 20 min, ou o inchaço é seguido por recuperação (. Analogamente ao que foi descrito para o dendrotomy laser, ver Allegra Mascaro et al 23), tente novamente com novas verificações aumentando o o tempo de paragem e / ou a potência do laser. </li><li> Monitorar a eventual remodelação do axônio, nos dias que se seguem à axotomia laser com dois fótons de imagem in vivo (Figuras 2 e 3). O padrão vascular é utilizado como padrão de referência para recuperar o axônio dissecado nos dias seguintes. </li><li> Subcutânea administrar Carprofen (5 mg / kg) a cada sessão de imagens para toda a duração do experimento. </li></ol>

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We have nothing to disclose.

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.

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			<td>Lab standard stereotaxic, rat and mouse</td>
			<td>Stoelting&#160;</td>
			<td>51670</td>
			<td></td>
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		<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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			<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>
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			<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>
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			<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>
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</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 ...

laser-nanosurgery-of-cerebellar-axons-in-vivo

Research

JoVE Journal

Neuroscience

Laser Nanosurgery of Cerebellar Axons In Vivo

8.1K visualizações.  University of Florence.  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. 

Vídeo: 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.

Em Visualization vivo das vesículas sinápticas Dentro Drosophila larval Axônios segmentar

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

Neurociência

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.

Transfecção de células de camundongo por Ganglionares da Retina In vivo Eletroporação

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.

In vivo Laser axotomia em 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.

Neuroimagem Funcional Usando rompimento da barreira hemato-encefálica ultra-sônico e Manganês-RM

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.

Manipulação genética de Cerebelar Granule Neurônios<em&gt; In Vitro</em&gt; E<em&gt; In Vivo</em&gt; Estudar Neuronal Morfologia e Migração

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 de imagens de pós-natal Cerebelar Granule migração celular Usando confocal Macroscopia

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.

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

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.

Modelagem de morte Neuronal e a degeneração no Mouse grânulo cerebelar primária de neurônios

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.

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

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.