Este estudo foi financiado em parte pela Fundação IPSEN. IMV é um Professor Sociedade Americana do Câncer da Biologia Molecular, e detém o Irwin Jacobs e Joan Presidente em Ciências da Vida Exemplar. Este trabalho foi financiado em parte por concessões do NIH, Fundação Leducq, Fundação Meriaux, Medicina Ellison Foundation, Ipsen / Biomeasure, Sanofi Aventis, Câncer de Próstata Foundation, eo HN e Frances C. Berger Foundation. PAS é financiado pelo Fundo de Dotação McKnight de Neurociências e do Instituto Nacional sobre Abuso de Drogas (R01 DA019022). Os autores gostariam de agradecer a Mark Schmitt. Arte gráfica foi desenhada por Jamie T. Simon. Patrocínio foi gentilmente cedido pela Invitrogen.

1. Organotípicas Culturas Cerebelar: <ol><li> Para conseguir gerar organotípicas culturas cerebelar usar mouses entre P0-P3 ou P8-P12. Para nosso experimento utilizamos ratos na faixa P8-P12, porque nesta fase de desenvolvimento era mais apropriado para o nosso estudo. </li><li> Prepare a dissecção eo meio de cultura: <ol><li> Dissecção do cérebro e preparação fatia são realizados em baixa de sódio líquido cefalorraquidiano artificial (ACSF) contendo 1mM de cloreto de cálcio, 10mM D-Glicose, 4 mM de cloreto de potássio, cloreto de magnésio 5mM, bicarbonato de sódio 26mm, 246 mm de sacarose e solução de vermelho de fenol (1:1000) , pH 7,3. Para esterilizar a solução, filtrá-la através de um filtro 0,22 mm e armazenar a 4 ° C. </li><li> Fatias cerebelar são cultivadas em 75% Mínimo Essencial Medium Eagle (MEM), soro de cavalo 25% inativado pelo calor, HEPES 25 mM, 1mM glutamina, 5mg / mL de glicose, penicilina (100 U / mL) e estreptomicina (100 U / mL). Filtrar o meio através de um filtro de 0,22 mm e armazenar a 4 ° C para um máximo de duas semanas. </li></ol></li><li> Dissecar o cérebro na gelada médio ACSF previamente aeradas com carbogênio (95% de oxigênio e dióxido de carbono de 5%) Note, que a saturação com carbogênio vai mudar o pH da solução para que ele irá mudar de vermelho para laranja. A dissecção deve ser realizado, no montante mínimo de tempo possível tomando cuidado para não danificar a estrutura do cérebro. </li><li> Fatia do cérebro em cortes sagitais de 400 m usando uma vibratome. <ol><li> Faça um corte sagital com uma lâmina de barbear em um hemisfério, tentando cortar o mínimo possível do cerebelo, o que irá proporcionar uma superfície plana para corte. O córtex é apenas usado para apoio para fazer o corte mais fácil, mas como o tempo é uma questão importante, é mais conveniente para cortar a metade rostral do córtex com uma lâmina de barbear. </li><li> Use cianoacrilato (Superglue) para fixar a posição do cérebro no prato vibratome metal. É importante para refrigerar a placa de gelo antes de adicionar a cola e espalhe bem com a ponta do tubo para formar uma camada adesiva fina. Se houver um excesso de Superglue, ele irá retirar do prato quando entra em contato com a ACSF ea amostra será perdido. </li><li> Uma vez que o cérebro está ligado à placa vibratome, adicionar mídia o suficiente para cobrir ACSF o cérebro e colocar gelo abaixo da placa para mantê-lo frio. </li><li> Cortar fatias 400 mm e transferi-los com um plástico Pasteur pipeta (cortar a ponta para alargá-la e evitar danos às fatias) para uma placa de cultura de 6 bem célula que contém ACSF no gelo. </li><li> Separar o cerebelo do resto do cérebro com a ajuda de duas seringas de insulina (seringas com diâmetro pequeno calibre 28 agulhas) sob o microscópio de dissecação. Algumas fatias terá Superglue anexado às bordas: este é o momento para tentar removê-lo, pois ele pode afetar a viabilidade da fatia. É importante fazê-lo cuidadosamente sem danificar a estrutura do cerebelo. </li></ol></li><li> Transferir as fatias cerebelar a 6 bem-placas contendo insere placa 30 milímetros cultura com 0,4 mM poros. Adicionar 1 mL de meios de cultura por poço, que foi pré-condicionado por incubação por pelo menos 2 horas a 37 ° C e 5% CO 2 (na cultura de células incubadora). Coloque o insere no topo da mídia certificando-se que não há bolhas de ar presas debaixo e em seguida, coloque as fatias de cerebelo (1-3 por pastilha) em cima das inserções ter certeza que eles estão totalmente estendida, e que o líquido não está cobrindo-los. Aspirar o excesso de meios de comunicação, se necessário. Incubar a 37 ° C e 5% CO 2 mudando o meio de cultura a cada 2 ou 3 dias. O melhor é substituir apenas uma parte do meio (por exemplo, volume de ½) de uma só vez, desde meio utilizado contém fatores fatia derivados tróficos que são importantes para a sobrevivência da célula. </li></ol> 2. Tratamento Fas e coloração das fatias Cerebelar: <ol><li> Para o experimento desafio apoptose, primeira cultura que as fatias por 5 dias em mudança de mídia no dia in vitro 3 (Div3). Em DIV5 culturas são tratadas com 0,5 mg / mL do anticorpo Jo2 Fas agonista, adicionando-o diretamente para a mídia, a fim de ter um fundo limpo de morte celular desde as mudanças dos meios de comunicação podem induzir alguma mortalidade nas culturas. Metade dos poços são deixados sem tratamento como controle. </li><li> Após 24 horas remova a mídia sob as inserções por sucção. Para corrigir as fatias, adicione 1 mL de gelo frio PFA 4% abaixo e acima da 1 mL de inserção. Incubar por 10 minutos em temperatura ambiente. </li><li> Após 10 minutos retire a PFA e lave brevemente com PBS frio. </li><li> Em seguida remova o PBS e adicionar 1 mL por baixo e 1 mL acima da inserção de 20% de metanol gelado em PBS e incubar por 5 minutos em temperatura ambiente. </li><li> Lave novamente com PBS e adicionar 1 mL e 1 mL por baixo acima da inserção de 0,5% de 100 Triton-X em PBS para permeabilização. Incubar por um mínimo de 12 horas a 4 ° C. </li><li> Depois de permeabilização, bloco com 20% BSA em PBS por um mínimo de4 horas à temperatura ambiente ou durante a noite a 4 ° C. Nesta fase as fatias podem ser mantidos em solução de bloqueio por pelo menos 3 dias a 4 ° C. </li><li> De modo a manchar as fatias de corte cuidadosamente o pedaço da membrana inserir anexado à fatia do cerebelo e transferi-la para placas de 24 poços contendo PBS. </li><li> A imunocoloração é feito sequencialmente, primeiro com o reagente TUNEL por uma hora e, em seguida, com anticorpo Calbidin durante a noite. <ol><li> Aspirar o PBS e adicionar 250 mL de reagente off TUNEL em cada poço ter certeza que ele cobre toda a superfície da fatia. Incubar a 37 ° C por uma hora no escuro. </li><li> Após uma hora retire a mistura TUNEL e lavar com PBS três vezes por 10 minutos. </li><li> Após a última lavagem, remover o PBS e adicionar 250 mL de uma diluição 1 / 1000 do anticorpo calbindina em PBS e incubar overnight a 4 ° C no escuro. </li><li> Remover a solução de anticorpo primário e lavar com PBS três vezes por 10 minutos. </li><li> Adicionar mL 250 de anticorpo secundário diluído 1 / 500 em PBS e incubar por pelo menos três horas à temperatura ambiente no escuro. O kit TUNEL usamos é rotulado com TMR vermelho, pois o nosso anticorpo secundário para calbindina é conjugado com Alexa-488. </li><li> Lavar três vezes por 10 minutos com PBS e monte as fatias em uma lâmina de vidro usando uma mídia de montagem que contém DAPI. </li><li> Imagem as fatias com um microscópio confocal usando a 488 nm e 561 nm comprimento de onda de excitação para calbindina e TUNEL, respectivamente. </li></ol></li></ol> 3. Resultados representativos: O desafio principal deste protocolo deve ser capaz de gerar saudável culturas fatia cerebelar, especialmente desde a nossa leitura final será a morte celular. Uma cultura saudável aparece como aquele onde a camada de células do corpo é translúcido e permite que você veja a estrutura foliated do cerebelo sob o microscópio (Figura 1). Depois de alguns dias em cultura as fatias de começar a células finas e não-neuronal pode ser observado a migração para fora das margens da fatia. As células redondas escuras (mais provável macrófagos) que cobrem as fatias depois de alguns dias em cultura, acabará por diminuir em número após 10-14 dias in vitro (Figura 2) 5. A prova final para a viabilidade das fatias é a mancha para diferentes marcadores celulares, como calbindina para as células de Purkinje. A Figura 3 mostra uma imagem de microscópio confocal representante da camada de células de Purkinje com núcleos TUNEL várias positivo. É importante considerar que, mesmo se algumas fatias recebeu um estímulo apoptótico, eles só foram expostas por 24 horas. Isto permitiu tempo suficiente para observar fragmentação do DNA nas células morrendo com a coloração TUNEL, mas uma camada de células Purkinje definido ainda estava presente. <img src="/files/ftp_upload/2564/2564fig1.jpg" alt="Figura 1" /> Figura 1. Fatia cerebelar saudável em div2. Esta imagem mostra a camada de células do corpo translúcido ea estrutura foliated cerebelar em uma fatia saudável. <img src="/files/ftp_upload/2564/2564fig2.jpg" alt="Figura 2" /> Figura 2. Fatia cerebelar saudável em DIV 7. Nesta imagem ainda podemos observar a estrutura foliated do cerebelo e do aparecimento de corpos celulares escuro. <img src="/files/ftp_upload/2564/2564fig3.jpg" alt="Figura 3" /> Figura 3. Imagem representativa de uma fatia Fas-tratados cerebelar. Esta imagem confocal mostra as células Purkinje corados com calbindina (verde) e as células apoptóticas TUNEL positivas para a coloração (vermelho). Células de Purkinje não aparecem em uma única linha, mas estão agrupados formando uma estrutura folium-like.

<ol>
	<li>Hogue, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+fetal+ependymal+cells+in+tissue+cultures.">Human fetal ependymal cells in tissue cultures.</a> Anat Rec. 99, 523-529 (1947).</li><li>Hogue, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+studies+of+human+fetal+brain+cells+in+tissue+cultures.">Review of studies of human fetal brain cells in tissue cultures.</a> Etudes Neonatales. 1, 1-13 (1952).</li><li>Gogolla, N., Galimberti, I., DePaola, V., Caroni, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staining+protocol+for+organotypic+hippocampal+slice+cultures.">Staining protocol for organotypic hippocampal slice cultures.</a> Nat Protoc. 1, 2452-2456 (2006).</li><li>Stoppini, L., Buchs, P. A., Muller, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+organotypic+cultures+of+nervous+tissue.">A simple method for organotypic cultures of nervous tissue.</a> J Neurosci Methods. 37, 173-182 (1991).</li><li>Beat, H., G&#228;hwiler, S. M. T., McKinney, R. A., Debanne, D., Robertson, R. T., Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Organotypic Slice Cultures of Neural Tissue in Culturing Nerve Cells. , 461-498 (1998).</li></ol>

Todo o trabalho foi feito com animais conduzidos de acordo com as políticas estabelecidas pelo IACUC. O Instituto Salk é uma facilidade AAALAC credenciados.

Este método descreve uma das muitas aplicações possíveis de culturas organotípicas neuronal e nos permitiu estudar o efeito dos estímulos apoptóticos no tipo selvagem e mutante fatias de cerebelo. A parte mais crítica desta experiência é ser capaz de gerar consistentemente saudável culturas fatia cerebelar. Os factores chave são dissecção e técnica de corte e a capacidade de executar o protocolo no menor tempo possível, mas ao mesmo tempo, tomando cuidado para não danificar as fatias. Outro fator crucial é a preparação ea composição do meio de cultura, essas culturas são muito sensíveis, por isso tivemos de testar várias receitas e marcas de mídia. Mesmo diferentes lotes de soro ou quaisquer outros componentes dos meios de comunicação podem afetar a viabilidade das fatias. Nós temos os melhores resultados com o soro de cavalo de Invitrogen Lot # 480116. Uma vez que as fatias de cerebelo são gerados pode-se estudar o desenvolvimento do cerebelo, eletrofisiologia, a sobrevivência da célula isolada ou em resposta a estímulos apoptóticos, excitotoxicidade ou privação de oxigênio. Estudos comparativos utilizando tipo selvagem e mutante fatias cerebelar foram muito perspicaz em muitos aspectos da função cerebelar e desenvolvimento.

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		<th>Catalogue Number</th>
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<td>MEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11090</td>
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<td>Horse Serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16050</td>
<td>&nbsp;</td>
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<td>Hepes</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15630</td>
<td>&nbsp;</td>
<tr>
<td>Glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25030</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/ Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140</td>
<td>&nbsp;</td>
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<td>Superglue</td>
<td>&nbsp;</td>
<td>Krazy Glue</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>6 well / 24 well cell culture plates</td>
<td>&nbsp;</td>
<td>Corning Incorporated</td>
<td>3506/ 3527</td>
<td>&nbsp;</td>
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<td>30mm culture plate inserts (0.4-&mu;m pore)</td>
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<td>Millipore</td>
<td>PICM03050</td>
<td>&nbsp;</td>
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<td>Purified NA/LE Hamster anti-mouse CD95 (Jo2)</td>
<td>&nbsp;</td>
<td>BD Pharmingen</td>
<td>554254</td>
<td>&nbsp;</td>
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<td>In situ cell death detection kit, TMR red</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>12 156 792 910</td>
<td>&nbsp;</td>
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<td>Rabbit anti-Calbindin D-28k antibody</td>
<td>&nbsp;</td>
<td>Swant</td>
<td>CB-38a</td>
<td>&nbsp;</td>
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<td>Alexa 488-goat anti-rabbit IgG secondary antibody</td>
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<td>Invitrogen (Molecular probes)</td>
<td>A-11008</td>
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<td>Vectashield Mounting Medium with DAPI</td>
<td>&nbsp;</td>
<td>Vector labs</td>
<td>H-1200</td>
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<td>Dissecting Microscope</td>
<td>&nbsp;</td>
<td>Zeiss Stemi SV 11</td>
<td>&nbsp;</td>
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<td>Vibratome</td>
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<td>Leica</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Confocal Microscope</td>
<td>&nbsp;</td>
<td>Leica TCS SP2 AOBS</td>
<td>&nbsp;</td>
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organotypic cerebellar cultures: apoptotic challenges and detection

Organotípicas culturas de tecido neuronal foram introduzidas pela primeira vez em 1947 1,2 Hogue, que constituíram um grande avanço no campo da neurociência. Desde então, a técnica foi desenvolvida e, atualmente, existem muitas maneiras diferentes de preparar culturas organotípicas. O método apresentado aqui foi adaptada da descrita por Stoppini et al. Para a preparação das fatias e de Gogolla et al. Para o procedimento de coloração 3,4. Uma característica exclusiva desta técnica é que ele permite que você estude as diferentes partes do cérebro, como hipocampo ou cerebelo em sua estrutura original, proporcionando uma grande vantagem sobre as culturas dissociadas em que toda a organização celular e redes neuronais são interrompidos. No caso do cerebelo é ainda mais vantajosa, pois permite o estudo das células de Purkinje, extremamente difícil de obter como cultura principal dissociados. Este método pode ser usado para estudar algumas características de desenvolvimento do cerebelo in vitro, bem como para experimentos eletrofisiológicos e farmacológicas, tanto do tipo selvagem e camundongos mutantes. O método descrito aqui foi projetado para estudar o efeito dos estímulos apoptóticos, tais como ligante Fas no cerebelo em desenvolvimento, usando coloração TUNEL para medir a morte celular por apoptose. Se a coloração TUNEL é combinada com o tipo de marcadores celulares específicos, como calbindina de células de Purkinje, é possível avaliar a morte celular de uma forma população de células específicas. A coloração calbindina também serve o propósito de avaliar a qualidade das culturas de cerebelo.

Watch this Scientific Journal Video about Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection at JoVE.com

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

Este método descreve a geração de organotípicas culturas cerebelar eo efeito de determinados estímulos apoptóticos sobre a viabilidade de diferentes tipos de células do cerebelo.

Organotípicas Culturas Cerebelar: Desafios Apoptotic e Detecção

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. ...

organotypic-cerebellar-cultures-apoptotic-challenges-and-detection

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JoVE Journal

Neuroscience

18.1K visualizações.  The Salk Institute for Biological Studies. Este método descreve a geração de organotípicas culturas cerebelar eo efeito de determinados estímulos apoptóticos sobre a viabilidade de diferentes tipos de células do cerebelo.

Vídeo: Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

Organotípicas Hippocampal Culturas Slice

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

Neurociência

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. 
Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. 
Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.
In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

Multieletrodos Recordings Array of Avalanches Neuronal em culturas organotípicas

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

Organotípicas culturas de mesencéfalo ventral Slice Embrionárias: Um Sistema para o Estudo de Desenvolvimento Neuronal dopaminérgicos In vitro</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11 are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.

A Análise de Purkinje Morfologia de células dendríticas em Culturas fatia organotípicas

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

Culturas Organotípicas fatia para Estudar Oligodendrocyte Dynamics e Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in&#160;vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury.

A Análise de Neurovascular Remodelação em Entorhino-hipocampal culturas Organotípicas Fatia

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester

Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating innate immunity. Primary rodent cell culture models have greatly advanced our understanding of neuronal-glial interactions, but only recently have methods to specifically eliminate microglia from mixed cultures been utilized. One such technique &#8211; described here &#8211; is the use of L-leucine methyl ester, a lysomotropic agent that is internalized by macrophages and microglia, wherein it causes lysosomal disruption and subsequent apoptosis13,14. Experiments using L-leucine methyl ester have the power to identify the contribution of microglia to the surrounding cellular environment under diverse culture conditions. Using a protocol optimized in our laboratory, we describe how to eliminate microglia from P5 rodent cerebellar granule cell culture. This approach allows one to assess the relative impact of microglia on experimental data, as well as determine whether microglia are playing a neuroprotective or neurotoxic role in culture models of neurological conditions, such as stroke, Alzheimer&#8217;s or Parkinson&#8217;s disease.

Microglia can influence neurons and other glia in culture by various non-cell autonomous mechanisms. Here, we present a protocol to selectively deplete microglia from primary neuronal cultures. This method has the potential to elucidate the role of microglial-neuronal interactions, with implications for neurodegenerative conditions where neuroinflammation is a hallmark feature.

A depleção seletiva de Microglia de Cerebelar Granule culturas de células utilizando L-leucina éster metílico

Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating ...

Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a promising target for intervention to improve functional regeneration. So far, no in vitro model exists that grants the possibility to examine functional recovery of propriospinal fibers. Therefore, a representative model that is based on two organotypic spinal cord sections of embryonic rat, cultured next to each other on multi-electrode arrays (MEAs) was developed. These slices grow and, within a few days in vitro, fuse along the sides facing each other. The design of the used MEAs permits the performance of lesions with a scalpel blade through this fusion site without inflicting damage on the MEAs. The slices show spontaneous activity, usually organized in network activity bursts, and spatial and temporal activity parameters such as the location of burst origins, speed and direction of their propagation and latencies between bursts can be characterized. Using these features, it is also possible to assess functional connection of the slices by calculating the amount of synchronized bursts between the two sides. Furthermore, the slices can be morphologically analyzed by performing immunohistochemical stainings after the recordings. 
Several advantages of the used techniques are combined in this model: the slices largely preserve the original tissue architecture with intact local synaptic circuitry, the tissue is easily and repeatedly accessible and neuronal activity can be detected simultaneously and non-invasively in a large number of spots at high temporal resolution. These features allow the investigation of functional regeneration of intraspinal connections in isolation in vitro in a sophisticated and efficient way.

Here we present a protocol that is based on spinal cord slices cultured on multi-electrode arrays to study functional regeneration of propriospinal connections in vitro.

Investigando regeneração funcional em organotï¿½icas Medula Espinhal Co-culturas cultivadas em Arrays Multi-eletrodo

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

Organotypic fatias Hippocampal culturas como um modelo para estudo neuroproteção e invasão de células tumorais

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Here, we provide a low-cost and reliable method to generate electroporated brain organotypic slice cultures from mouse embryos suitable for confocal microscopy and live-imaging techniques.

Ex Utero Eletroporação e culturas Organotypic fatia do cérebro embrionário Mouse para imagens ao vivo de migração gabaérgica interneurônios

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...