Pesquisa financiada pelo Departamento de Defesa (Defense Advanced Research Projects Agency, Prêmio Faculdade Young, Grant Número N66001-09-1-2117) e NINDS (R15NS070734).

1. Preparação Cirúrgica de Animais 1. Assuntos experimentais Utilizar ratos da B6.Cg-Tg (Thy1-COP4/eYFP) linha transgénica 18Gfng / J 9; JAX estirpe # 7612), ou outros ratos expressando o canal de catião sensível à luz azul, channelrodopsina-2, em neurónios corticais cerebrais. Aplicação de luz azul, para o córtex cerebral do B6.Cg-Tg (Thy1-COP4/eYFP) linha transgénica 18Gfng / J faz com que os neurónios piramidais que expressam channelrodopsina-2 para despolarizar e sofrem a acção potenciais 9,10. Como consequência da activação de células piramidais, interneurónios locais são activados, e a mudança de potencial é propagada para neurónios para além do local de estimulação 10. Realizar a cirurgia em condições estéreis em adesão a diretrizes regulamentares aplicáveis: autoclave instrumentos cirúrgicos e softpacks (campo cirúrgico, compressas de gaze); quente talão esterilizar todos tolerantes ao calor dispositivos metálicos implantados (cânulas, parafusos e fios)para 30 seg; Desinfetar calor intolerantes dispositivos implantáveis ​​(cabo de fibra óptica conector, plástico), com um mergulho de 10 segundos em etanol; sopro etanol-mergulhados itens seco pela aplicação de ar comprimido. 2. Colocação de anestesia, Estereotáxica e Liquidação da Caveira Use o IMPAC 6 integrado multi Paciente sistema (Vet Equip Inc) para anestesiar o rato. Utilizar 5% de oxigénio Isoflurane/95% para indução e 3% de oxigénio Isoflurane/97% para manutenção durante a cirurgia. Colocar o animal sobre uma manta de circulação de água-ajustado para 37 ° C. Não é necessário controlar a temperatura do corpo, se a manta é mantida a esta temperatura. 3. Preparando o crânio para Implantação Preparar o local da incisão com todas as peles de barbear a partir do topo do crânio. A área depilada deve se estender lateralmente de orelha a orelha e ântero-posterior dos olhos até a parte posterior do crânio. Desinfectar a pele exposta com três CONSECUTIVOSCotonetes de e de betadine seguido por etanol. Fazer uma incisão mediana na parte superior do crânio, de entre os olhos para a parte posterior do crânio. Limpar o crânio com peróxido de hidrogénio e solução salina estéril (NaCl a 0,9%). Controlar o sangramento com uma ferramenta de cauterização. Localizar e marcar bregma e lambda para determinar coordenadas estereotáxica. Coordenadas estereotáxicas para configurações de eléctrodos / optogenética estímulo que temos utilizado são apresentados na Tabela 1, e mostrado esquematicamente na Figura 1A. 4. Preparação dos locais de implantação no crânio Use uma broca dental de alta velocidade com um pouco de rebarbas 0,5 milímetros bola para estabelecer cada local de implante no crânio. Chanfrar a margem externa de cada furo com um 0,7 milímetros bola bit rebarba para facilitar a inserção do parafuso. 5. Inserção e Proteção de cânulas, EMG Leads Leads e EEG Insira os parafusos de EEG (0,8 milímetros de comprimento, com 2 cm de 3 não revestidos0 calibre de cobre estanhado-ônibus fio pré-soldada à cabeça do parafuso) nos furos e conduzi-los com uma chave de fenda entalhada a mão revoluções aproximadamente 4-5 para obter a profundidade desejada. Segurar cânulas guia no lugar com um suporte de cânula estereotáxico e depois fixá-las no crânio e âncoras com cimento acrílico. Cada cânula guia deve conter uma cânula manequim / estilete (Plastics One, parte # C312 DC \ 1) desde o momento da cirurgia para o tempo de experimentação (10-14 dias), para manter a desobstrução. 6. Fechando Sítio Cirúrgico Uma vez EEGs e cânulas guia estão posicionados no crânio, bond-los com cimento acrílico dental (Lang Dental - Ortho-Jet cimento). Depois de conjuntos de cimento, posicionar o conector de plástico (Pinnacle parte Tecnologia # 8402) acima do monte de cimento seco. Soldar as extremidades dos fios condutores provenientes de EEG para os contactos do conector de plástico. Cubra o fio leva em cimento. Em seguida, desenhe os fios EMG através do músculo da nucas deslizando-as para dentro do tambor de uma agulha 21ga perfurado através do músculo. Amarre um nó cirurgião dupla de náilon 5-0 em torno desses fios apenas distal para onde eles saem do músculo. Suture junto a pele que foi recolhido para acessar o tecido muscular com o único cirurgião nós interrompidos, usando um corte reverso P-3 agulha e náilon 5-0. 7. Analgesia pós-operatória e recuperação Administrar a buprenorfina como analgésico (0,1 mg / kg, subcutânea) e flunixin meglumine (0,1 mg / kg, subcutânea), como um anti-inflamatório, imediatamente após a cirurgia, e diariamente nos dias 1-2 após a cirurgia. Permitir que os animais se recuperar em condições de habitação padrão de colônia por pelo menos sete dias antes da experimentação. Com nenhum cuidado adicional para além da habitação colónia padrão, cânulas pode ser esperado para permanecer patente por pelo menos duas semanas, com os estiletes indwelling garantidos no lugar. Sensores de EEG e EMG pode também ser esperado para reterfuncionalidade para esta duração. 2. Geração de Pulsos Temporário Luz 1. Programação de MC unidade de estímulo Ligue a unidade MC-estímulo (Multi-Channel Systems) através de uma conexão USB a um computador desktop. Use a planilha como MC Estímulo-interface de usuário para programar a unidade de estímulo MC. Digite o TTL desejado (transistor-transistor logic) de tensão para o período em diante, as durações de estímulo e fora períodos, o número de ciclos por segundo desejados, eo número total de ciclos repetidos para a sessão de estímulo inteiro. Para mais detalhes, consulte MC-estímulo manual técnico: 3. Fabricação / Montagem de Fibra Óptica PrEFCs para entrega Luz para o córtex cerebral <ol><li> Suprimentos e equipamentos necessários são encontrados em: <a href="http://www.thorlabs.com/Thorcat/1100/1166-D02.pdf" target="_blank">http://www.thorlabs.com/Thorcat/1100/1166-D02.pdf</a> </li><li> Corte e polimento de cabos de fibra óptica. <a href="http://www.thorlabs.com/Thorcat/1100/1166-D02.pdf" target="_blank">http://www.thorlabs.com/Thorcat/1100/1166-D02.pdf</a> </li></ol> 4. Assembleia de Roedor sistema de estimulação optogenética 1. Interface do MC-Estímulo Unidade e Laser Uma vez que a unidade MC-estímulo é programado, execute-o como um gerador de sinal independente de um binário de 5 V ON / OFF de sinal. Ligue a unidade MC estímulo para a unidade de potência TTL habilitado do laser com conectores BNC. 2. Produzir Laser saída via cabo de fibra óptica Utilizar o mostrador digital e com ligação de saída de potência variável da unidade de potência do laser para manually ajustar a potência de saída do laser. Ligue a unidade de potência do laser para o laser através de um cabo de fita. Ligue o FC fêmea (ponteira do conector) do adaptador de fibra do laser para o conector macho FC no cabo de fibra óptica (a 3 m de comprimento, 200 um de diâmetro de fibra de vidro revestido de condução de luz refletindo acrilato de revestimento de polímero). 3. Transmitindo a Laser para o Animal: comutador rotativo Conectar o cabo de fibra óptica de origem (3 m de comprimento), e o cabo de fibra óptica de entrega (45 cm de comprimento) de extremidades opostas do conjunto óptico rotativo que encerra duas lentes de colimação (lentes dóricas). A junta de fibra óptica rotativo serve como um comutador: enquanto o animal se move sobre a sua gaiola, a junta rotativa roda para impedir a ruptura dos cabos de fibra óptica. Use braçadeiras plásticas para fixar o comutador de um metal ficar colocado por cima do compartimento cilíndrico em que o animal está alojado. 4. Colocar Entrega de fibra ópticaCabo Direto Contenha o mouse usando uma mão para prender o mouse sob uma palma em concha. Orientar a cabeça do meio entre o experimentador eo dedo indicador. Remova a cânula simulado a partir da cânula guia colocado para a cânula de fibra óptica. Desmarque a cânula guia de detritos usando uma agulha estéril 25ga. Insira o cabo de fibra óptica com a mão e prendê-lo ao cânula guia de fibra óptica com uma tampa de rosca de rosca recuperado de uma cânula manequim. Controlar a profundidade de inserção do cabo de fibra óptica no cérebro por um nó de sutura ligada no cabo de fibra óptica a uma distância fixa a partir da extremidade plana clivada. Todo o aparelho experimental é apresentado na Figura 1B. 5. Medição de EEG-definida do sono e da concentração de lactato durante a estimulação optogenética 1. Pré-calibragem do sensor Lactato Pré-calibração do sensor de lactato é realizada com um in vitrokit (Pinnacle parte Tecnologia # 7000-K1-T). O sensor é equilibrada em solução salina tamponada com fosfato (pH 7,4), em seguida, expostos a três concentrações de L-lactato de forma gradual, por protocolos do fabricante. 2. Inserção do sensor de lactato e cabo EEG Inserir o sensor pré-calibrado no crânio montado cânula guia de lactato de forma idêntica ao procedimento de inserção do cabo de fibra óptica (secção 4.4). Ligue o sensor de lactato para o pré-amplificador do 8400 Pinnacle biossensor com conectores bipolares ficha. Anexe este pré-amplificador para o conector de 8 pinos no Headmount cirurgicamente implantado. 3. Estabelecer optogenética intensidade do estímulo Antes de coletar dados, use o botão de controle de laser intensidade para ajustar a intensidade do estímulo optogenética. A amplitude da resposta de EEG irá variar entre animais, devido a factores que ainda não foram estudadas sistematicamente, uma<html> d deve ser verificada por inspecção visual no início da estimulação. Na prática, uma intensidade de 80-120 μWatts tipicamente produzir um aumento na amplitude do EEG de aproximadamente 50% na frequência de estimulação. Post-hoc de análise rápida de Fourier Transform (ver secção 5.5) é necessário quantificar a magnitude absoluta da resposta. 4. Coleta de Dados Coletar dados usando o Pinnacle 8400 sistema: <a href="http://pinnaclet.com/pal-8400.html" target="_blank">http://pinnaclet.com/pal-8400.html</a> . 5. Processamento de Dados com Neuroscore Classificar dormir estados por inspeção visual dos dados de EEG e EMG com a interface Neuroscore (DataSciences, Internacional: <a href="http://www.datasci.com/products/software/NeuroScore_CNS_Software.asp" target="_blank">http://www.datasci.com/products/software/NeuroScore_CNS_Software.asp</a> ) ou a interface Sirenia (Pinnacle Tecnologia:f = &quot;http://www.pinnaclet.com/sirenia.html&quot; target = &quot;_blank&quot;&gt; http://www.pinnaclet.com/sirenia.html). Dados do processo em 10 segundos épocas como sono-vigília, o movimento não-rápido dos olhos, ou sono do movimento rápido dos olhos baseada em EEG e EMG. Salve dados como planilhas do Microsoft Excel para análise e testes adicionais estatística.

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Os métodos aqui apresentados permitem medir a relação entre o sono e as alterações na concentração do lactato cérebro intermediário glicolítico numa escala de tempo não era possível anteriormente. Animais sofrem transições espontâneas entre vigília, NREMS e REMS. Além disso, somos capazes de aplicar estímulos optogenética enquanto que os animais submetidos a estas transições. Os dados recolhidos até à data demonstram que tanto espontânea e induzida por ondas de impacto sobre a leitura de um bioss...

<table border="1"><tbody><tr><td> Componente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários (opcional) </td></tr><tr><td> Basi Rato Guia Cânula </td><td> Pinnacle Tecnologia / Basi Inc </td><td> 7032 </td><td></td></tr><tr><td> Biosensor lactato </td><td> Pinnacle Tecnologia </td><td> 7004 </td><td></td></tr><tr><td> Cabeça de Montagem </td><td> Pinnacle Tecnologia </td><td> 8402 </td><td></td></tr><tr><td> Sleep / Sistema de gravação Biosensor </td><td> Pinnacle Tecnologia </td><td> 8400-K1-SL </td><td> 2 canais de EEG, EMG um canal, e um biossensor </td></tr><tr><td> Rato Tethered in-vitro kit de calibração </td><td> Pinnacle Tecnologia </td><td> 7000-K1-T </td><td></td></tr><tr><td> Fibra Óptica Guia Cânula </td><td> Um plásticos </td><td> C312G </td><td> 21 Guia do calibre Cânula </td></tr><tr><td> Cânula manequim </td><td> Um plásticos </td><td> C312DC </td><td> 21 Manequim Calibre </td></tr><tr><td> Diamante Scribe Fibra </td><td> Thorlabs </td><td> S90W </td><td></td></tr><tr><td> Conector de fibra de crimpagem </td><td> Thorlabs </td><td> CT042 </td><td></td></tr><tr><td> Tubulação de furca </td><td> Thorlabs </td><td> FT030 </td><td> 03.0 milímetros </td></tr><tr><td></td><td> Thorlabs </td><td> T10S13 </td><td> Max Dia. 0,012 </td></tr><tr><td> Stripper Tubo furca </td><td> Thorlabs </td><td> FTS3 </td><td></td></tr><tr><td> Fibra Revestimento nua rígido Multimodo </td><td> Thorlabs </td><td> BFL37-200 </td><td> Núcleo 200 uM, 0,37 NA </td></tr><tr><td> Fio Snips / Kevlar Tesouras </td><td> Thorlabs </td><td> T865 </td><td></td></tr><tr><td> Epoxy da fibra óptica </td><td> Thorlabs </td><td> F112 </td><td></td></tr&gt; <tr><td> Ferramenta Stripper fibra </td><td> Thorlabs </td><td></td><td></td></tr><tr><td> Plate Glass Polishing </td><td> Thorlabs </td><td> CTG913 </td><td></td></tr><tr><td> Borracha Polir </td><td> Thorlabs </td><td> NRS913 </td><td></td></tr><tr><td> Olho Lupa </td><td> Thorlabs </td><td> JEL10 </td><td></td></tr><tr><td> Kim Wipes </td><td> Thorlabs </td><td> KW32 </td><td></td></tr><tr><td> Ar Comprimido </td><td> Thorlabs </td><td> CA3 </td><td></td></tr><tr><td> Polimento Puck </td><td> Thorlabs </td><td> D50-xx </td><td></td></tr><tr><td> Âmbito Inspeção de fibra </td><td> Thorlabs </td><td> CL-200 </td><td></td></tr><tr><td> Filmes de polimento </td><td> Thorlabs </td><td> LFG5P, LFG3P, LFG1P, LFG03P </td><td></td></tr><tr><td> FC / PC extremidade do conector </td><td> Thorlabs </td&gt; <td> 30126G2-240 </td><td> 240 Bore um, SS virola </td></tr><tr><td> MC unidade de estímulo </td><td> Multi-Canal de Sistemas </td><td> STG-4002 </td><td></td></tr><tr><td> MC Software de estímulo </td><td> Multi-Canal de Sistemas </td><td> MC-Estímulo V 2.1.5 </td><td></td></tr><tr><td> Blue Laser </td><td> CrystaLaser </td><td> CL473-050-0 </td><td></td></tr><tr><td> Laser Alimentação </td><td> CrystaLaser </td><td> CL2005 </td><td></td></tr><tr><td> Conjunto de Fibra Óptica Rotary </td><td> Lentes dóricas </td><td> FRJ-v4 </td><td></td></tr><tr><td></td><td></td><td></td><td> Tabela 2. Suprimentos e equipamentos. </td></tr></tbody></table>

simultaneous electroencephalography, real-time measurement of lactate concentration and optogenetic manipulation of neuronal activity in the rodent cerebral cortex

Embora o cérebro representa menos de 5% da massa corporal por, utiliza cerca de um quarto da glucose utilizada pelo corpo em repouso 1. A função do sono movimento rápido dos olhos não (NREMS), a maior porção do sono pelo tempo, é incerto. No entanto, uma característica saliente do NREMS é uma redução significativa da taxa de utilização de glicose cerebral em relação ao estado de vigília 2-4. Esta e outras descobertas levaram à crença de que o sono tem uma função relacionada ao metabolismo cerebral. No entanto, os mecanismos subjacentes à redução no metabolismo da glicose cerebral durante NREMS permanecem por ser elucidados. Um fenômeno associado com NREMS que podem afetar a taxa metabólica cerebral é a ocorrência de ondas lentas, oscilações em freqüências menos de 4 Hz, no eletroencefalograma 5,6. Estas ondas lentas detectados ao nível do crânio ou da superfície cortical cerebral reflectir aoscilações de neurônios subjacentes entre um estado despolarizado / up e um estado hiperpolarizada / baixo 7. Durante o estado para baixo, as células não sofrem potenciais de ação em intervalos de até vários milissegundos cem. Restauração dos gradientes de concentração iônica subseqüentes para os potenciais de ação representa uma carga significativa metabólica na célula 8; ausência de potenciais de ação durante os estados para baixo associados NREMS pode contribuir para um metabolismo reduzido em relação ao acordar. Dois desafios técnicos tiveram que ser abordadas para que essa hipotética relação a ser testada. Em primeiro lugar, foi necessário medir o metabolismo glicolítico cerebral com uma resolução temporal de reflexo da dinâmica do EEG cerebral (isto é, ao longo de segundos em vez de minutos). Para o fazer, foi medida a concentração de lactato, o produto da glicólise aeróbica, e, portanto, uma leitura da taxa do metabolismo da glucose no cérebro dos ratos. Lactato foimedida utilizando um sensor de lactato-oxidase com base em tempo real, incorporado no córtex frontal. O mecanismo de detecção é constituído por um eléctrodo de platina-irídio rodeado por uma camada de moléculas de oxidase de lactato. O metabolismo do lactato pela lactato oxidase produz peróxido de hidrogénio, o qual produz uma corrente no eléctrodo de platina-irídio. Assim, uma rampa para cima da glicólise cerebral proporciona um aumento na concentração do substrato para a oxidase de lactato, que, em seguida, reflecte-se no aumento da corrente no eléctrodo de detecção. Era ainda necessário medir estas variáveis ​​enquanto manipulando a excitabilidade do córtex cerebral, a fim de isolar esta variável de outras facetas NREMS. Criámos um sistema experimental para a medição simultânea de actividade neuronal por meio do elecetroencephalogram, a medição do fluxo glicolítico através de um biossensor de lactato, e a manipulação da actividade neuronal cortical cerebral através optogenética activação de pyraneurônios MIDAL. Foi utilizado este sistema para documentar a relação entre o sono relacionada electroencefalográfico formas de onda e a dinâmica momento-a-momento da concentração de lactato no córtex cerebral. O protocolo pode ser útil para qualquer indivíduo interessados ​​em estudar, em livremente comportando roedores, a relação entre a actividade neuronal medido ao nível EEG e energetics celulares dentro do cérebro.

<html> Como mostrado na Figura 2, um mouse equipado para optogenética estimulação e lactato / EEG / EMG dados foram submetidos à coleta de transições espontâneas sono / vigília do estado enquanto EEG, EMG e concentração de lactato cerebral foram monitorados continuamente. Corrente para o sensor de lactato aumentou durante os períodos de baixa amplitude do EEG e diminuiu durante os períodos de alta amplitude EEG. Como mostrado na Figura 3, ambos os canais de...

Watch this Scientific Journal Video about Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral C

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Um procedimento é descrito para manipular a actividade de neurónios piramidais corticais cerebrais optogenetically enquanto o electroencefalograma, electromiograma, e concentração do lactato cerebral são monitorados. Gravações experimentais são realizados em ratinhos de cabo-cativos, enquanto eles sofrem espontâneas do sono / vigília ciclos. Optogenética equipamento é montado no nosso laboratório, o equipamento de gravação encontra-se comercialmente disponível.

Eletroencefalografia simultânea, em tempo real de medição da concentração de lactato e Manipulação optogenética da atividade neuronal do córtex cerebral de roedores

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

simultaneous-electroencephalography-real-time-measurement-lactate

Research

JoVE Journal

Neuroscience

20.1K visualizações.  Washington State University. Um procedimento é descrito para manipular a actividade de neurónios piramidais corticais cerebrais optogenetically enquanto o electroencefalograma, electromiograma, e concentração do lactato cerebral são monitorados. Gravações experimentais são realizados em ratinhos de cabo-cativos, enquanto eles sofrem espontâneas do sono / vigília ciclos. Optogenética equipamento é montado no nosso laboratório, o equipamento de gravação encontra-se comercialmente dispon?...

Vídeo: Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Simultaneous fMRI and Electrophysiology in the Rodent Brain

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in which functional MRI data and in vivo intracortical recording can be performed simultaneously. The combination of MRI and electrical recording is technically challenging because the electrodes used for recording distort the MRI images and the MRI acquisition induces noise in the electrical recording. To minimize the mutual interference of the two modalities, glass microelectrodes were used rather than metal and a noise removal algorithm was implemented for the electrophysiology data. In our studies, two microelectrodes were separately implanted in bilateral primary somatosensory cortices (SI) of the rat and fixed in place. One coronal slice covering the electrode tips was selected for functional MRI. Electrode shafts and fixation positions were not included in the image slice to avoid imaging artifacts. The removed scalp was replaced with toothpaste to reduce susceptibility mismatch and prevent Gibbs ringing artifacts in the images. The artifact structure induced in the electrical recordings by the rapidly-switching magnetic fields during image acquisition was characterized by averaging all cycles of scans for each run. The noise structure during imaging was then subtracted from original recordings. The denoised time courses were then used for further analysis in combination with the fMRI data. As an example, the simultaneous acquisition was used to determine the relationship between spontaneous fMRI BOLD signals and band-limited intracortical electrical activity. Simultaneous fMRI and electrophysiological recording in the rodent will provide a platform for many exciting applications in neuroscience in addition to elucidating the relationship between the fMRI BOLD signal and neuronal activity.

We have developed a method for simultaneous functional magnetic resonance imaging and electrophysiological recording in the rodent brain, providing a platform for the investigation of the relationship between neural activity and the blood oxygenation level dependent (BOLD) MRI signal.

FMRI simultânea e Eletrofisiologia no cérebro de roedores

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in ...

Neurociência

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the first born neurons remain closer to the ventricle while the last born neurons migrate past the first born neurons towards the surface of the brain1. In addition to neuronal migration2, a key process for normal cortical function is the regulation of neuronal morphogenesis3. While neuronal morphogenesis can be studied in vitro in primary cultures, there is much to be learned from how these processes are regulated in tissue environments.
We describe techniques to analyze neuronal migration and/or morphogenesis in organotypic slices of the cerebral cortex4,6. A pSilencer modified vector is used which contains both a U6 promoter that drives the double stranded hairpin RNA and a separate expression cassette that encodes GFP protein driven by a CMV promoter7-9. Our approach allows for the rapid assessment of defects in neurite outgrowth upon specific knockdown of candidate genes and has been successfully used in a screen for regulators of neurite outgrowth8. Because only a subset of cells will express the RNAi constructs, the organotypic slices allow for a mosaic analysis of the potential phenotypes. Moreover, because this analysis is done in a near approximation of the in vivo environment, it provides a low cost and rapid alternative to the generation of transgenic or knockout animals for genes of unknown cortical function. Finally, in comparison with in vivo electroporation technology, the success of ex vivo electroporation experiments is not dependant upon proficient surgery skill development and can be performed with a shorter training time and skill.

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth.

Métodos de Estudo da morfogênese neuronal:<em&gt; Ex vivo</em&gt; Eletroporação RNAi no Cortex Cerebral Murina embrionárias

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

Visualização e manipulação genética de dendritos e espinhas no córtex cerebral e hipocampo do rato usando<em&gt; In utero</em&gt; Eletroporação

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Dois registros eletrofisiológicos de sinapticamente evocadas Respostas astroglial e neuronal em fatias hipocampais agudas

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

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 ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

Induzir Plasticidade de astrocítica Receptores de Manipulação de Neuronal queima de moeda

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

In vivo Optogenetic estimulação do sistema nervoso central de roedores

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools to selectively manipulate brain rhythms. Here we describe an approach combining projection-specific optogenetics with extracellular electrophysiology for high-fidelity control of hippocampal theta oscillations (5-10 Hz) in behaving mice. The specificity of the optogenetic entrainment is achieved by targeting channelrhodopsin-2 (ChR2) to the GABAergic population of medial septal cells, crucially involved in the generation of hippocampal theta oscillations, and a local synchronized activation of a subset of inhibitory septal afferents in the hippocampus. The efficacy of the optogenetic rhythm control is verified by a simultaneous monitoring of the local field potential (LFP) across lamina of the CA1 area and/or of neuronal discharge. Using this readily implementable preparation we show efficacy of various optogenetic stimulation protocols for induction of theta oscillations and for the manipulation of their frequency and regularity. Finally, a combination of the theta rhythm control with projection-specific inhibition addresses the readout of particular aspects of the hippocampal synchronization by efferent regions.

We describe the use of optogenetics and electrophysiological recordings for selective manipulations of hippocampal theta oscillations (5-10 Hz) in behaving mice. The efficacy of the rhythm entrainment is monitored using local field potential. A combination of opto- and pharmacogenetic inhibition addresses the efferent readout of hippocampal synchronization.

Optogenetic de arrastamento de Theta Hippocampal oscilações em comportar-se ratos

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools ...

Combined Transcranial Magnetic Stimulation and Electroencephalography of the Dorsolateral Prefrontal Cortex

Transcranial magnetic stimulation (TMS) is a non-invasive method that produces neural excitation in the cortex by means of brief, time-varying magnetic field pulses. The initiation of cortical activation or its modulation depends on the background activation of the neurons of the cortical region activated, the characteristics of the coil, its position and its orientation with respect to the head. TMS combined with simultaneous electrocephalography (EEG) and neuronavigation (nTMS-EEG) allows for the assessment of cortico-cortical excitability and connectivity in almost all cortical areas in a reproducible manner. This advance makes nTMS-EEG a powerful tool that can accurately assess brain dynamics and neurophysiology in test-retest paradigms that are required for clinical trials. Limitations of this method include artifacts that cover the initial brain reactivity to stimulation. Thus, the process of removing artifacts may also extract valuable information. Moreover, the optimal parameters for dorsolateral prefrontal (DLPFC) stimulation are not fully known and current protocols utilize variations from the motor cortex (M1) stimulation paradigms. However, evolving nTMS-EEG designs hope to address these issues. The protocol presented here introduces some standard practices for assessing neurophysiological functioning from stimulation to the DLPFC that can be applied in patients with treatment resistant psychiatric disorders that receive treatment such as transcranial direct current stimulation (tDCS), repetitive transcranial magnetic stimulation (rTMS), magnetic seizure therapy (MST) or electroconvulsive therapy (ECT).

The protocol presented here is for TMS-EEG studies utilizing intracortical excitability test-retest design paradigms. The intent of the protocol is to produce reliable and reproducible cortical excitability measures for assessing neurophysiological functioning related to therapeutic interventions in the treatment of neuropsychiatric diseases such as major depression.

Estimulação magnética transcraniana combinada e Eletroencefalografia do córtex pré-frontal Dorsolateral

Transcranial magnetic stimulation (TMS) is a non-invasive method that produces neural excitation in the cortex by means of brief, time-varying magnetic ...

Simultaneous Cryosectioning of Multiple Rodent Brains

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In neuroscience, as well as a plethora of other scientific fields, these techniques are used. Immunohistochemistry can be done on slide mounted tissue or free-floating sections. Preparing slide-mounted samples is a time intensive process. The following protocol for a technique, called the Megabrain, reduced the time taken to cryosection and mount brain tissue by up to 90% by combining multiple brains into a single frozen block. Furthermore, this technique reduced variability seen between staining rounds, in a large histochemical study. The current technique has been optimized for using rodent brain tissue in downstream immunohistochemical analyses; however, it can be applied to different scientific fields that use cryosectioning.

Here, we present a protocol to freeze and section brain tissue from multiple animals as a timesaving alternative to processing single brains. This reduces staining variability during immunohistochemistry and reduces time cryosectioning and imaging.

Cryosectioning simultânea de vários cérebros de roedores

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In ...