Este trabalho foi apoiado pelos Institutos Nacionais de Subsídios à Saúde (R01NS085215 a K.F., T32 GM107000 e F30MH122146 a A.C.). Os autores agradecem à Sra. Naoe Watanabe por assistência técnica hábil.

Todos os protocolos animais foram revisados e aprovados pelo Comitê Institucional de Cuidados e Uso de Animais (IACUC) da Faculdade de Medicina da Universidade de Massachusetts. A preparação da cultura de fatias, a preparação plasmida e a eletroporação também estão detalhadas em nossos métodos publicados anteriormente e podem ser encaminhadas para informações adicionais10.
1. Preparação para cultura de fatias
<ol><li>Prepare culturas de fatias hipocampais organotípicas do rato como descrito anteriormente11, usando camundongos pós-natal de 6 a 7 dias de idade de ambos os sexos.<ol>
<li>Preparar a mídia de dissecção para a cultura de fatia organotipípica composta (em mM): 238 sacarose, 2,5 KCl, 1 CaCl2, 4 MgCl2, 26 NaHCO3, 1 NaH2PO4, e 11 glicose em água desionizada, depois gás com 5% DE CO2/95% O2 a um pH de 7,4.</li>
		<li>Prepare a mídia de cultura de fatia organotipípica composta por: 78,8% (v/v) Águia Média Essencial Mínima, 20% (v/v) soro de cavalo, 17,9 mM NaHCO3, 26,6 mM de glicose, 2 M CaCl2, 2 M MgSO4,30 mM HEPES, insulina (1 μg/mL) e 0,06 mM ácido ascórbico, pH ajustado para 7,3. Ajuste a osmolaridade para 310-330 osmol usando um osmômetro.</li>
		<li>Disseque hipocampi de todo o cérebro usando duas espátulas, e corte (400 μm) usando um helicóptero de tecido. Separe as fatias usando duas fórceps e transfira para inserções de cultura celular de 30 mm em uma placa de 6 poços cheia de mídia de cultura (950 μL) sob as pastilhas.</li>
	</ol>
</li>
	<li>Armazene culturas de fatias organotipíticas em uma incubadora de cultura de tecidos (35°C, 5% CO2) e mude a mídia de cultura de fatias a cada dois dias.</li>
</ol>2. Preparação plasmid
<ol><li>Prepare o plasmídeo para o gene de interesse.<ol>
<li>Subclone aprimorou o gene da proteína fluorescente verde (EGFP) em um vetor pCAG.</li>
		<li>Purifique o plasmídeo pCAG-EGFP com um kit de purificação livre de endotoxinas e dissolva-se em uma solução interna que consiste em água tratada com pirocarbonato de dietilo contendo 140 mM K-metanosulfonato, 0,2 mM EGTA, 2 mM MgCl2e 10 mM HEPES, ajustado para pH 7.3 com KOH (concentração plasmida: 0,1 μg/μL).</li>
	</ol>
</li>
</ol>3. Preparação de pipeta de vidro
<ol><li>Puxe pipetas de vidro borossilicato (4,5 – 8 MΩ) em um puxador de micropipette(Figura 1A). NOTA: As pipetas de vidro usadas para gravações de grampos de remendo de células inteiras são ideais para eletroporação.</li>
	<li>Asse pipetas de vidro durante a noite a 200°C para esterilizar.</li>
	<li>Verifique o tamanho da ponta da pipeta sob um microscópio de dissecção para aproximar a resistência elétrica.<ol>
<li>Opcional:Verifique a resistência da pipeta anexando a pipeta ao eletrodo de eletroporação e use o micromanipulador para manobrar a ponta da pipeta em fluido cerebrospinal artificial esterilizado por filtro (aCSF) contendo (em mM): 119 NaCl, 2,5 KCl, 0,5 CaCl2, 5 MgCl2, 26 NaHCO3, 1 NaH2PO4 e 11 glicose em água desionizada, gaseado com 5% de CO2/95% O2 a um pH de 7,4. Confirme a resistência real usando a leitura no eletroporador. NOTA: Quanto mais nítida a ponta da pipeta, maior a resistência elétrica. A resistência à pipeta deve estar abaixo de 10 MΩ. Pipetas de vidro com alta resistência à pipeta(Figura 1B) muitas vezes entupem na ponta durante a eletroporação repetida.</li>
	</ol>
</li>
</ol>4. Configuração da plataforma de eletroporação
<ol><li>Instale o eletroporador em uma plataforma padrão de eletrofisiologia de células inteiras, equipada com um microscópio vertical montado em uma mesa de mudança com uma bomba micromanipuladora e peristáltica.</li>
	<li>Instale o headstage do eletroporador em um micromanipulador e conecte um par de alto-falantes ao eletroporador. Conecte o eletroporador a um pedal de pé que pode ser usado para enviar um pulso quando estiver pronto. NOTA: Os alto-falantes emitem um tom quando ligados, que é um indicador da resistência elétrica no eletrodo. Isso permite determinar mudanças relativas na resistência sem afastar a atenção do procedimento.</li>
</ol>5. Preparação para eletroporação
<ol><li>Transfira as pastilhas de cultura de fatias de 6 placas de poço para 3 cm de placas de Petri carregadas com 900 μL de mídia cultural e armazene em uma incubadora de CO2 até estar pronta para realizar a eletroporação.<ol>
<li>Pré-inser em que a cultura fresca insira com meios de cultura fatia (1 mL) por pelo menos 30 min em uma placa de Petri de 3,5 cm para fatias de cultura após a eletroporação.</li>
	</ol>
</li>
	<li>Limpe e prepare a plataforma para eletroporação.<ol>
<li>Perfunda as linhas com alvejante de 10% por 5 minutos para esterilizar o tubo e a câmara antes de iniciar o experimento para o dia.</li>
		<li>Perfumar as linhas com água autoclavada deionizada por pelo menos 30 minutos para enxaguar completamente.</li>
		<li>Perfunda as linhas com aCSF esterilizado por filtro contendo tetrodotoxina de 0,001 mM (TTX). NOTA: O TTX minimiza a toxicidade celular e a morte devido à superexcitação dos interneurons10.</li>
	</ol>
</li>
	<li>Defina os parâmetros de pulso do eletroporador: amplitude de –5 V, pulso quadrado, trem de 500 ms, frequência de 50 Hz e uma largura de pulso de 500 μs.</li>
	<li>Encha a pipeta de vidro com 5 μL de solução interna contendo plasmídeos.<ol>
<li>Remova quaisquer bolhas de ar presas da ponta da pipeta piscando e tocando suavemente a ponta várias vezes.</li>
		<li>Verifique se há danos na ponta visualizando-a sob um microscópio de dissecção ou repetindo o passo 3.3.1 para verificar a resistência da pipeta. NOTA: Se a ponta estiver danificada, a pipeta de vidro deve ser descartada, e esta etapa deve ser repetida com uma nova pipeta de vidro previamente preparada na etapa 3.</li>
	</ol>
</li>
	<li>Conecte com segurança a ponta da pipeta ao eletrodo e ligue os alto-falantes. Registo a leitura (resistência da pipeta) do eletroporador quando a ponta tiver feito contato com o meio aCSF.</li>
	<li>Corte a membrana de inserção de cultura usando uma lâmina afiada e isole uma cultura de fatia. Transfira cuidadosamente a cultura da fatia para a câmara de eletroporação usando fórceps angulares afiados e fixe sua posição com uma âncora de fatia.<ol>
<li>Não mantenha a cultura da fatia fora da incubadora por mais de 30 minutos de cada vez para evitar efeitos colaterais, como alterações na saúde neuronal ou função12.</li>
	</ol>
</li>
</ol>6. Células eletroporadas de interesse
<ol><li>Aplique pressão positiva na pipeta com boca ou usando uma seringa de 1 mL (pressão de 0,2 - 0,5 mL) presa à tubulação.</li>
	<li>Use os controles de botão tridimensional do micromanipulador para manobrar a ponta da pipeta perto da superfície da cultura da fatia.</li>
	<li>Escolha uma célula-alvo e aproxime-a, mantendo a pressão positiva aplicada até que uma covinha se forme na superfície da célula, visível no microscópio.</li>
	<li>Realize ciclos de pressão.<ol>
<li>Aplique rapidamente uma leve pressão negativa pela boca de modo que uma vedação solta se forme entre a ponta da pipeta e a membrana plasmática, indicada visualmente pela membrana subindo um pouco na ponta da pipeta. Observe um aumento (~2,5x a resistência inicial) na resistência à pipeta ouvindo um aumento no tom proveniente dos alto-falantes. Aplice rapidamente a pressão positiva para que a covinha se reeme.</li>
		<li>Imediatamente complete pelo menos mais dois ciclos de pressão sem pausar, em seguida, mantenha pressão negativa para 1 s. NOTA: Pausar entre ciclos, aplicar muita pressão ou segurar a pressão negativa por muito tempo pode causar danos celulares significativos e possivelmente causar a morte da célula durante a eletroporação.</li>
	</ol>
</li>
	<li>Pulso rápido do eletroporador uma vez usando o pedal do pé quando o tom dos alto-falantes atinge um ápice estável no tom, indicando pico de resistência elétrica. Não espere no pico de resistência por mais de 1 s antes de enviar o pulso. NOTA: Não observamos eletroporação fora do alvo ao usar este protocolo10. Apenas as células em contato com a pipeta de vidro durante os ciclos de pressão foram transfecidas. Posicionar a pipeta perto de outros neurônios não resulta em transfecção genética.</li>
	<li>Retire suavemente a pipeta aproximadamente 100 μm da célula sem aplicar pressão.</li>
	<li>Reaplousa a pressão positiva, verificando se a resistência é semelhante à leitura registrada na etapa 5.5 e, em seguida, aproxime-se da célula seguinte.<ol>
<li>Remova os potenciais tamancos, indicados visualmente ou por uma resistência significativa (>15% maior) à pipeta após a eletroporação, aplicando pressão positiva. NOTA: Se não houver entupimento visível e a resistência ainda for significativamente maior, descarte a pipeta e use uma nova. Em média, uma pipeta pode ser usada para até 20 eventos de eletroporação se o usuário tivercuidado 10.</li>
	</ol>
</li>
	<li>Após a eletroporação, transfira a cultura da fatia para uma inserção de cultura fresca e incubar a 35°C na incubadora por até 3 dias.</li>
</ol>7. Fixação, coloração e imagem de culturas de fatias hipocampais organotípicas
<ol><li>Fixar culturas de fatia organofíspica eletroporada 2 – 3 dias após a transfecção com 4% de paraformaldeído e 4% de sacarose em 0,01 M de salina tamponada de fosfato (1x PBS) por 1,5 h à temperatura ambiente.</li>
	<li>Remova as fatias fixativas e incubadas em 30% de sacarose em 0,1 M tampão fosfato (1x PB) por 2 h.</li>
	<li>Coloque fatias em um copo de slides e congele-as colocando o vidro de deslizamento em cima de gelo seco esmagado. Descongele as fatias em temperatura ambiente e transfira-as para uma placa de 6 poços cheia de 1x PBS.</li>
	<li>Manche as fatias com anticorpos anti-GFP e coelhinho em tampão GDB (0,1% gelatina, 0,3% Triton X-100, 450 mM NaCl e 32% 1x PB, pH 7,4) por 2h a temperatura ambiente.</li>
	<li>Lave as fatias com 1x PBS três vezes à temperatura ambiente por 10 minutos cada lavagem.</li>
	<li>Incubar fatias com anticorpo secundário alexa 488 conjugado e anticorpo secundário alexa 594 conjugados em tampão GDB por 1 h à temperatura ambiente. Incubar fatias com DAPI (4 μg/mL) em 1x PBS por 10 min a temperatura ambiente.</li>
	<li>Lave as fatias com 1x PBS três vezes à temperatura ambiente por 3 minutos cada lavagem.</li>
	<li>Monte fatias em lâminas de vidro usando meio de montagem e realize imagens de fluorescência.</li>
</ol>

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Os autores declaram que não têm conflitos de interesse.

Descrevemos aqui um método de eletroporação que transfecta neurônios excitatórios e inibitórios com alta eficiência e precisão. Nosso protocolo de eletroporação otimizado tem três avanços inovadores para alcançar transfecção genética altamente eficiente. Nossa primeira modificação foi aumentar o tamanho da pipeta em comparação com os protocolos publicados anteriormente3,5,6. Essa mudança nos permitiu eletrop...

Na biologia molecular, uma das considerações mais importantes para um pesquisador é como entregar um gene de interesse em uma célula ou população de células para elucidar sua função. Os diferentes métodos de parto podem ser categorizados como biológicos (por exemplo, um vetor viral), químicos (por exemplo, fosfato de cálcio ou lipídico) ou físicos (por exemplo, eletroporação, microinjeção ou biolística)1,2. Os métodos biológicos são altamente eficientes e podem ser específicos do tipo celular, mas são limitados pelo desenvolvimento de ferramentas genéticas específicas. Abordagens químicas são muito poderosas in vitro, mas transfecções são geralmente aleatórias; além disso, essas abordagens são principalmente reservadas apenas para células primárias. Das abordagens físicas, a biolística é a mais simples e fácil do ponto de vista técnico, mas novamente produz resultados aleatórios de transfecção com uma eficiência relativamente baixa. Para aplicações que requerem transferência para células específicas sem a necessidade de desenvolver ferramentas genéticas, buscamos eletroporação unicelular3,4.
Considerando que a eletroporação usada para se referir apenas à eletroporação de campo, nos últimos vinte anos, vários protocolos de eletroporação unicelular in vitro e in vivo foram desenvolvidos para melhorar a especificidade e eficiência5,6,7, demonstrando que a eletroporação pode ser usada para transferir genes para células individuais e pode, portanto, ser extremamente precisa. No entanto, os procedimentos são tecnicamente exigentes, demorados e relativamente ineficientes. De fato, trabalhos mais recentes têm investigado a viabilidade das plataformas de eletroporação mecanizadas8,9, que podem ajudar a eliminar várias dessas barreiras para pesquisadores interessados em instalar tal robótica. Mas para aqueles que procuram meios mais simples, os problemas com eletroporação, ou seja, morte celular, falha de transfecção e entupimento de pipetas, permanecem uma preocupação.
Recentemente desenvolvemos um método de eletroporação que usa pipetas de vidro de ponta maior, parâmetros de pulso elétrico mais suaves e uma etapa de ciclismo de pressão única, que gerou uma eficiência de transfeção muito maior em neurônios excitatórios do que os métodos anteriores, e nos permitiu pela primeira vez transfectar genes em interneurônios inibitórios, incluindo interneurônios inibidores expressivos na região hipocampal ca1 da cultura organotipica do rato10. No entanto, a confiabilidade deste método de eletroporação em diferentes tipos inibitórios e estágios de desenvolvimento neuronal não foi abordada. Aqui, demonstramos que essa técnica de eletroporação é capaz de transfetar genes tanto em neurônios excitatórios quanto em diferentes classes de interneurônios. É importante ressaltar que a eficiência da transfecção foi alta independentemente dos dias in vitro (DIV) da idade da cultura da fatia testada. Esta técnica estabelecida e fácil de usar é altamente recomendada para qualquer investigador interessado em usar eletroporação unicelular para diferentes tipos de células no contexto do tecido cerebral in vitro do rato.

<table>
	<tbody>
		<tr>
			<td>Plasmid preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid Purification Kit</td>
			<td>Qiagen</td>
			<td>12362</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Organotypic slice culture preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>6 Well Plates</td>
			<td>GREINER BIO-ONE</td>
			<td>657160</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5/45 Forceps</td>
			<td>FST</td>
			<td>#5/45</td>
			<td>Angled dissection forceps for organotypic slice culture preparation</td>
		</tr>
		<tr>
			<td>Flask Filter Unit</td>
			<td>Millipore</td>
			<td>SCHVU02RE</td>
			<td>Filtration and storage of culture media</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Binder</td>
			<td>BD C150-UL</td>
			<td></td>
		</tr>
		<tr>
			<td>McIlwain Tissue Chopper</td>
			<td>TED PELLA, INC.</td>
			<td>10180</td>
			<td>Tissue chopper for organotypic slice culture preparation</td>
		</tr>
		<tr>
			<td>Millicell Cell Culture Insert, 30 mm</td>
			<td>Millipore</td>
			<td>PIHP03050</td>
			<td>Organotypic slice culture inserts</td>
		</tr>
		<tr>
			<td>Osmometer</td>
			<td>Precision Systems</td>
			<td>OSMETTE II</td>
			<td></td>
		</tr>
		<tr>
			<td>PTFE coated spatulas</td>
			<td>Cole-Parmer</td>
			<td>SK-06369-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>FST</td>
			<td>14958-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo Microscope</td>
			<td>Olympus</td>
			<td>SZ61</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Vacuum Filtration System</td>
			<td>Millipore</td>
			<td>SCGPT01RE</td>
			<td>Filtration and storage of aCSF</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Electrode preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Capillary Glasses</td>
			<td>Warner Instruments</td>
			<td>640796</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipetter Puller</td>
			<td>Sutter Instrument</td>
			<td>P-1000</td>
			<td>Puller</td>
		</tr>
		<tr>
			<td>Oven</td>
			<td>Binder</td>
			<td>BD (E2)</td>
			<td></td>
		</tr>
		<tr>
			<td>Puller Filament</td>
			<td>Sutter Instrument</td>
			<td>FB330B</td>
			<td>Puller</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Single-cell electroporation and fluorescence imaging #1</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3.5 mm Falcon Petri Dishes</td>
			<td>BD Falcon</td>
			<td>353001</td>
			<td></td>
		</tr>
		<tr>
			<td>Airtable</td>
			<td>TMC</td>
			<td>63-7512E</td>
			<td></td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td>Q Imaging</td>
			<td>Retiga-2000DC</td>
			<td>Camera</td>
		</tr>
		<tr>
			<td>Electroporation System</td>
			<td>Molecular Devices</td>
			<td>Axoporator 800A</td>
			<td>Electroporator</td>
		</tr>
		<tr>
			<td>Fluorescence Illumination System</td>
			<td>Prior</td>
			<td>Lumen 200</td>
			<td></td>
		</tr>
		<tr>
			<td>Manipulator</td>
			<td>Sutter Instrument</td>
			<td>MPC-385</td>
			<td>Manipulator</td>
		</tr>
		<tr>
			<td>Metamorph software</td>
			<td>Molecular Devices</td>
			<td></td>
			<td>Image acquisition</td>
		</tr>
		<tr>
			<td>Peristaltic Pump</td>
			<td>Rainin</td>
			<td>Dynamax, RP-2</td>
			<td>Perfusion pump</td>
		</tr>
		<tr>
			<td>Shifting Table</td>
			<td>Luigs &#38; Neuman</td>
			<td>240 XY</td>
			<td></td>
		</tr>
		<tr>
			<td>Speaker</td>
			<td>Unknown</td>
			<td></td>
			<td>Speakers connected to the electroporator</td>
		</tr>
		<tr>
			<td>Stereo Microscope</td>
			<td>Olympus</td>
			<td>SZ30</td>
			<td></td>
		</tr>
		<tr>
			<td>Table Top Incubator</td>
			<td>Thermo Scientific</td>
			<td>MIDI 40</td>
			<td></td>
		</tr>
		<tr>
			<td>Upright Microscope</td>
			<td>Olympus</td>
			<td>BX61WI</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence imaging #2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>All-in-One Fluorescence Microscope</td>
			<td>Keyence</td>
			<td>BZ-X710</td>
			<td></td>
		</tr>
	</tbody>
</table>

single-cell electroporation across different organotypic slice culture of mouse hippocampal excitatory and class-specific inhibitory neurons

A eletroporação estabeleceu-se como um método crítico para transferir genes específicos para as células para entender sua função. Aqui, descrevemos uma técnica de eletroporação unicelular que maximiza a eficiência (~80%) da transfesão genética in vitro em neurônios inibitórios excitatórios e específicos de classe na cultura de fatia hipocampal organotipada do camundongo. Usando grandes eletrodos de vidro, fluido cerebrospinal artificial contendo tetrodotoxina e pulsos elétricos leves, entregamos um gene de interesse em neurônios piramimatais de CA1 hipocampal e interneurônios inibitórios. Além disso, a eletroporação poderia ser realizada em fatias hipocampais cultivadas até 21 dias in vitro sem redução na eficiência da transfecção, permitindo o estudo de diferentes estágios de desenvolvimento da cultura de fatias. Com o interesse crescente em examinar as funções moleculares dos genes em uma variedade diversificada de tipos celulares, nosso método demonstra uma abordagem confiável e direta para a transfecção genética in vitro no tecido cerebral do camundongo que pode ser realizada com equipamentos e técnicas de eletrofisiologia existentes.

Nossa eletroporação unicelular é capaz de fornecer genes precisamente em neurônios excitatórios e inibitórios visualmente identificados. Eletroporamos três tipos diferentes de células neuronais em três pontos de tempo diferentes. Linhas de glutamato parvalbumin (Pv) ou glutamato vesicular tipo 3 (VGT3) expressando neurônios foram visualizados cruzando linhas decre Pv (JAX #008069) ou VGT3cre (JAX #018147) com linhas repórter TdTomato (uma variante de proteína fluorescente vermelha) (Jax ...

Watch this Scientific Journal Video about Single-Cell Electroporation across Different Organotypic Slice Culture of Mouse Hippocampal Excitatory and Class-Specific Inhibitory Neurons at JoVE.com

Single-Cell Electroporation across Different Organotypic Slice Culture of Mouse Hippocampal Excitatory and Class-Specific Inhibitory Neurons

Apresentado aqui é um protocolo para eletroporação unicelular que pode fornecer genes em neurônios excitatórios e inibitórios em uma gama de idades in vitro de cultura de fatias hipocampais. Nossa abordagem fornece uma expressão precisa e eficiente de genes em células individuais, que podem ser usadas para examinar funções células autônomas e intercelulares.

Eletroporação unicelular em diferentes culturas organotipápicas de neurônios inibitórios hipocampais e inibidores específicos de classe

Electroporation has established itself as a critical method for transferring specific genes into cells to understand their function. Here, we describe a ...

single-cell-electroporation-across-different-organotypic-slice

Research

JoVE Journal

Neuroscience

3.6K visualizações.  University of Massachusetts Medical School. Apresentado aqui é um protocolo para eletroporação unicelular que pode fornecer genes em neurônios excitatórios e inibitórios em uma gama de idades in vitro de cultura de fatias hipocampais. Nossa abordagem fornece uma expressão precisa e eficiente de genes em células individuais, que podem ser usadas para examinar funções células autônomas e intercelulares.

Vídeo: Single-Cell Electroporation across Different Organotypic Slice Culture of Mouse Hippocampal Excitatory and Class-Specific Inhibitory Neurons

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of the hippocampus is relatively preserved 1. The organotypic culture system allows for the examination of neuronal, astrocytic and microglial effects, but as an ex vivo preparation, does not address effects of blood flow, or recruitment of peripheral inflammatory cells. To that end, this culture method is frequently used to examine excitotoxic and hypoxic injury to pyramidal neurons of the hippocampus, but has also been used to examine the inflammatory response. Herein we describe the methods for generating hippocampal slice cultures from postnatal rodent brain, administering toxic stimuli to induce neuronal injury, and assaying and quantifying hippocampal neuronal death.

The organoptypic hippocampal slice culture model is an in vitro model used to examine neuronal injury in a variety of paradigms. In this article, we describe the methods for generating slice cultures and quantifying neuronal injury.

O modelo de Cultura organotípicas Hippocampal Slice para o exame das lesões neuronais

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of ...

Neurociência

Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in which the enhanced version of the green fluorescent protein (EGFP) is exclusively expressed in all neurons of the developing central and peripheral nervous system1, allowing the possibility to both film the innervation of the forelimb and to manipulate this process with pharmacological and genetic techniques2. The most critical parameter in the successful cultivation of such slice cultures is the method by which the slices are prepared. After extensive testing of a variety of methods, we have found that a vibratome is the best possible device to slice the embryos such that they routinely result in a culture that demonstrates viability over a period of several days, and most importantly, develops in an age-specific manner. For mid-gestation embryos, this includes the normal outgrowth of spinal nerves from the spinal cord and the dorsal root ganglia to their targets in the periphery and the proper determination of skeletal and muscle tissue. 
In this work, we present a method for processing whole embryos of embryonic day (E) E10 to E12 into 300 - 400 micrometer slices for cultivation in a standard tissue culture incubator, which can be studied for up to two days after slice preparation. Critical for the success of this approach is the use of a vibratome to slice each agarose-embedded embryo. This is followed by the cultivation of the slices upon Millicell culture membrane inserts placed upon a small volume of medium, resulting in an interface culture technique. One litter with an average of 7 embryos routinely produces at least 14 slices (2-3 slices of the forelimb region per embryo), which varies slightly due to the age of the embryos as well as to the thickness of the slices. About 80% of the cultured slices show nerve outgrowth, which can be measured througout the culturing period2. Representative results using the tauGFP mouse line are demonstrated.

We present a method to prepare organotypic slices of mid-gestation mouse embryos for the cultivation and time-lapse imaging of peripheral nerve outgrowth.

Cultura Slice organotípicas de GFP-expressando embriões de camundongos para imagens em tempo real de crescimento dos nervos periféricos

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in ...

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Nucleofection e Cultura Primária de Neurônios do hipocampo do rato embrionárias e Cortical

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

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 ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

Isolamento e Cultura de neurônios do hipocampo de ratos pré-natal

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

Single-cell Profiling of Developing and Mature Retinal Neurons

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and gene expression programs for extremely rare cell subsets has been a challenge using standard whole-tissue approaches. Gene expression profiling of individual cells allows for unprecedented access to cell types that comprise only a small percentage of the total tissue1-7. In addition, this technique can be used to examine the gene expression programs that are transiently expressed in small numbers of cells during dynamic developmental transitions8.
This issue of cellular diversity arises repeatedly in the central nervous system (CNS) where neuronal connections can occur between quite diverse cells9. The exact number of distinct cell types is not precisely known, but it has been estimated that there may be as many as 1000 different types in the cortex itself10. The function(s) of complex neural circuits may rely on some of the rare neuronal types and the genes they express. By identifying new markers and helping to molecularly classify different neurons, the single-cell approach is particularly useful in the analysis of cell types in the nervous system. It may also help to elucidate mechanisms of neural development by identifying differentially expressed genes and gene pathways during early stages of neuronal progenitor development.
As a simple, easily accessed tissue with considerable neuronal diversity, the vertebrate retina is an excellent model system for studying the processes of cellular development, neuronal differentiation and neuronal diversification. However, as in other parts of the CNS, this cellular diversity can present a problem for determining the genetic pathways that drive retinal progenitors to adopt a specific cell fate, especially given that rod photoreceptors make up the majority of the total retinal cell population11. Here we report a method for the identification of the transcripts expressed in single retinal cells (Figure 1). The single-cell profiling technique allows for the assessment of the amount of heterogeneity present within different cellular populations of the retina2,4,5,12. In addition, this method has revealed a host of new candidate genes that may play role(s) in the cell fate decision-making processes that occur in subsets of retinal progenitor cells8. With some simple adjustments to the protocol, this technique can be utilized for many different tissues and cell types.

A method for the isolation of single retinal cells and subsequent amplification of their cDNAs is described. Single-cell transcriptomics reveals the degree of cellular heterogeneity present in a tissue and uncovers new marker genes for rare cell populations. The accompanying protocol can be adjusted to suit many different cell types.

De uma única célula perfil de imaturos e maduros neurônios da retina

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and ...

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

Cultura primária de neurônios dopaminérgicos mouse

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

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 ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

Emparelhados Recordings células inteiras em organotï¿½icas fatias do hipocampo

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

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 ...