Este trabalho foi financiado pela Universidade de Basel, Departamento de Biomedicina, ea National Science Foundation suíço (31003A_141007). Markus Saxer prestou apoio técnico.

Experimentos em animais foram realizados de acordo com a Directiva do Conselho Comunidades Europeias de 22 de Setembro de 2010 (2010/63 / UE) e foram analisados ​​e autorizados pelas autoridades suíças. 1 Configurando culturas fatia Organotípicas Entorhino-hipocampo <ol><li> Prepare entorhino-hipocampal culturas organotípicas (EHOSCs) de pós-natal filhotes dia 4 (P4) do mouse usando o método de incubação 4,11 estática. Aqui, utilizam ratinhos C57BL6 e CB6F1. <ol><li> Tome cerca de 30 min por rato filhote para a preparação de culturas fatia, ou seja, 3 horas para uma ninhada de seis filhotes de rato. Realizar todas as etapas em condições estéreis em uma bancada de fluxo laminar com instrumentos cirúrgicos esterilizados. </li><li> A fim de evitar uma possível interferência a partir da posição do corte ao longo do eixo do septo-temporal, usar apenas o hipocampo fatias de metade do septo. Se não for indicado de outra forma, realizar todos os passos à temperatura ambiente. </li></ol></li><li> Corte tele cabeça de um filhote de cachorro P4 rato com uma tesoura cirúrgica. Sem anestesia é necessário para este passo. <ol><li> Remover o crânio em volta do cérebro e identificar a fenda entre a extremidade caudal dos hemisférios cerebrais e mesencéfalo. Cuidadosamente remover a parte do cérebro para rostral do mesencéfalo do crânio e colocá-lo no meio de preparação de gelado consistindo em MEM suplementado com uma forma estabilizada de L-glutamina. </li></ol></li><li> Continue a dissecção sob microscópio estereoscópico com o cérebro estar imerso no gelo médio preparação frio. Remover meninges remanescentes dos hemisférios corticais. Identificar o hipocampo na superfície caudal medial do hemisfério e separá-lo em conjunto com o córtex entorrinal adjacente do resto do cérebro. <ol><li> Transferir o tecido de um cortador de tecido e cortado 400 um de espessura, sob condições assépticas, transversal ao eixo septo-temporal do hipocampo. </li></ol></li><li> Coloque as fatias sobre ceinserções de cultura II (aproximadamente 6 fatias por pastilha) e incubar em placas de 6 poços com 1 ml por poço de meio de incubação (47 ml de MEM, 25 ml de Meio Eagle, 25 ml de soro de cavalo, 1 ml I glutamax, 1 ml de uma solução estéril de glicose a 10%, pH 7,3) numa atmosfera húmida com 5% de CO 2, a 37 ° C. Troca da mídia no dia seguinte e, em seguida, a cada dois dias até uma semana. </li></ol> 2 In Vitro hipóxia e oxigênio-glicose Privação com reperfusão <ol><li> Ver Tabela 1 para um horário exato momento do procedimento. EHOSCs de cultura para 5 dias (DIV5) antes da exposição a hipoxia ou POG. Neste ponto de tempo mudar a forma de meio de incubação contendo 25% de soro de cavalo para meio isento de soro, com a seguinte composição: meio Neurobasal com suplemento B27 a 2%, 1% de glucose e glutamax adicional de 0,1%. </li><li> Preparar o meio OGD como detalhado em 7 de referência .As concentrações finais dos componentes é a seguinte: 0,3mM de CaCl2, 70 mM de NaCl, 5,25 mM de NaHCO3, 70 mM de KCl, 1,25 mM NaH 2 PO 4, 2 mM de MgSO4 e 10 mM de sacarose a pH 6.8. Adicionar 1 ml de meio a cada poço OGD de duas placas de cultura de tecidos de 6 poços. <ol><li> Perfundir a forma OGD com N 2 durante 1 hora, em uma câmara de hipoxia e, em seguida, vedar e manter S / N em uma incubadora a 37 ° C. </li><li> Use tiras de anaeróbios como indicadores de hipóxia. Considere o meio estar livre de oxigénio quando a cor das tiras mudou de rosa para branco. </li><li> Para a indução da hipóxia, utilizar meio isento de soro com glucose, em vez de meio de OGD. Antes da utilização, o meio de perfundir com N 2, em uma câmara de hipoxia como descrito acima para o meio de OGD. </li></ol></li><li> Antes de as fatias são expostas a OGD, perfundir a forma OGD novamente com N 2 durante 30 min. Adicionar 2 mL de uma solução 1 mg / ml de iodeto de propídio (PI) de solução por cavidade (para uma concentração final de 2 ug / ml) para as fatias, durante 30 min e seleccionarapenas fatias saudáveis ​​para experimentação, conforme indicado por um reduzido número de células positivas para PI. </li><li> Utilizar o modelo de exposição OGD e reperfusão indicado na Figura 1. Privar fatias de oxigénio ou apenas (hipoxia) ou de oxigénio e glicose (POG), durante 15 min. <ol><li> Para a indução de hipoxia ou OGD, transferir as fatias em forma OGD e manter por 15 minutos em câmara de hipóxia. Certifique-se de que todos os fluidos que permanecem nos lados da peça inserta são aspirados para longe quando as culturas são comutados de forma regular para OGD médio e para trás. </li><li> Após a hipoxia ou OGD, substituir o meio de OGD com meio isento de soro e oxigenado culturas são deixados a recuperar durante 3, 24 ou 48 horas em meio isento de soro na incubadora normal a 37 ° C com 5% de CO 2. Adicionar PI novamente antes da fixação para a determinação da morte celular. </li></ol></li></ol> 3. tratamentos farmacológicos de Entorhino-hipocampal culturas Organotípicas Fatia <ol><li> Culture EHOSCs durante 5 dias (DIV5) em meio de incubação antes de exposição a tratamentos farmacológicos. Iniciar os tratamentos farmacológicos ou durante ou imediatamente após a OGD OGD quando fatias são transferidas para meio isento de soro. </li><li> Tratamentos para proteção contra POG. <ol><li> Adicionar 100 | iM 6-ciano-7-nitroquinoxalina-2.3-diona (CNQX) ou 1 uM a tetrodotoxina (TTX) ao meio de cultura durante a indução OGD (durante 15 min) ou imediatamente após a indução OGD durante 30 min. Em seguida, incuba-se meio isento de soro durante 48 horas normóxica antes da fixação. </li></ol></li><li> Os tratamentos para a indução da morte excitotóxica. <ol><li> Após 5 dias de incubação em culturas de meio interruptor para meio isento de soro contendo 100 uM (RS) -α-amino-3-hidroxi-5-metil-4-isoxazolpropiónico ácido (AMPA), durante 30 min. </li><li> Retire o meio de cultura contendo AMPA por aspiração (ter o cuidado de remover qualquer meio residual em inserções), em seguida, transferir as fatias para placas novas com acesso médio e soro normalmantê-los por 48 horas, até a fixação. </li></ol></li></ol> 4. Fixação e Imunocoloração de Entorhino-hipocampal culturas Organotípicas Fatia <ol><li> O iodeto de propídio (PI) a coloração. <ol><li> Adicionar PI durante 30 min para o meio de cultura de fatias de vida, a uma concentração de 2 ug / ml. Ver culturas vivas com um microscópio invertido equipado com óptica de fluorescência e selecione fatias para mais tratamentos. Para a coloração de PI em combinação com imuno-histoquímica, adicionar solução de PI 30 min antes da fixação das culturas. </li></ol></li><li> A fixação das fatias. <ol><li> Remover o meio de cultura da placa de 6 poços e adicionar 3 ml de preparado de fresco a 4% de paraformaldeído (PFA) a cada poço. Em seguida, colocar a placa de 6 cavidades em uma geladeira e fixar O / N a 4 ° C. </li><li> Remover a solução de PFA no dia seguinte e as fatias de lavar 3-4 vezes com tampão fosfato salino (PBS). </li></ol></li><li> A remoção das fatias a partir da cultura de células insert e procedimento de bloqueio para imuno-histoquímica de secções de livre flutuação. <ol><li> Retirar cuidadosamente e retire as fatias das inserções de cultura utilizando uma arte pincel do tamanho 0 ou menor. Transferir cada faixa individual num poço de uma placa de 96 poços preenchidos com tampão de bloqueio (PBS com 3% de soro de cabra normal (NGS) e 2% de Triton-X100). Manter as fatias em tampão de bloqueio durante 2 horas à temperatura ambiente, sob agitação constante por um agitador orbital. </li></ol></li><li> A imunocoloração das culturas de fatias <ol><li> Para a marcação dos vasos sanguíneos usar policlonal anti-laminina a uma diluição de 1: 200 em PBS contendo 1% NGS e 0,5% de Triton-X100. </li><li> Para os neurónios rotulagem utilizar anti-microtúbulo associado proteína 2 (MAP2), a uma diluição de 1: 500 em PBS contendo 1% NGS e 0,5% de Triton-X100. </li><li> Incubar as fatias com anticorpos primários durante 48 horas a 4 ° C com agitação constante. Após o período de incubação, lavá-las em PBS com 0,5% Triton-X100 em 3-4 vezes. </li&gt; <li> Dilui-se anticorpos secundários 1: 500 em PBS contendo 1% NGS e 0,5% de Triton-X100. Incubar as fatias durante 2 horas à temperatura ambiente no escuro, com os anticorpos secundários conjugados com Alexa fluoróforo. Use de cabra anti-coelho adequado ou anticorpo secundário de cabra anti-ratinho conjugado com Alexa 350 ou 488. </li><li> Remover a solução de anticorpo secundário e lava-se as fatias de três vezes com solução salina tamponada com Tris (TBS); montá-los em lâminas de vidro e lamela as lâminas com Mowiol. </li></ol></li><li> Microscopia das fatias coradas </li><li> Ver as fatias manchadas ou em um microscópio padrão com equipamentos de epifluorescência ou com um microscópio confocal. Tire fotos com uma câmera apropriada. Para algumas imagens, inverter o contraste de fluorescência, a fim de produzir vasos com uma coloração escura sobre um fundo claro (Figuras 5 e 6). </li></ol> 5 Determinação da densidade de vasos Regional nas culturas <ol><li> Quantificar o vessel com base na densidade de vasos passagens como descrito na Referência 8. </li><li> Use fatias manchadas para laminina para essas medições. Sobreponha as imagens gravadas de aumento de 10x com uma grade 6 x 6, onde cada grade é igual a 100 x 100 mm por metro quadrado, área total de 0,25 milímetros 2. </li><li> Sobreposição três dessas redes na região CA1, CA3, DG, e CE (Figura 2). Conte as embarcações que cruzam as linhas do grid. </li><li> Para a obtenção de resultados com uma boa relevância estatística, fazer medições em pelo menos três experiências independentes, cada uma das quais os dados a partir de 3 filhotes de rato com três fatias por ratinho filhote. </li><li> Determine o valor médio das passagens de navio da grades para cada região e realizar uma análise estatística por ANOVA com correção de Bonferroni como post hoc teste (p &lt;0,05 considerada significativa), utilizando software estatístico adequado. </li></ol>

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Experimentos em animais foram realizados de acordo com a Directiva do Conselho Comunidades Europeias de 24 de Novembro de 1986 (86/609 / CEE) e foram analisados ​​e autorizados pelas autoridades suíças. Os autores não têm nada a revelar.

Com os métodos aqui apresentados, hipocampo culturas organotípicas de fatias pode ser utilizado como uma ferramenta versátil para estudar alterações plásticas no tecido neural após a lesão. Enquanto as culturas organotípicas de fatias tenha sido utilizado no passado para o estudo de reacções neuronais após isquemia 6, 7, o novo aspecto do estudo simultâneo de alterações vasculares aumenta significativamente as aplicações potenciais do presente método. A indução de hipoxia ou OGD por si só...

O sistema nervoso central é particularmente sensível a uma perda ou redução do fornecimento de oxigénio e glicose pela vasculatura. Mesmo um tanto curta interrupção do fornecimento de sangue para o cérebro pode induzir uma perda permanente da função das áreas cerebrais relevantes que levam às síndromes típicas tempos. Além da perda de neurônios nas áreas afetadas primários, há normalmente uma perda neuronal atraso adicional através de danos secundários. Infelizmente, até agora, nenhum tratamento neuroprotector para a redução da morte neuronal secundária estava disponível um. Os esforços de pesquisa para estudar os mecanismos de lesão secundária se baseiam na utilização de modelos animais de isquemia cerebral como a oclusão da artéria cerebral média e de várias técnicas de oclusão trombótica (para uma revisão recente ver 2). Em paralelo, também devido a limitações e preocupações éticas com o uso de modelos animais, cultura fatia organotípica de vários tecidos do sistema nervoso central têm sido utilizados que permitam o study de reações neuronais para vários tipos de lesões 3-5. Para estudar a reacção neuronais sob condições que imitam a lesão cerebral isquémica, o sistema modelo de privação de oxigénio glicose (OGD) foi desenvolvido. Neste modelo, as culturas de fatia são temporariamente expostos a um meio que carece de glicose, tendo sido equilibrada com o gás de azoto na ausência de oxigénio. Com um tal tratamento, é possível induzir a lesão neuronal e perda, que é bastante semelhante ao observado após lesão isquémica in vivo de 6, 7. No hipocampo, um tal tratamento induz a perda neuronal em CA1 especificamente, mas não no área CA3 ou do giro dentado do hipocampo. Em contraste, o estudo de reacções vasculares em culturas de fatias até agora não tem sido amplamente realizadas. Uma razão óbvia é a falta de circulação e de vasos sanguíneos de perfusão no modelo de cultura de fatia. Contudo, mostrámos previamente que é possível manter bvasos Lood em CNS fatia culturas por vários dias 8, 9. O objetivo geral deste método é monitorar não só o destino dos neurônios após POG, mas estender o estudo para o destino e remodelação da vasculatura que é uma parte importante da resposta a uma lesão. Até agora, os estudos têm exigido a utilização de experiências com animais (De Jong et al, 1999;. Cavaglia et al, 2001.). No protocolo apresentado aqui, vamos detalhar como tais estudos podem ser feitos em culturas fatia organotypic entorhino-hipocampal desafiou tanto a hipóxia ou por lesões excitotóxicos seguida da análise de ambos os sobrevivência e vasos sanguíneos reações neuronais. Este protocolo é baseado em um estudo publicado anteriormente sobre o assunto 10 e pode ser útil para qualquer laboratório interessado em interações neurovasculares no SNC.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Minimum Essential Medium MEM</td>
			<td style="width:71px;">Gibco</td>
			<td>11012-044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glutamax</td>
			<td style="width:71px;">Gibco</td>
			<td>35050-061</td>
			<td>stabilized form of L-glutamine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Millicell cell culture inserts</td>
			<td style="width:71px;">Millipore</td>
			<td style="width:119px;">PICM03050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Basal medium Eagle&#160;</td>
			<td style="width:71px;">Gibco</td>
			<td>41010-026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Horse serum</td>
			<td style="width:71px;">Gibco</td>
			<td>26050-088</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Neurobasal medium</td>
			<td style="width:71px;">Gibco</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">B27 supplement</td>
			<td style="width:71px;">Gibco</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Anaerobic strips</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td align="right">59886</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Propidium iodide solution</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td>P4864</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">AMPA</td>
			<td style="width:71px;">R&#38;D systems</td>
			<td>0169-10</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CNQX</td>
			<td style="width:71px;">R&#38;D systems</td>
			<td style="width:119px;">0190/10</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TTX</td>
			<td style="width:71px;">R&#38;D systems</td>
			<td style="width:119px;">1078/1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">polyclonal anti-laminin</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td>L9393&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti-MAP2</td>
			<td style="width:71px;">Abcam</td>
			<td style="width:119px;">ab11267</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Alexa anti mouse 350</td>
			<td style="width:71px;">Molecular Probes</td>
			<td>A11045</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Alexa anti mouse 488</td>
			<td style="width:71px;">Molecular Probes</td>
			<td>A11001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Alexa anti rabbit 350</td>
			<td style="width:71px;">Molecular Probes</td>
			<td>A11046</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Alexa anti rabbit 488</td>
			<td style="width:71px;">Molecular Probes</td>
			<td>A11008</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Statistics software</td>
			<td style="width:71px;">GraphPad Software</td>
			<td style="width:119px;">GraphPad Prism</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">McIlwain tissue chopper</td>
			<td style="width:71px;">Ted Pella</td>
			<td align="right" style="width:119px;">10180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hypoxia chamber</td>
			<td>Billups-Rothenberg</td>
			<td style="width:119px;">MIC-101</td>
			<td></td>
		</tr>
	</tbody>
</table>

the analysis of neurovascular remodeling in entorhino-hippocampal organotypic slice cultures

Lesão cerebral isquêmica é uma das condições mais comuns e devastadoras que comprometem a função cerebral adequada e muitas vezes leva a persistir déficits funcionais nos pacientes afetados. Apesar dos esforços de pesquisa intensiva, ainda não existe uma opção de tratamento eficaz disponível, que reduz a lesão neuronal e protege os neurônios nas áreas isquêmicas da morte secundário atrasado. A investigação nesta área, tipicamente envolve a utilização de modelos animais e elaborados problemáticos. Culturas fatia organotípica Entorhino-hipocampal desafiados com privação de oxigênio e glicose (POG) são estabelecidos em modelos in vitro que mimetizam isquemia cerebral. O aspecto inovador deste estudo é que as alterações dos vasos sanguíneos do cérebro são estudadas, além de alterações neuronais ea reação tanto do compartimento neuronal e compartimento vascular podem ser comparados e correlacionados. Os métodos apresentados neste protocolo ampliar substancialmente as aplicações potenciais da ouabordagem da cultura fatia ganotypic. A indução de hipoxia ou OGD por si só pode ser aplicado por meios bastante simples em culturas organotípicas de fatias e leva a danos de confiança e reprodutível no tecido neural. Isto está em contraste com os experimentos em animais complexos e problemáticos induzindo acidente vascular cerebral e isquemia in vivo. Ao ampliar a análise para incluir o estudo da reação da vasculatura poderia fornecer novas maneiras de como preservar e restaurar as funções cerebrais. A abordagem da cultura fatia apresentado aqui pode se transformar em uma ferramenta atrativa e importante para o estudo da lesão isquêmica cerebral e pode ser útil para testar medidas terapêuticas potenciais com vistas a neuroproteção.

Oxygen privação de glicose e hipóxia induzir a morte neuronal e redução dos vasos sanguíneos, especificamente na região CA1 do hipocampo. OGD ou privação de oxigénio e durante 15 min, sozinho induziu uma forte indução de morte celular como observado por coloração com iodeto de propídio, especificamente na área CA1 do hipocampo (Figura 3), semelhante ao descrito anteriormente 7. Com marcadores para visualizar a rede de vasos sanguíneos, verificou-se que a densi...

Watch this Scientific Journal Video about The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures at JoVE.com

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

Um protocolo para culturas fatia organotípica entorhino-hipocampal, que permite a reprodução de muitos aspectos da lesão cerebral isquêmica, é apresentado. Ao estudar as mudanças do neurovasculature além de alterações nos neurônios, este protocolo é uma ferramenta versátil para estudar mudanças plásticas no tecido neural após lesão.

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 ...

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JoVE Journal

Neuroscience

9.7K visualizações.  University of Basel.  Um protocolo para culturas fatia organotípica entorhino-hipocampal, que permite a reprodução de muitos aspectos da lesão cerebral isquêmica, é apresentado. Ao estudar as mudanças do neurovasculature além de alterações nos neurônios, este protocolo é uma ferramenta versátil para estudar mudanças plásticas no tecido neural após lesão. 

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

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

Slice Preparation, Organotypic Tissue Culturing and Luciferase Recording of Clock Gene Activity in the Suprachiasmatic Nucleus

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the anterior hypothalamus. The clock is directly synchronized by light via the retina and optic nerve. Circadian oscillations are generated by interacting negative feedback loops of a number of so called "clock genes" and their protein products, including the Period (Per) genes. The core clock is also dependent on membrane depolarization, calcium and cAMP 1. The SCN shows daily oscillations in clock gene expression, metabolic activity and spontaneous electrical activity. Remarkably, this endogenous cyclic activity persists in adult tissue slices of the SCN 2-4. In this way, the biological clock can easily be studied in vitro, allowing molecular, electrophysiological and metabolic investigations of the pacemaker function.
The SCN is a small, well-defined bilateral structure located right above the optic chiasm 5. In the rat it contains ~8.000 neurons in each nucleus and has dimensions of approximately 947 &mu;m (length, rostrocaudal axis) x 424 &mu;m (width) x 390 &mu;m (height) 6. To dissect out the SCN it is necessary to cut a brain slice at the specific level of the brain where the SCN can be identified. Here, we describe the dissecting and slicing procedure of the SCN, which is similar for mouse and rat brains. Further, we show how to culture the dissected tissue organotypically on a membrane 7, a technique developed for SCN tissue culture by Yamazaki et al. 8. Finally, we demonstrate how transgenic tissue can be used for measuring expression of clock genes/proteins using dynamic luciferase reporter technology, a method that originally was used for circadian measurements by Geusz et al. 9. We here use SCN tissues from the transgenic knock-in PERIOD2::LUCIFERASE mice produced by Yoo et al. 10. The mice contain a fusion protein of PERIOD (PER) 2 and the firefly enzyme LUCIFERASE. When PER2 is translated in the presence of the substrate for luciferase, i.e. luciferin, the PER2 expression can be monitored as bioluminescence when luciferase catalyzes the oxidation of luciferin. The number of emitted photons positively correlates to the amount of produced PER2 protein, and the bioluminescence rhythms match the PER2 protein rhythm in vivo 10. In this way the cyclic variation in PER2 expression can be continuously monitored real time during many days. The protocol we follow for tissue culturing and real-time bioluminescence recording has been thoroughly described by Yamazaki and Takahashi 11.

The procedure of preparing slices containing the adult mouse hypothalamic suprachiasmatic nucleus (SCN), and a rapid way to culture the SCN tissue in organotypic culture condition, are reported. Further, the measurement of oscillatory clock gene protein expression using dynamic luciferase reporter technology is described.

Preparação fatia, cultura de tecidos organotípicas e Gravação luciferase de Atividade Gene Relógio no núcleo supraquiasmático

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the ...

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 ...

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

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. Since then, the technique was developed further and currently there are many different ways to prepare organotypic cultures. The method presented here was adapted from the one described by Stoppini et al. for the preparation of the slices and from Gogolla et al. for the staining procedure 3,4.
A unique feature of this technique is that it allows you to study different parts of the brain such as hippocampus or cerebellum in their original structure, providing a big advantage over dissociated cultures in which all the cellular organization and neuronal networks are disrupted. In the case of the cerebellum it is even more advantageous because it allows the study of Purkinje cells, extremely difficult to obtain as dissociated primary culture. This method can be used to study certain developmental features of the cerebellum in vitro, as well as for electrophysiological and pharmacological experiments in both wild type and mutant mice.
The method described here was designed to study the effect of apoptotic stimuli such as Fas ligand in the developing cerebellum, using TUNEL staining to measure apoptotic cell death. If TUNEL staining is combined with cell type specific markers, such as Calbindin for Purkinje cells, it is possible to evaluate cell death in a cell population specific manner. The Calbindin staining also serves the purpose of evaluating the quality of the cerebellar cultures.

This method describes the generation of organotypic cerebellar cultures and the effect of certain apoptotic stimuli on the viability of different cerebellar cell types.

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. ...

Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex. Because of the lack of specific pharmacological agents for these channels, we have taken a genetic approach to manipulating channel expression. We use an organotypic culture preparation (16) in order to maintain cell morphology and the laminar pattern of cortex. We typically isolate acute neocortical slices at postnatal days 8-10 and maintain the slices in culture for 3-7 days. This allows us to study neurons at a similar age to those in our work with acute slices and minimizes the development of exuberant excitatory connections in the slice. We record from visually-identified pyramidal neurons in layers II/III or V using infrared illumination (IR-) and differential interference contrast microscopy (DIC) with whole cell patch clamp in current- or voltage-clamp. We use biolistic (Gene gun) transfection of wild type or mutant potassium channel DNA to manipulate expression of the channels to study their function. The transfected cells are easily identified by epifluorescence microscopy after co-transfection with cDNA for green fluorescent protein (GFP). We compare recordings of transfected cells to adjacent, untransfected neurons in the same layer from the same slice.

This is a protocol to prepare and maintain a neocortical slice preparation in organotypic culture for the purpose of making electrical recordings from pyramidal neurons.

Gravação de células inteiras a partir de uma preparação Slice organotípicas de Neocortex

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex.  Because of the ...

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 ...

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. ...