This work was supported by a research grant MOP 119271 to JMW by the Canadian Institute of Health Research. The authors would like to thank Ms. Yao Fang Tan for her outstanding technical assistance.

NOTA: Todos os procedimentos com animais conformados com as orientações de saúde e bem-estar animal do Departamento de Medicina Comparativa da Universidade de Toronto. 1. Preparação de fatias do hipocampo <ol><li> Esterilizar os seguintes instrumentos usando o autoclave seco a 125 ° C: cabo de bisturi (# 3) (2), fórceps padrão standard, grande (1), Small dissector tesoura (dobrado para o outro) (1), Micro colher (colher e plana extremidades espátula) (1), Micro-espátulas (arredondados e extremidades arredondadas cônicos) (2), pincel fino (1), Fogo polido Pasteur pipeta (2), praças de gaze, 2 x 2 polegadas (5). </li><li> Quando estéril, colocar os instrumentos em um recipiente estéril e manter coberto até o uso. Imediatamente antes da dissecação, mergulhe os instrumentos em uma solução de etanol a 70%. </li><li> Prepara-se uma placa de cultura de 6 poços com inserção de cultura antes de iniciar o procedimento de dissecção por adição de 1 ml de meio de cultura / poço e armazenando a placa na incubadora a 35 ° C, umand 5% de CO 2. </li></ol> 2. Organizar instrumentos de dissecação em estéril de fluxo laminar <ol><li> Pulveriza-se a câmara de fluxo laminar com etanol a 70% e remover instrumentos esterilizados dissecção a partir de álcool. Permitir que os instrumentos para secar enquanto descansa em uma placa de Petri esterilizada para evitar o contato entre o álcool e o tecido do cérebro dissecado. </li><li> Depósito 5-7 ml de solução de dissecação gelada esterilizados em 2 grandes placas de Petri estéreis. Um prato vai relaxar e limpar a cabeça (sujo), o outro para o resfriamento e enxaguar a esvaziado cérebro (limpo). Coloque um papel de filtro esterilizado numa das tampas de placas de Petri para dissecar o cérebro. </li><li> Colocar uma pequena quantidade de papel de filtro, esterilizado em uma pequena tampa da caixa de Petri para dissecar o hipocampo. Depósito 3-5 ml de solução esterilizada dissecação gelada em 2 pequenos, placas de Petri estéreis. Um prato vai realizar o hipocampo escavado para fora, ninguém vai segurar seções durante a separação das secções do hipocampodissecção sob microscópio. </li><li> Prepare o cortador de tecidos gravando um pedaço de papel de filtro esterilizado para a fase de corte e a montagem de uma lâmina de barbear estéril. Molhar o papel de filtro com solução de dissecação esterilizado. </li><li> Spray de um bio-saco limpo com álcool e coloque-o em fluxo laminar bancada limpa. </li></ol> 3. Hippocampal Dissection <ol><li> Pulverizar o filhote de rato P7 Sprague Dawley com etanol 70% fora do fluxo laminar bancada limpa e rapidamente decapitar o animal usando grandes tesouras cirúrgicas estéreis dentro do banco de fluxo laminar. Deixe a cabeça cair na solução de dissecação gelada em uma das placas de Petri. </li><li> Na placa de Petri, enxaguar o sangue e rapidamente transferir a cabeça para papel de filtro esterilizado, lado ventral para baixo. </li><li> Usando o bisturi, cortar ao longo da superfície dorsal no plano sagital para expor o crânio subjacente. Cortar através da pele, mas não o osso subjacente, que é macio e facilmente penetrável em ratos de tsua idade. Separe este bisturi &quot;sujo&quot; e não usar em tecido cerebral. </li><li> Usando as pequenas tesouras de dissecção (anguladas para o outro) e uma pinça, cortado o crânio ao longo da sutura sagital do crânio para bregma, o ponto na anatomia do crânio onde a sutura coronal é entrecortada perpendicularmente pela sutura sagital. Use uma pinça para puxar abas crânio para cima e longe da linha média do crânio. </li><li> Coloque a colher micro no lado inferior do cérebro, por baixo do tronco cerebral, para levantar suavemente para fora do cérebro do crânio. Levante o cérebro para expor os nervos ópticos e bulbo olfatório na superfície basal do cérebro. Corte essas estruturas com uma tesoura pequena para separar totalmente o cérebro de crânio. Remover e transferir o cérebro intacto para o outro prato de Petri contendo solução grande dissecação gelada. </li><li> Usando o micro colher, transferir o cérebro a uma tampa pequena placa de Petri contendo papel de filtro estéril. Com uma pipeta de Pasteur estéril, lavar o cérebro com algumas gotas de dissecting solução para manter o tecido úmido. </li><li> Usando uma lâmina de bisturi &quot;clean&quot; cortar os dois hemisférios separados. Transfira o hemisfério esquerdo volta a grande placa de Petri com micro colher e coloque o lado pia hemisfério baixo na solução de dissecação gelada para posterior utilização. </li><li> Ver a face medial do hemisfério direito e identificar o fornix borda, uma banda de destaque da matéria branca ao longo da borda medial do hipocampo. Usando um bisturi estéril, faça um corte sagital através do fundo de saco, mas tome cuidado, pois apenas 0,5 cm da ponta do bisturi vai ser suficiente para cortar o fundo de saco. </li><li> Usando 2 micro-espátulas, remova o primeiro hipocampo do hemisfério direito, colocando a mão-espátula direita no tronco cerebral e levantando o córtex sobreposta com a espátula esquerdo. Suavemente levantar o córtex para revelar o ventrículo lateral e medial da superfície do hipocampo. A linha curva branca, a fímbria, agora deve estar visível. </li><li> Alinhe a curvatura da espátula com a curvatura da fímbria e pressione suavemente a espátula sob a fímbria. Deslize a espátula esquerda e passeio ao longo do eixo rostral-caudal e levante espátula na direção dorsal para remover hipocampo. </li><li> Transferir o hipocampo de uma 2ª pequena placa de Petri com solução de dissecção gelado. Repita o mesmo procedimento no hemisfério esquerdo para remover o hipocampo esquerdo. </li><li> Usando uma espátula micro, transferir cuidadosamente o hipocampo para o palco cortador de tecidos. Dispor-los adjacentes e paralelos um ao outro e perpendiculares ao eixo da lâmina de helicóptero. Utilização de um pincel para posicionar o tecido e adição de algumas gotas de solução de dissecção no topo do hipocampos. </li><li> Corte o tecido em 400 mm fatias sem parar para remover fatias individuais (geralmente eles não vão aderir à lâmina). Depois de todo o hipocampo foi cortado, use o pincel para transferir delicadamente as seções para a 2ª pequena placa de Petri com solução de dissecção. </li><li> De acordo com anúncioissecting microscópio, separe cuidadosamente as fatias flutuante usando amicro-espátula e pincel. </li><li> Remova a placa de cultura de pré-preparados com inserção de cultura do incubador e lugar numa câmara de fluxo laminar. </li><li> Usando uma pipeta de Pasteur polida ao fogo, desenhar 4-5 fatias para a pipeta de transferência e fatias à superfície apical de membrana inserção de cultura. Em seguida, ajuste o posicionamento com um pincel e deixe espaço entre as seções individuais e da fronteira da inserção da cultura. </li><li> Usando uma pipeta de Pasteur estéril, remover o excesso de solução de dissecação da superfície apical de membrana. NOTA: Ao remover solução evitar desenhar cortes de tecido para a pipeta. Como alternativa, use uma pipeta regular (P200 ou P1000) com pontas de pipetas esterilizadas para remover lentamente solução. </li><li> Colocar a placa de cultura com meio de cultura contendo soro e as fatias de hipocampo de volta na incubadora a 35 ° C e 5% de CO 2. NOTA: Se as chamadas experiênciapara a geração de culturas de vários animais, limpar completamente o fluxo laminar banco limpo entre dissecções. Retorno instrumentos para solução de álcool e substituir todas as placas de Petri, papéis de filtro e lâminas de barbear estéril, antes da segunda dissecção. </li></ol> 4. alimentação e manutenção de Organotípicas Slices <ol><li> Culturas para alimentação animal em um fluxo laminar estéril bancada limpa. </li><li> Realize a primeira alimentação das seções de cultura dois dias pós-dissecção. Aspirar meio de cultura de idade que usa a pipeta de vidro esterilizado. </li><li> Use estéreis 5 ml pipeta serológica para adicionar 1 ml de, meio contendo soro fresco, estéril aos poços. </li><li> Gentilmente substituir a inserção da cultura e tomar cuidado para remover as bolhas de ar que podem ter se formado debaixo da superfície da membrana. </li><li> Após a primeira alimentação, mudar meio todos os outros dias. </li></ol> 5. Incubar fatias de tecido com timidina Analogues para Label Newborn Neurônios <ol><li> A fim de estudara maturação e integração de células granulares do denteado do hipocampo, incubar as fatias organotípicas com um análogo da timidina, tal como bromodesoxiuridina (BrdU) ou Chlorodeoxyuridine (CldU). Aqui, nós utilizamos CldU devido à sua maior solubilidade em solução salina. </li><li> Após 3DIV, adicionar 1 ml de / ml de solução concentrada CldU 10 mg em solução salina a 1 ml de meio de cultura para uma concentração final de 10 ug / ml. Se BrdU é usado, corrigir a concentração para as diferenças de peso molecular. Adicionar meio com CldU para poços de cultura e incuba-se o tecido com CldU contendo meio durante 2 horas a 35 ° C. </li><li> Após 2 h de incubação a 35 ° C, remover o meio contendo CldU e substitua com meio de alimentação regular para retomar a programação de alimentação normal (descrito acima). </li></ol> 6. Os tecidos Fixation e Armazenamento <ol><li> Estabeleça um cronograma para a aplicação de tratamentos e fixação de amostras de tecido antes de iniciar experimentos de cultura (Figura 2B). </li> &lt;li&gt; A um dia predeterminado pós-dissecação, preparar o seguinte numa hotte de laboratório: uma proveta de 10-50 ml contendo 4% de paraformaldeído (PFA) em tampão fosfato salino (PBS); um copo vazio para o meio de cultura descartados; pequeno fórceps; e uma pipeta de 1000 ml com pontas descartáveis. </li><li> Remova a placa de cultura (s) a partir de câmara de incubação e transferência para uma coifa. Inclinação das inserções individuais de placas bem com fórceps. Em seguida, utilizar uma pipeta para remover o meio de cultura e transferência para uma proveta de eliminação. Inclinar a placa de cultura a um ângulo para ajudar a garantir que todo o meio de cultura é removido. </li><li> Concluir a remoção por meio de uma placa de cultura de cada vez. Ao meio foi removido, adicionar 1 ml de PFA 4% para cada poço de cultura e sele bem placa com parafilm. Repita o procedimento para o maior número de placas de cultura como placas necessárias e transferir para geladeira a 4 ° C, durante 24 horas. </li><li> Após 24 horas, prepare o seguinte em um exaustor: 10-50 taça ml contendo 0,1% de azida de sódio em PBS; um vazioTaça por PFA descartado (siga as precauções de segurança ao descartar substâncias tóxicas); pequeno fórceps; e 1.000 ml pipeta com pontas descartáveis. </li><li> Siga o procedimento descrito no ponto 6.4 para a adição de PFA, mas adicionar 1 ml de PBS com azida de sódio em vez disso. Uma vez terminada a transmissão, placas de poços de vedação para uso futuro por envolvimento bordas em parafilm e armazenar na geladeira a 4 ° C. </li></ol> 7. O corte do tecido por imuno-histoquímica <ol><li> Realizar tecido corte usando um vibratome. A seguinte série de passos ajudar a maximizar o rendimento de secções de tecido utilizáveis ​​a partir de culturas organotípicas. NOTA: Imediatamente após dissecação do hipocampo e plaqueamento das fatias, o tecido tem uma espessura de aproximadamente 400 um. No entanto, depois de 2-3 semanas na câmara de incubação, fatias de tecido começará a achatar, resultando numa espessura de secção final de 150-300 | im. </li><li> Prepare os seguintes itens para a seção de tecido cultivado: a # 11 bisturi blade e manusear, um prato de vidro Petri contendo PBS gelado, um micro-dissecação fórceps, e uma fase de corte vibratome limpa para montar tecido. </li><li> Usar um bisturi para cuidadosamente cortada ao longo do perímetro da membrana inserção circular de modo que ele pode ser separado da carcaça inserção de plástico. Deixar um amplo espaço entre a fatia culta e o bisturi. </li><li> Transferir a membrana inserção individual para uma placa de Petri contendo PBS. Após lavagem em PBS, utilize uma pinça para transferir a membrana para a fase de montagem vibratome. </li><li> Em seguida, usar o bisturi para eliminar o excesso de membrana que envolve fatias cultivadas e cortar o excesso de material para criar bordas limpas. Este passo irá garantir a membrana é plana e pode aderir facilmente à superfície de corte. </li><li> Colocar 1-2 gotas de adesivo sobre a fase de corte vibratome e espalhar em camada uniforme utilizando uma agulha 22 G. Espalhe adesiva em uma forma retangular com a borda longa paralela à lâmina de corte do vibratome. Execute esta etaparapidamente, para evitar que o adesivo de secagem. </li><li> Use uma pinça para transferir a membrana aparadas contendo fatias do hipocampo para o estágio de corte e gentilmente posicionar a membrana na cola e garantir que não haja bolhas de ar. </li><li> Como o superglue seca, transferir o palco vibratome com a membrana colada de volta para a PBS contendo placa de Petri. Prepare a lâmina vibratome e uma azida de sódio contendo 48 poços para armazenar seções do tecido. </li><li> Use vibratome para gerar 30 mm secções do tecido fatia organotípicas e transferência para uma placa de 48 poços contendo azida de sódio para armazenamento e subsequente coloração imuno-histoquímica. </li><li> Consulte os protocolos de imuno-histoquímica 26,29 coloração pré-existentes. </li></ol>

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G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoskeletal,+synaptic,+and+nuclear+protein+changes+associated+with+rat+interface+organotypic+hippocampal+slice+culture+development.">Cytoskeletal, synaptic, and nuclear protein changes associated with rat interface organotypic hippocampal slice culture development.</a> Developmental Brain Research. 160 (2), 275-286 (2005).</li><li>Fabian-Fine, R., Volknandt, W., Fine, A., Stewart, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-dependent+pre-+and+postsynaptic+distribution+of+AMPA+receptors+at+synapses+in+CA3+stratum+radiatum+of+hippocampal+slice+cultures+compared+with+intact+brain.">Age-dependent pre- and postsynaptic distribution of AMPA receptors at synapses in CA3 stratum radiatum of hippocampal slice cultures compared with intact brain.</a> European Journal of Neuroscience. 12 (10), 3687-3700 (2000).</li><li>Laplagne, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+convergence+of+neurons+generated+in+the+developing+and+adult+hippocampus.">Functional convergence of neurons generated in the developing and adult hippocampus.</a> PLoS Biology. 4 (12), e409 (2006).</li><li>McDonald, H. Y., Wojtowicz, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+neurogenesis+in+the+dentate+gyrus+of+adult+rats.">Dynamics of neurogenesis in the dentate gyrus of adult rats.</a> Neuroscience Letters. 385 (1), 70-75 (2005).</li><li>Stone, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+convergence+of+developmentally+and+adult-generated+granule+cells+in+dentate+gyrus+circuits+supporting+hippocampus-dependent+memory.">Functional convergence of developmentally and adult-generated granule cells in dentate gyrus circuits supporting hippocampus-dependent memory.</a> Hippocampus. 21 (12), 1348-1362 (2011).</li><li>Wang, S., Scott, B. W., Wojtowicz, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogenous+properties+of+dentate+granule+neurons+in+the+adult+rat.">Heterogenous properties of dentate granule neurons in the adult rat.</a> Journal of Neurobiology. 42 (2), 248-257 (2000).</li><li>Wojtowicz, J. M., Kee, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BrdU+assay+for+neurogenesis+in+rodents.">BrdU assay for neurogenesis in rodents.</a> Nature Protocols. 1 (3), 1399-1405 (2006).</li><li>Fritsch, R. S. E. R., Weibel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereological+Methods,+Vol.+1:+Practical+Methods+for+Biological+Morphometry.">Stereological Methods, Vol. 1: Practical Methods for Biological Morphometry.</a> Zeitschrift f&#252;r Allgemeine Mikrobiologie. 21 (8), 630-630 (1981).</li><li>Bunk, E. C., Konig, H. G., Bonner, H. P., Kirby, B. P., Prehn, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMDA-induced+injury+of+mouse+organotypic+hippocampal+slice+cultures+triggers+delayed+neuroblast+proliferation+in+the+dentate+gyrus:+an+in+vitro+model+for+the+study+of+neural+precursor+cell+proliferation.">NMDA-induced injury of mouse organotypic hippocampal slice cultures triggers delayed neuroblast proliferation in the dentate gyrus: an in vitro model for the study of neural precursor cell proliferation.</a> Brain Research. 1359, 22-32 (2010).</li><li>Kolb, B., Pedersen, B., Ballermann, M., Gibb, R., Whishaw, I. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+and+postnatal+injections+of+bromodeoxyuridine+produce+age-dependent+morphological+and+behavioral+abnormalities.">Embryonic and postnatal injections of bromodeoxyuridine produce age-dependent morphological and behavioral abnormalities.</a> The Journal Of Neuroscience : The Official Journal Of The Society For Neuroscience. 19 (6), 2337-2346 (1999).</li><li>Morris, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetic+toxicology+of+5-bromodeoxyuridine+in+mammalian+cells.">The genetic toxicology of 5-bromodeoxyuridine in mammalian cells.</a> Mutation Research. 258 (2), 161-188 (1991).</li><li>Bannigan, J., Langman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cellular+effect+of+5-bromodeoxyuridine+on+the+mammalian+embryo.">The cellular effect of 5-bromodeoxyuridine on the mammalian embryo.</a> Journal Of Embryology And Experimental Morphology. 50, 123-135 (1979).</li><li>Breunig, J. J., Arellano, J. I., Macklis, J. D., Rakic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Everything+that+glitters+isn't+gold:+a+critical+review+of+postnatal+neural+precursor+analyses.">Everything that glitters isn't gold: a critical review of postnatal neural precursor analyses.</a> Cell Stem Cell. 1 (6), 612-627 (2007).</li><li>Duque, A., Rakic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+effects+of+bromodeoxyuridine+and+[3H]thymidine+incorporation+into+DNA+on+cell+proliferation,+position,+and+fate.">Different effects of bromodeoxyuridine and [3H]thymidine incorporation into DNA on cell proliferation, position, and fate.</a> The Journal Of Neuroscience : The Official Journal Of The Society For Neuroscience. 31 (42), 15205-15217 (2011).</li><li>Namba, T., Mochizuki, H., Onodera, M., Namiki, H., Seki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+neurogenesis+in+hippocampal+slice+cultures:+early+in+vitro+labeling+of+neural+precursor+cells+leads+to+efficient+neuronal+production.">Postnatal neurogenesis in hippocampal slice cultures: early in vitro labeling of neural precursor cells leads to efficient neuronal production.</a> Journal of Neuroscience Research. 85 (8), 1704-1712 (2007).</li></ol>

The authors declare that they have no competing financial interests.

Seguindo CldU (ou BrdU) a administração, o calendário de aplicação de agentes farmacológicos podem ser escolhidas como alvo para PED-nascidos durante janelas específicas do desenvolvimento. Por exemplo, um agente hipotético pode ser aplicada durante a segunda semana pós-injecção CldU, o que é proposto para coincidir com a idade de neurónios imaturos que se encontram numa fase de desenvolvimento em que o GABA é despolarizantes. Estudos futuros utilizando este protocolo poderá adaptar o agente farmacológic...

Adult neurogenesis in the mammalian hippocampus represents a remarkable example of the brain&#8217;s innate capacity for adaptability and plasticity. Dentate granule cells (DGCs) are generated from a renewable pool of neural progenitor cells in the hippocampal dentate gyrus, which is one of the two presently well-characterized neurogenic regions in the mammalian brain, and is thought to be particularly important for learning and memory. The hippocampus is part of the limbic system and has a deep location within the mammalian brain, which makes it a difficult target for precise pharmacological manipulation. Additionally, aberrant neurogenesis has been implicated in conditions, such as epilepsy, schizophrenia, and Alzheimer&#8217;s disease, which has prompted interest in understanding the influence of various pharmacological agents during the maturation and survival of newborn neurons. The distinction between postnatal and adult neurogenesis is blurred and previous studies have shown that many features of in vivo neuronal development in the early postnatal period and adulthood are similar25. Here we emphasize postnatal neurogenesis and suggest possible applications to adult neurogenesis.
Organotypic slice cultures provide an efficient in vitro method for studying various physiological properties of the mammalian hippocampus. The value of slice cultures prepared from rodent brains can be summarized in three main qualities: 1) the protocol is straightforward and requires readily available materials; 2) slice cultures allow for pharmacological studies that eliminate complex variables such as the deep anatomic location of the hippocampus and circumvents the blood brain barrier1; and 3) the well characterized structure of the hippocampus and tri-synaptic circuit is preserved2. Previous investigators have used the organotypic hippocampal culture to study synaptic development and physiology3,4, gliogenesis5-7, ischemic brain damage8,9, neuroprotection and neurorepair10-12 as well as epilepsy13-15.The slice cultures could also provide a useful model system allowing for the monitoring of cell development in conjunction with labeling of cells with green fluorescent protein (GFP) or other vital markers.
Slice cultures have also been previously employed to study postnatal hippocampal neurogenesis16-19, but one important factor in the majority of these studies is the well-characterized degeneration that results from explanting tissue from adult animals after approximately 2 weeks in vitro20,21. For this reason, slice cultures are typically prepared from early post-natal (P5-P10) mice or rat pups, which utilizes the improved viability of early postnatal brain tissue for culturing22. While previous studies have shown that the early postnatal and adult hippocampus differ with regards to synaptic physiology and the expression of specific neuronal subtypes23,24, there is substantial conservation of the choreographed developmental program that newborn dentate granule cells proceed through during maturation25. Additionally, recent studies have suggested that the physiological characteristics of newborn DGCs in culture are very similar to immature neurons in the acute hippocampal slice preparation16.

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			<td rowspan="2" style="width:131px;">Company</td>
			<td rowspan="2" style="width:135px;">Catalog Number</td>
			<td rowspan="2" style="width:239px;">Comments/Description</td>
		</tr>
		<tr height="20">
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:265px;">5-chloro-2&#39;-deoxyuridine (CldU)</td>
			<td style="width:131px;">MP Biomedicals</td>
			<td align="right" style="width:135px;">105478</td>
			<td style="width:239px;">Hazardous, Carcinogenic</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:265px;">Cell culture inserts, 30mm diameter, 0.4&#181;m pore size</td>
			<td style="width:131px;">Thermo scientific&#160;</td>
			<td align="right" style="width:135px;">140660</td>
			<td style="width:239px;">Nuclon delta coating on these inserts provides better tissue adhesion and improves slice quality.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Conical Centrifuge tubes (sterile)</td>
			<td style="width:131px;">Fisher Scientific</td>
			<td style="width:135px;">14-432-22</td>
			<td style="width:239px;"></td>
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			<td height="40" style="height:40px;width:265px;">Dissector scissors (angled to side)</td>
			<td style="width:131px;">Fine Science Tools&#160;</td>
			<td style="width:135px;">14082-09</td>
			<td style="width:239px;"></td>
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			<td height="20" style="height:20px;width:265px;">Minimum essential medium (MEM)</td>
			<td style="width:131px;">Gibco</td>
			<td style="width:135px;">11095; liquid</td>
			<td style="width:239px;">Store at 4&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Eclipse Ni-U fluorescent microscope</td>
			<td style="width:131px;">Nikon</td>
			<td style="width:135px;"></td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:265px;">Glue for tissue</td>
			<td style="width:131px;">Krazy Glue</td>
			<td style="width:135px;">KG585</td>
			<td style="width:239px;">Use minimum amount of glue to achieve adhesion as any tissue exposed to glue will be unusable for IHC.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Hank&#8217;s Balanced Salt Solution (HBSS) (500 mL)</td>
			<td style="width:131px;">Gibco</td>
			<td style="width:135px;">14025-092</td>
			<td style="width:239px;">Store at 4&#176;C</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Horse Serum Heat Inactivated (500 mL)</td>
			<td style="width:131px;">Gibco</td>
			<td style="width:135px;">16050-122</td>
			<td style="width:239px;">Make 50 mL aliquots and store at -20&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:265px;">Kimwipes</td>
			<td style="width:131px;">Kimberly-Clarke</td>
			<td style="width:135px;">TW 31KYPBX</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:265px;">Modified glass pipettes (bottom of Pasteur pipette removed and edge smoothed with Bunsen flame)</td>
			<td style="width:131px;"></td>
			<td style="width:135px;"></td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Petri Dish (100mm x 15mm) and (60mm x 15mm)</td>
			<td style="width:131px;">Fisher Brand</td>
			<td style="width:135px;">FB0875712 and FB0875713A</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Scalpel blades #11</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:135px;">10011-00</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Scalpel handle #3</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:135px;">10003-12</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Serological Pipettes</td>
			<td style="width:131px;">Sorfa Medical Plastic Co.</td>
			<td style="width:135px;">P8050</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Standard Pattern forceps</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:135px;">11000-12</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Sterile vacuum filter</td>
			<td style="width:131px;">Thermo-Scientific</td>
			<td style="width:135px;">565-0020</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Surgical Scissors</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:135px;">14054-13</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Syringe driven filter unit</td>
			<td style="width:131px;">Millipore-Millex</td>
			<td style="width:135px;">SLGP033RS</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Tissue chopper with moveable stage</td>
			<td style="width:131px;">Stoelting&#160;</td>
			<td align="right" style="width:135px;">51425</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:265px;">Fine tip paintbrush</td>
			<td style="width:131px;"></td>
			<td style="width:135px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

organotypic slice cultures for studies of postnatal neurogenesis

Aqui nós descrevemos uma técnica para estudar a neurogênese pós-natal do hipocampo no cérebro de roedores utilizando a técnica de cultura fatia organotípica. Este método mantém a morfologia topográfica característica do hipocampo, enquanto que permite a aplicação directa de agentes farmacológicos para o desenvolvimento do giro dentado do hipocampo. Adicionalmente, culturas de fatias pode ser mantido durante até 4 semanas, e assim, permitir uma para estudar o processo de maturação de neurónios granulares-nascidos. Culturas de fatias para permitir a manipulação farmacológica eficaz de fatias de hipocampo, enquanto excluindo variáveis ​​complexas, tais como as incertezas relativas à localização anatómica profunda do hipocampo, bem como a barreira de sangue do cérebro. Por estas razões, buscou-se otimizar culturas fatia organotypic especificamente para pesquisa neurogênese pós-natal.

Determinar se as culturas organotypic seria adequado para a investigação a neurogênese adulta necessário que preencham dois critérios principais: 1) que fatias manter características morfológicas características de fatias do hipocampo após 10-21 dias in vitro (DIV), e 2) que PED-nascidos pode ser quantificada utilizando técnicas de imuno-histoquímica padrão comumente empregados na pesquisa neurogênese adulta. Relativamente ao primeiro critério, a Figura 1A e 1B de...

Watch this Scientific Journal Video about Organotypic Slice Cultures for Studies of Postnatal Neurogenesis at JoVE.com

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Aqui nós descrevemos uma técnica para estudar a neurogênese pós-natal do hipocampo utilizando a técnica de cultura fatia organotípica. Este método permite a manipulação in vitro da neurogénese adulto e permite a aplicação directa de agentes farmacológicos para o hipocampo cultivadas.

Cultures Organotípicas fatia de Estudos de Pós-natal Neurogênese

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

organotypic-slice-cultures-for-studies-of-postnatal-neurogenesis

Research

JoVE Journal

Developmental Biology

12.2K visualizações.  University of Toronto.  Aqui nós descrevemos uma técnica para estudar a neurogênese pós-natal do hipocampo utilizando a técnica de cultura fatia organotípica. Este método permite a manipulação in vitro da neurogénese adulto e permite a aplicação directa de agentes farmacológicos para o hipocampo cultivadas. 

Vídeo: Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical and molecular methods provides information of potential target genes and signaling pathways. To further elucidate specific regulatory mechanisms requires a system allowing the manipulation of only a small number of cells of a specific tissue by either overexpression, ablation or re-introduction of specific genes and follow their fate during development. To achieve this ex utero electroporation of hippocampal structures, especially the dentate gyrus, followed by organotypic slice culture provides such a tool. Using this system to generate mosaic deletions allows determining whether the gene of interest regulates cell-autonomously developmental processes like progenitor cell proliferation or neuronal differentiation. Furthermore it facilitates the rescue of phenotypes by re-introducing the deleted gene or its target genes. In contrast to in utero electroporation the ex utero approach improves the rate of successfully targeting deeper layers of the brain like the dentate gyrus. Overall ex utero electroporation and organotypic slice culture provide a potent tool to study regulatory mechanisms in a semi-native environment mirroring endogenous conditions.

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Here we present a protocol providing a tool to examine regulatory mechanisms of specific genes during hippocampal development. Employing ex utero electroporation and organotypic slice culture allows the up- and down-regulation of the expression of genes of interest in single cells and follow their fate during development.

Ex Utero Eletroporação e Organotípicas Cultura Slice of Mouse Hippocampal Tissue

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical ...

Biologia do Desenvolvimento

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

Geração de Culturas Geneticamente Modificados Pele organotï¿½icas Usando desvitalizado derme humana

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues

Primary cells are derived directly from tissue and are thought to be more representative of the physiological state of cells in vivo than established cell lines. However, primary cell cultures usually have a finite life span and need to be frequently re-established. Fibroblasts are an easily accessible source of primary cells. Here, we discuss a simple and quick experimental procedure to establish primary fibroblast cultures from ears and tails of mice. The protocol can be used to establish primary fibroblast cultures from ears stored at RT for up to 10 days. When the protocol is carefully followed, contaminations are unlikely to occur despite the use of non-sterile tissue stored for extended time in some cases. Fibroblasts proliferate rapidly in culture and can be expanded to substantial numbers before undergoing replicative senescence.

We describe a simple and quick experimental procedure for generating primary fibroblasts from the ears and tails of mice. The procedure does not require special animal training and can be used for the generation of fibroblast cultures from ears stored at RT for up to 10 days.

Culturas de geração primária de fibroblastos de rato orelha e da cauda Tecidos

Primary cells are derived directly from tissue and are thought to be more representative of the physiological state of cells in vivo than established cell ...

Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, and allow for the study of the cellular and molecular basis of normal and abnormal cardiac morphogenesis. Recent emerging technologies of retrospective single clonal analyses make the study of cardiac morphogenesis at single cell resolution feasible. However, tissue opacity and light scattering of the heart as imaging depth is increased hinder whole-heart imaging at single cell resolution. To overcome these obstacles, a whole-embryo clearing system that can render the heart highly transparent for both illumination and detection must be developed. Fortunately, in the last several years, many methodologies for whole-organism clearing systems such as CLARITY, Scale, SeeDB, ClearT, 3DISCO, CUBIC, DBE, BABB and PACT have been reported. This lab is interested in the cellular and molecular mechanisms of cardiac morphogenesis. Recently, we established single cell lineage tracing via the ROSA26-CreERT2; ROSA26-Confetti system to sparsely label cells during cardiac development. We adapted several whole embryo-clearing methodologies including Scale and CUBIC (clear, unobstructed brain imaging cocktails and computational analysis) to clear the embryo in combination with whole mount staining to image single clones inside the heart. The heart was successfully imaged at single cell resolution. We found that Scale can clear the embryonic heart, but cannot effectively clear the postnatal heart, while CUBIC can clear the postnatal heart, but damages the embryonic heart by dissolving the tissue. The methods described here will permit the study of gene function at a single clone resolution during cardiac morphogenesis, which, in turn, can reveal the cellular and molecular basis of congenital heart defects.

We describe a protocol to volumetrically image fluorescent protein labeled cells deep inside intact embryonic and postnatal hearts. Utilizing tissue-clearing methods in combination with whole mount staining, single fluorescent protein-labeled cells inside an embryonic or postnatal heart can be imaged clearly and accurately.

Imagiologia Cleared embrionário e pós-natal Corações na resolução de célula única

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, ...

Visualization of Cell Cycle Variations and Determination of Nucleation in Postnatal Cardiomyocytes

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and cytokinesis) and acytokinetic mitosis (karyokinesis but no cytokinesis). Such atypical cell cycle variations result in polyploid and multinucleated cells rather than in cell division. Therefore, to determine cardiac turnover and regeneration, it is of crucial importance to correctly identify cardiomyocyte nuclei, the number of nuclei per cell, and their cell cycle status. This is especially true for the use of nuclear markers for identifying cell cycle activity, such as thymidine analogues Ki-67, PCNA, or pHH3. Here, we present methods for recognizing cardiomyocytes and their nuclearity and for determining their cell cycle activity. We use two published transgenic systems: the Myh6-H2B-mCh transgenic mouse line, for the unequivocal identification of cardiomyocyte nuclei, and the CAG-eGFP-anillin mouse line, for distinguishing cell division from cell cycle variations. Combined together, these two systems ease the study of cardiac regeneration and plasticity.

To distinguish cell division from cell cycle variations in cardiomyocytes, we present protocols using two transgenic mouse lines: Myh6-H2B-mCh transgenic mice, for the unequivocal identification of cardiomyocyte nuclei, and CAG-eGFP-anillin mice, for distinguishing cell division from cell cycle variations.

Visualização de Variações do ciclo celular e Determinação de Nucleação no pós-natais Cardiomyocytes

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and ...

Dissection and Coronal Slice Preparation of Developing Mouse Pituitary Gland

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate embryonic origins and functions &#8212; the neurohypophysis and the adenohypophysis. The developing mouse pituitary gland is tiny and delicate with an elongated oval shape. A coronal section is preferred to display both the adenohypophysis and neurohypophysis in a single slice of the mouse pituitary.
The goal of this protocol is to achieve proper pituitary coronal sections with well-preserved tissue architectures from developing mice. In this protocol, we describe in detail how to dissect and process pituitary glands properly from developing mice. First, mice are fixed by transcardial perfusion of formaldehyde prior to dissection. Then three different dissecting techniques are applied to obtain intact pituitary glands depending on the age of mice. For fetal mice aged embryonic days (E) 17.5 - 18.5 and neonates up to 4 days, the entire sella regions including the sphenoid bone, gland, and trigeminal nerves are dissected. For pups aged postnatal days (P) 5 - 14, the pituitary glands connected with trigeminal nerves are dissected as a whole. For mice over 3 weeks old, the pituitary glands are carefully dissected free from the surrounding tissues. We also display how to embed the pituitary glands in a proper orientation by using the surrounding tissues as landmarks to obtain satisfying coronal sections. These methods are useful in analyzing histological and developmental features of pituitary glands in developing mice.

We present a protocol to dissect pituitary glands and prepare pituitary coronal sections from developing mice.

Dissecação e preparação Coronal fatia de desenvolver Mouse hipófise

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate ...

Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. Moreover, although existing culture techniques allow biochemical perturbations, these methods do not provide a means to study the effects of mechanical force and tissue stiffness during development of the inner ear sensory organs. Here we describe a method for three-dimensional organotypic culture of the intact murine utricle and cochlea that overcomes these limitations. The technique for adjustment of a three-dimensional matrix stiffness described here permits manipulation of the elastic force opposing tissue growth. This method can therefore be used to study the role of mechanical forces during inner ear development. Additionally, the cultures permit virus-mediated gene delivery, which can be used for gain- and loss-of-function experiments. This culture method preserves innate hair cells and supporting cells and serves as a potentially superior alternative to the traditional two-dimensional culture of vestibular and auditory sensory organs.

Three-dimensional organotypic cultures of the murine utricle and cochlea in optically clear collagen I gels preserve innate tissue morphology, allow for mechanical stimulation through adjustment of matrix stiffness, and permit virus-mediated gene delivery.

Culturas Organotypic tridimensional de órgãos sensoriais auditivas e vestibulares

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. ...

Single-cell Transcriptomic Analyses of Mouse Pancreatic Endocrine Cells

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, including insulin-secreting &#946; cells, are differentiated from common endocrine progenitors during the embryonic stage. Immature endocrine cells expand via cell proliferation and mature during a long postnatal developmental period. However, the mechanisms underlying these processes are not clearly defined. Single-cell RNA-sequencing is a promising approach for the characterization of distinct cell populations and tracing cell lineage differentiation pathways. Here, we describe a method for the single-cell RNA-sequencing of isolated pancreatic &#946; cells from embryonic, neonatal and postnatal pancreases.

We describe a method for the isolation of endocrine cells from embryonic, neonatal and postnatal pancreases followed by single-cell RNA sequencing. This method allows analyses of pancreatic endocrine lineage development, cell heterogeneity and transcriptomic dynamics.

Análises de célula única Transcriptomic de células endócrinas no pâncreas de rato

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, ...

Neurogenesis Using P19 Embryonal Carcinoma Cells

The P19 cell line derived from a mouse embryo-derived teratocarcinoma has the ability to differentiate into the three germ layers. In the presence of retinoic acid (RA), the suspension cultured P19 cell line is induced to differentiate into neurons. This phenomenon is extensively investigated as a neurogenesis model in vitro. Therefore, the P19 cell line is very useful for molecular and cellular studies associated with neurogenesis. However, protocols for neuronal differentiation of P19 cell line described in the literature are very complex. The method developed in this study are simple and will play a part in elucidating the molecular mechanisms in neurodevelopmental abnormalities and neurodegenerative diseases.

The P19 mouse embryonic carcinoma cell line (P19 cell line) is widely used for studying the molecular mechanism of neurogenesis with great simplification compared to in vivo analysis. Here, we present a protocol for retinoic acid-induced neurogenesis in the P19 cell line.

Neurogênese usando células de Carcinoma embrionário P19

The P19 cell line derived from a mouse embryo-derived teratocarcinoma has the ability to differentiate into the three germ layers. In the presence of ...

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic expression. Notch signaling effector Hes1 and the Notch ligand Delta-like1 (Dll1) are expressed in an oscillatory manner in neural stem/progenitor cells. Because the period of the oscillatory expression of these genes is very short (2 h), it is difficult to monitor their cyclic expression. To examine such rapid changes in the gene expression or protein dynamics, fast response reporters are required. Because of its fast maturation kinetics and high sensitivity, the bioluminescence reporter luciferase is suitable to monitor rapid gene expression changes in living cells. We used a destabilized luciferase reporter for monitoring the promoter activity and a luciferase-fused reporter for visualization of protein dynamics at single cell resolution. These bioluminescence reporters show rapid turnover and generate very weak signals; therefore, we have developed a highly sensitive bioluminescence imaging system to detect such faint signals. These methods enable us to monitor various gene expression dynamics in living cells and tissues, which are important information to help understand the actual cellular states.

Neural stem/progenitor cells exhibit various expression dynamics of Notch signaling components that lead to different outcomes of cellular events. Such dynamic expression can be revealed by real-time monitoring, not by static analysis, using a highly sensitive bioluminescence imaging system that enables visualization of rapid changes in gene expressions.

Imagens de bioluminescência em tempo real da dinâmica de sinalização de notch durante a neurogênese de Murine

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic ...