Este trabalho foi financiado pelo sueco Medical Research Council (K2009-75SX-21028-01-3, K2008-61X-20700-01-3); FONCICYT 000000000091984, os fundamentos dos Jeansson, Königska Söderström sjukhemmet, Lundqvist Marta e och Sigurd Elsa Goljes Minne e Sociedade Sueca de Medicina SLS-95151. Professor D. Gene Block, UCLA, é reconhecido agradecimento pelos comentários valiosos sobre o manuscrito. Agradecemos ao Dr. Michael Andäng e Dr Helena Johard para o bloqueador dos canais de HCN, e Prof Abdel El-Manira para a prestação de vídeo-microscópio. Considerações éticas: Todos os experimentos em animais foram realizados de acordo com as diretrizes e regulamentos estabelecidos pelo Karolinska Institutet e &quot;de Estocolmo Norra Djurförsöksetiska Nämnd&quot;. Todos os experimentos com animais são realizadas com a intenção de minimizar qualquer possível estresse ou desconforto ao animal.

<ol>
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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+response+of+Period+1+expression+within+the+suprachiasmatic+nucleus.">Differential response of Period 1 expression within the suprachiasmatic nucleus.</a> J Neurosci. 25, 5481-5487 (2005).</li><li>Davidson, A. J., Yamazaki, S., Arble, D. M., Menaker, M., Block, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resetting+of+central+and+peripheral+circadian+oscillators+in+aged+rats.">Resetting of central and peripheral circadian oscillators in aged rats.</a> Neurobiol Aging. 29, 471-477 (2008).</li><li>Croning, M. D., Haddad, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+brain+slice+chamber+designs+for+investigations+of+oxygen+deprivation+in+vitro.">Comparison of brain slice chamber designs for investigations of oxygen deprivation in vitro.</a> J Neurosci Methods. 81, 103-111 (1998).</li><li>Burgoon, P. W., Boulant, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-sensitive+properties+of+rat+suprachiasmatic+nucleus+neurons.">Temperature-sensitive properties of rat suprachiasmatic nucleus neurons.</a> Am J Physiol Regul Integr Comp Physiol. 281, 706-715 (2001).</li><li>Nishide, S. Y., Honma, S., Honma, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+circadian+pacemaker+in+the+cultured+suprachiasmatic+nucleus+from+pup+mice+is+highly+sensitive+to+external+perturbation.">The circadian pacemaker in the cultured suprachiasmatic nucleus from pup mice is highly sensitive to external perturbation.</a> Eur J Neurosci. 27, 2686-2690 (2008).</li><li>Yoshikawa, T., Sellix, M., Pezuk, P., Menaker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Timing+of+the+ovarian+circadian+clock+is+regulated+by+gonadotropins.">Timing of the ovarian circadian clock is regulated by gonadotropins.</a> Endocrinology. 150, 4338-4347 (2009).</li><li>Kohn, R. A., Dunlap, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calculation+of+the+buffering+capacity+of+bicarbonate+in+the+rumen+and+in+vitro.">Calculation of the buffering capacity of bicarbonate in the rumen and in vitro.</a> J Anim Sci. 76, 1702-1709 (1998).</li><li>Wilsbacher, L. D., Yamazaki, S., Herzog, E. D., Song, E. J., Radcliffe, L. A., Abe, M., Block, G., Spitznagel, E., Menaker, M., Takahashi, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photic+and+circadian+expression+of+luciferase+in+mPeriod1-luc+transgenic+mice+in+vivo.">Photic and circadian expression of luciferase in mPeriod1-luc transgenic mice in vivo.</a> Proc Natl Acad Sci U S A. 99, 489-494 (2002).</li><li>Sellix, M. 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Não há conflitos de interesse declarados.

Vantagens e desvantagens com a tecnologia repórter luciferase Em contraste com métodos ex vivo, como RT-PCR, hibridização in situ e Western blot que exigem amostras de tecido em muitos momentos diferentes (dando um tempo de resolução baixa, normalmente de 2-4 horas dependendo da freqüência de amostragem) a fim de estudar diurna variações na expressão dos genes e proteínas, a tecnologia permite que o repórter luciferase de alta resolução (1-10 min) estudos de oscilações circadiano por muitos dias na mesma preparação. Estudos, assim, o número de animais utilizados é minimizado e detalhada dos efeitos sobre a fase e período do ritmo são viáveis, o que normalmente não é possível usar técnicas de amostragem convencional, com tempo de resolução baixa. No entanto, embora as medidas de amplitude relativa do ritmo são possíveis, deve ser enfatizado que a tecnologia repórter não é quantitativa e não pode, portanto, ser utilizada para medir quantidades de genes transcritos ou traduzidos proteínas. As gravações do tecido pode ser iniciado e analisadas imediatamente após o cultivo, uma grande vantagem para estudar diretamente os efeitos do estresse molecular da vivo, em turnos vivo induzida pela luz fase etc A cultura SCN organotípicas permite a manipulação de longo prazo (semanas) farmacológica do cérebro adulto tecido que contém uma rede madura intacta sináptica ea tecnologia repórter luciferase permite gravações estáveis ​​durante muitas semanas. Assim, o tecido não precisa ser pós-neonatal ou cedo, a fim de obter culturas saudáveis, e em contraste com a aguda crônica fatia tratamentos farmacológicos pode ser realizada. Em contraste com o repórter-plasmídeo transfections em culturas de células, o que não fazer levar a incorporação de DNA no genoma, os transgênicos luciferase-modelos animais (bem como lenti-vírus transfections) são favoráveis ​​se alterações epigenéticas estão a ser estudado. Deve, neste contexto, ser mencionado que a imagem de luminescência é hoje possível com câmeras de alta sensibilidade CCD 11, permitindo gravações mesmo em neurônios SCN única (~ 6-10 mm) e outros tipos de células, utilizado por grandes laboratórios estudando ritmos circadianos. Finalmente, vários investigadores circadianos usam geralmente monitoramento de expressão molecular utilizando outros repórteres, como a Proteína Fluorescente Verde, a fim de estudar gene clock / oscilações de proteína 16-19. Aspectos de espessura de corte A espessura recomendada aqui da fatia SCN (200-300 mm) pode ser reduzido ou aumentado, se desejar. No entanto, embora o tecido aplaina para fora da membrana depois de alguns dias em cultura, não é recomendável exceder 500 mM espessura da fatia cortada inicialmente, a fim de preservar a viabilidade do tecido 7. A fatia mais fina do que 100 mm é de razões mecânicas e prática difícil de lidar. Porque o tecido SCN é heterogênea a espessura da fatia pode afetar a fase do sinal de saída luciferase, uma vez que diferentes regiões dentro do SCN (por exemplo, o &quot;core&quot; versus o &quot;shell&quot;, e as regiões dorsal contra ventral) oscilam com diferentes fases 20 e diferencialmente re-sincronizar após a fase de mudanças 21. A espessura da fatia também afeta a linha de base de contagem de fótons, já que mais tecido emite um número maior de fótons. A amplitude das oscilações moleculares podem desta forma ser indiretamente afetada pelo tamanho do explante. Por estas razões, e controle de culturas tratadas deve ser sempre do mesmo tamanho, espessura e contendo a mesma região do SCN, a fim de oscilar na mesma fase e com a mesma amplitude. Os dois núcleos unilateral, por outro lado, oscilam em fase uns com os outros e com amplitudes semelhantes, desde que o corte é vibratome horizontal e não em ângulo (que por sua vez é dependente de que a separação entre corte cerebelo e os dois hemisférios é perpendicular para a superfície da mesa). Um investigador que realiza cultura SCN pode experimentar que as culturas SCN não oscilam em fase uns com os outros. Praticando e padronização da técnica de fatiamento, ou seja, as fatias são sempre cortadas no nível do rostro-caudal mesma do SCN, vai melhorar o resultado. Etapas críticas <ol><li> Suprimento de oxigênio é fundamental. Porque o cérebro requer uma grande quantidade de oxigênio 22, o procedimento de corte precisa ser rápido (Quanto mais próximo 3,1-3,10 é ~ 5-6 minutos o que é melhor para manter os tecidos saudáveis). </li><li> O SCN é sensível às mudanças de temperatura e troca de meio. O SCN é sensível à temperatura e mudanças de temperatura grandes podem mudar a fase de pacemaker e causar artefatos experimentais 23. Se médio ou soluções sejam trocados ou adicionados durante um experimento em andamento, por exemplo, quando a aplicação de uma droga depois de alguns dias em cultura, o meio ou solution precisa ser pré-aquecido a 36-37 ° C (a mesma temperatura na câmara) antes da adição. Em experimentos farmacológicos que envolvem meio de intercâmbios, é importante sempre trabalhar com pré-condicionados meio de cultura celular. Adição / wash-out com frescos, médio incondicionado pode, potencialmente, de mudança de fase do ritmo SCN molecular 24, assim como outras culturas de células de tipo 25. </li><li> Composição do meio. É importante para a cultura do tecido no pH correto e osmolalidade. O meio de ar-buffering contém uma grande quantidade de HEPES, que tem uma ótima capacidade tampão na faixa de pH 6,8-8,2 e também é adequado para o armazenamento temporário de mudanças de pH que podem ocorrer como resultado da respiração celular. HEPES, por outro lado, é mais sensível às mudanças de temperatura em comparação com bicarbonato de sódio, que está incluído em pequenas quantidades no meio de cultura. Bicarbonato de sódio tem uma maior capacidade de buffer no baixo pH (5,1-7,1) Gama 26, portanto, apropriado em uma atmosfera de CO 2. No entanto, aumento da quantidade de bicarbonato de sódio no meio ar-buffering (DMEM 2902) aumenta significativamente a osmolalidade e não podem ser adicionados sem, simultaneamente, diluindo o meio, que por sua vez resulta na composição incorreta de aminoácidos, vitaminas e íons. Conselhos para preparar o meio de: <ol><li> Ser preciso e cuidadoso ao misturar o meio. </li><li> Use soluções estoque fresco ou recém-descongelado. </li><li> Em caso de osmolalidade muito alto não vale a pena diluindo. Mais do que 5-6% de diluição média muito provavelmente não funcionar. Use D-glicose para aumentar a osmolalidade se for muito baixa. </li></ol></li><li> Fase de mudanças devido ao tempo de preparação O tempo de preparação fatia é crítica. Efeito zero ou mínimo na fase é obtido se o procedimento de corte é realizada entre ZT dezembro 06-12. Todos dissecações fatia deve ser realizada na mesma fase do ciclo diurno ou circadiano, a fim de minimizar o erro devido à fase de variação entre as preparações. </li></ol> Possíveis modificações <ol><li> Para eletrofisiologia SCN aguda, o corte tem que ser realizada com uma campainha (Fluid Cerebro Artificial Spinal; ACSF) buffer, saturada com oxigênio (95% O 2, 5% de CO 2) antes do início do corte. Além disso, o oxigênio precisa ser adicionados continuamente ao buffer durante todo o procedimento de corte, como a atividade elétrica e sináptica está diretamente dependentes do fornecimento de oxigênio 22. </li><li> Fatias horizontais e sagital também pode ser preparado com êxito e culta do SCN. Os autores têm experiência com gravações luciferase em fatias horizontais, que de acordo com nossa experiência dar oscilações circadiano com menor amplitude, em comparação com o corte coronal. </li><li> O SCN é de aproximadamente 1 mm de comprimento rostro-caudal e da técnica de corte descrito aqui é voltado para a aquisição de uma seção SCN na região central. No entanto, mais de uma seção SCN pode ser obtido a partir do rato e cérebro de ratos, permitindo a investigação da expressão do gene / proteína circadiano em níveis mais rostral contra caudal do núcleo SCN. </li><li> Culturas fatia de animais mais jovens, os filhotes ou mesmo pós-natal de embriões, também pode ser preparado. Por favor, note que o cérebro eo crânio em filhotes muito jovens são muito macios e sensíveis. Além disso, o nervo óptico é fino e não completamente desenvolvidos. Tome cuidado ao dissecar o cérebro dos filhotes para que a região SCN não está danificado. Por exemplo, ferramentas de micro rongeur não pode ser usado para abrir o crânio de filhotes de rato, porque eles são grandes demais. Tesouras finas são o suficiente para cortar os tecidos moles em filhotes jovens. </li><li> A tampa de vidro técnica de cultura baseada descrito aqui também pode ser usado para luminescência imagem utilizando uma câmera CCD / EM-CCD, também em neurônios SCN único. </li><li> Outras regiões do sistema nervoso central, e os tecidos de órgãos periféricos de transgênicos PER2:: LUC animais pode ser facilmente obtida, culta e analisados ​​em termos de oscilações molecular, como o PER2:: LUC expressão continua a oscilar durante muitos dias também em uma série de outros tecidos e tipos de células 10. A maioria destes tecidos periféricos, por exemplo, o fígado, não precisam de uma membrana para a sobrevivência mas são cultivadas diretamente banhado em 11 médias. Por favor note que, devido à mudança de fase de corte, cultura e troca de mídia não pode seguir os mesmos princípios para SCN. </li></ol> Significado Em camundongos transgênicos e cepas de ratos (mPER2:: LUC; mPer1-luc 8, 10, 27) atividade da enzima luciferase reflete proteína ou ritmos expressão dos genes e pode ser avaliada através da gravação de bioluminescência. A bioluminescência luciferase gerado dá um sinal fraco, mas a luminescência de fundo está perto de zero, tornando este método vantajosa. Além disso, porque a molécula luciferase é instável e rapidamente degradada não há phototoxicidade, que podem aparecer durante a longo prazo iluminação excitatórios 28. Juntas, essas propriedades permitem experimentos de longa duração tornando a tecnologia repórter luciferase altamente vantajoso em pesquisa circadiano.

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>B27 supplement 50x</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>17504-044</td>
<td>&nbsp;</td>
<tr>
<td>Cover glasses</td>
<td>&nbsp;</td>
<td>Menzel-Gl&auml;ser</td>
<td>40#1</td>
<td>&#8776; 40 mm</td>
</tr>
<tr>
<td>Culture membranes</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>PICMORG50</td>
<td>0.4 &mu;m</td>
</tr>
<tr>
<td>DMEM low glucose w/o phenol red</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D2902</td>
<td>Powder for 1L</td>
</tr>
<tr>
<td>Filter paper</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>1003 055</td>
<td>&#8776; 55 mm</td>
</tr>
<tr>
<td>Filtration set</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>431097</td>
<td>Pore size 0.22 &mu;m
Polyethersulfone</td>
</tr>
<tr>
<td>Forceps </td>
<td>&nbsp;</td>
<td>Allgaier Instrumente</td>
<td>0203-7-PS</td>
<td>Dumant #7</td>
</tr>
<tr>
<td>HEPES</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>15630-056</td>
<td>100 ml</td>
</tr>
<tr>
<td>HBSS 10x</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco </td>
<td>14065-049</td>
<td>500 ml</td>
</tr>
<tr>
<td>NaOCH3 7.5%</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>25080-060</td>
<td>50 ml</td>
</tr>
<tr>
<td>Luciferin </td>
<td>&nbsp;</td>
<td>Promega</td>
<td>E1602</td>
<td>Beetle luciferin, potassium salt</td>
</tr>
<tr>
<td>Micro rongeur tool</td>
<td>&nbsp;</td>
<td>Allgaier Instrumente</td>
<td>332-097-140</td>
<td>&nbsp;</td>
<tr>
<td>PenStrep 10,000 U/ml</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>15140-122</td>
<td>100 ml</td>
</tr>
<tr>
<td>Petri dishes</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>430165</td>
<td>&#8776; 35 mm</td>
</tr>
<tr>
<td>PMT</td>
<td>&nbsp;</td>
<td>Hamamatsu</td>
<td>H9319-11MOD</td>
<td>&nbsp;</td>
<tr>
<td>Disposable scalpels</td>
<td>&nbsp;</td>
<td>Paragon</td>
<td>P503</td>
<td>Size 11</td>
</tr>
<tr>
<td>Vacuum filter</td>
<td>&nbsp;</td>
<td>Fisher </td>
<td>09-761-5</td>
<td>&nbsp;</td>
<tr>
<td>Vacuum grease</td>
<td>&nbsp;</td>
<td>Dow Corning</td>
<td>&nbsp;</td>
<td>50 g</td>
</tr>
<tr>
<td>Vibratome</td>
<td>&nbsp;</td>
<td>Campden Instruments</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

slice preparation, organotypic tissue culturing and luciferase recording of clock gene activity in the suprachiasmatic nucleus

A central de relógio (~ 24 h) circadiano coordenação ritmos diários na fisiologia e comportamento reside no núcleo supraquiasmático (SCN), localizado no hipotálamo anterior. O relógio está diretamente sincronizada com a luz através da retina e nervo óptico. Oscilações circadiano são geradas pela interação loops de feedback negativo de um número dos chamados &quot;genes do relógio&quot; e seus produtos protéicos, incluindo o período (por) genes. O clock do núcleo também é dependente despolarização da membrana, cálcio e cAMP 1. O SCN apresenta oscilações diárias na expressão dos genes, atividade metabólica e atividade elétrica espontânea. Notavelmente, esta atividade cíclica endógena persiste em fatias de tecido adulto do SCN 2-4. Desta forma, o relógio biológico pode facilmente ser estudados in vitro, permitindo investigação molecular, eletrofisiológica e metabólicas da função marca-passo. O SCN é uma pequena estrutura bem definida bilateral localizada logo acima do quiasma óptico 5. No rato que ele contém ~ 8,000 neurônios em cada núcleo e tem dimensões de cerca de 947 mM (eixo de comprimento, rostrocaudal) x 424 M (largura) x 390 M (altura) 6. Para dissecar o SCN é necessário cortar uma fatia do cérebro ao nível específico do cérebro onde o SCN podem ser identificadas. Aqui, descrevemos o processo de dissecação e corte do SCN, que é semelhante para o rato e cérebro de ratos. Além disso, mostramos como a cultura de tecidos dissecados organotypically em uma membrana 7, uma técnica desenvolvida para a cultura de tecidos SCN por Yamazaki et al. 8. Finalmente, demonstrar como tecidos transgênicos podem ser usados ​​para medir a expressão de genes relógio / proteínas usando a tecnologia dinâmico repórter luciferase, um método que originalmente era usado para as medições circadiano por Geusz et al. 9. Nós aqui usam tecidos SCN da transgênicos knock-in período2:: ratos luciferase produzido por Yoo et al 10.. Os ratos contêm uma proteína de fusão do período (PER) 2 ea luciferase enzima vaga-lumes. Quando PER2 se traduz na presença do substrato para a luciferase, ou seja, a luciferina, a expressão PER2 podem ser monitorados como bioluminescência quando luciferase catalisa a oxidação da luciferina. O número de fótons emitidos positivamente correlacionada com a quantidade de proteína produzida PER2, e os ritmos bioluminescência corresponder ao ritmo de proteínas PER2 in vivo 10. Desta forma, a variação cíclica na expressão PER2 pode ser continuamente monitorado em tempo real durante muitos dias. O protocolo que seguimos para cultura de tecidos e gravação em tempo real bioluminescência tem sido exaustivamente descrita por Yamazaki e Takahashi 11.

Watch this Scientific Journal Video about Slice Preparation, Organotypic Tissue Culturing and Luciferase Recording of Clock Gene Activity in the Suprachiasmatic Nucleus at JoVE.com

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

O processo de preparação de fatias contendo o adulto do rato hipotálamo núcleo supraquiasmático (SCN), e uma maneira rápida para a cultura do tecido SCN em condições organotípicas cultura, são relatados. Além disso, a medição da oscilação expressão da proteína do gene do relógio usando a tecnologia dinâmico repórter luciferase é descrito.

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 ...

slice-preparation-organotypic-tissue-culturing-luciferase-recording

Research

JoVE Journal

Neuroscience

34.9K visualizações.  Karolinska Institutet. O processo de preparação de fatias contendo o adulto do rato hipotálamo núcleo supraquiasmático (SCN), e uma maneira rápida para a cultura do tecido SCN em condições organotípicas cultura, são relatados. Além disso, a medição da oscilação expressão da proteína do gene do relógio usando a tecnologia dinâmico repórter luciferase é descrito.

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

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

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

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

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

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

Neurociência

Organotypic 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 ...

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 ...

Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. Pheromones are volatile or non-volatile short-lived molecules2 secreted and/or contained in biological fluids3,4, such as urine, a liquid known to be a main source of pheromones3. Pheromonal communication is implicated in a variety of key animal modalities such as kin interactions5,6, hierarchical organisations3 and sexual interactions7,8 and are consequently directly correlated with the survival of a given species9,10,11. In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO)10,12, a paired structure located at the base of the nasal cavity, and enclosed in a cartilaginous capsule. Each VNO has a tubular shape with a lumen13,14 allowing the contact with the external chemical world. The sensory neuroepithelium is principally composed of vomeronasal bipolar sensory neurons (VSNs)15. Each VSN extends a single dendrite to the lumen ending in a large dendritic knob bearing up to 100 microvilli implicated in chemical detection16. Numerous subpopulations of VSNs are present. They are differentiated by the chemoreceptor they express and thus possibly by the ligand(s) they recognize17,18. Two main vomeronasal receptor families, V1Rs and V2Rs19,20,21,22, are composed respectively by 24023 and 12024 members and are expressed in separate layers of the neuroepithelium. Olfactory receptors (ORs)25 and formyl peptide receptors (FPRs)26,27 are also expressed in VSNs.
Whether or not these neuronal subpopulations use the same downstream signalling pathway for sensing pheromones is unknown. Despite a major role played by a calcium-permeable channel (TRPC2) present in the microvilli of mature neurons28 TRPC2 independent transduction channels have been suggested6,29. Due to the high number of neuronal subpopulations and the peculiar morphology of the organ, pharmacological and physiological investigations of the signalling elements present in the VNO are complex.
Here, we present an acute tissue slice preparation of the mouse VNO for performing calcium imaging investigations. This physiological approach allows observations, in the natural environment of a living tissue, of general or individual subpopulations of VSNs previously loaded with Fura-2AM, a calcium dye. This method is also convenient for studying any GFP-tagged pheromone receptor and is adaptable for the use of other fluorescent calcium probes. As an example, we use here a VG mouse line30, in which the translation of the pheromone V1rb2 receptor is linked to the expression of GFP by a polycistronic strategy.

In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO). Here, an acute tissue slice preparation of VNO for performing calcium imaging is described. This physiological approach allows observations of subpopulations and/or individual neurons in a living tissue and is convenient for receptor-ligand identification.

Imagem Pheromone Sensing em um Preparação Slice rato Vomeronasal aguda do tecido

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. ...

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 ...

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).
Although circadian rhythms in running-wheel activity are typically linked to the SCN clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

Registro e análise dos ritmos circadianos em Running rodas Atividade em Roedores

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

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

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

Culturas Organotípicas fatia para Estudar Oligodendrocyte Dynamics e Myelination

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

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

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

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

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

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

Direct-current Stimulation and Multi-electrode Array Recording of Seizure-like Activity in Mice Brain Slice Preparation

Cathodal transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the stimulation parameters (e.g., orientation, field strength, and stimulation duration) need to be examined in mice brain slice preparations. Testing and arranging the orientation of the electrode relative to the position of the mice brain slice are feasible. The present method preserves the thalamocingulate pathway to evaluate the effect of DCS on anterior cingulate cortex seizure-like activities. The results of the multichannel array recordings indicated that cathodal DCS significantly decreased the amplitude of the stimulation-evoked responses and duration of 4-aminopyridine and bicuculline-induced seizure-like activity. This study also found that cathodal DCS applications at 15 min caused long-term depression in the thalamocingulate pathway. The present study investigates the effects of DCS on thalamocingulate synaptic plasticity and acute seizure-like activities. The current procedure can test the optimal stimulation parameters including orientation, field strength, and stimulation duration in an in vitro mouse model. Also, the method can evaluate the effects of DCS on cortical seizure-like activities at both the cellular and network levels.

Studies have shown that cathodal transcranial direct-current stimulation can produce suppressive effects on drug-resistant seizures. In this study, an in vitro experimental setup was devised in which the direct-current stimulation and multielectrode array recording of seizure-like activity were evaluated in mice brain slice preparation. The direct-current stimulation parameters were evaluated.