Name: collection of frozen rodent brain regions for downstream analyses
Uploaded: 2020-04-23T13:00:00
Description: Watch this Scientific Journal Video about Collection of Frozen Rodent Brain Regions for Downstream Analyses at JoVE.com

Este trabalho foi apoiado pelo NIH, DA043982 e DA046196.

Os animais utilizados neste estudo foram tratados de forma ética e humana, conforme estabelecido pelas diretrizes do Comitê Institucional de Cuidados e Uso de Animais (IACUC) e institutos nacionais de saúde (NIH).
NOTA: Todas as ferramentas e superfícies devem ser lavadas com um solvente apropriado para remover as nucleases18 antes de iniciar qualquer trabalho.
1. Armazenar cérebros
<ol><li>Remova rapidamente os cérebros de camundongo...

<ol>
	<li>. Allen Mouse Brain Atlas Available from: <a target="_blank" href="https://mouse.brain">https://mouse.brain</a> (2008)</li><li>Kuleshova, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetics+-+New+Potentials+for+Electrophysiology.">Optogenetics - New Potentials for Electrophysiology.</a> Neuroscience and Behavioral Physiology. 49 (2), 169-177 (2019).</li><li>Scanziani, M., H&#228;usser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiology+in+the+age+of+light.">Electrophysiology in the age of light.</a> Nature. 461, 930 (2009).</li><li>Shimono, M., Beggs, J. 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M., Lau, H. T., So, K. F., Chang, R. C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-dissection+of+Rat+Brain+for+RNA+or+Protein+Extraction+from+Specific+Brain+Region.">Micro-dissection of Rat Brain for RNA or Protein Extraction from Specific Brain Region.</a> JoVE. (7), e269 (2007).</li><li>Aring, L., S, S., Marcus, K., May, C., Murran, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laser Capture Microdissection. Methods in Molecular Biology. 1723, 2457 (2018).</li><li>Bj&#246;rk, K., 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=Glutathione-S-transferase+expression+in+the+brain:+possible+role+in+ethanol+preference+and+longevity.">Glutathione-S-transferase expression in the brain: possible role in ethanol preference and longevity.</a> The FASEB Journal. 20 (11), 1826-1835 (2006).</li><li>Jeong, H., 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=Gene+Network+Dysregulation+in+the+Trigeminal+Ganglia+and+Nucleus+Accumbens+of+a+Model+of+Chronic+Migraine-Associated+Hyperalgesia.">Gene Network Dysregulation in the Trigeminal Ganglia and Nucleus Accumbens of a Model of Chronic Migraine-Associated Hyperalgesia.</a> Frontiers in Systems Neuroscience. 12 (63), (2018).</li><li>Spijker, S., Li, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+Rodent+Brain+Regions.">Dissection of Rodent Brain Regions.</a> Neuroproteomics, Neuromethods. 57, 13-26 (2011).</li><li>Palkovits, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolated+removal+of+hypothalamic+or+other+brain+nuclei+of+the+rat.">Isolated removal of hypothalamic or other brain nuclei of the rat.</a> Brain Research. 59, 449-450 (1973).</li><li>Palkovits, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Enzymology. 103, 368-376 (1983).</li><li>Paxinos, G., Franklin, K. B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Mouse Brain in Stereotaxic Coordinates. 2 edn. , (2001).</li><li>Paxinos, G., Watson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Rat Brain in Stereotaxic Coordinates. 4 edn. , (1998).</li><li>RNaseZap. Ambion Available from: <a target="_blank" href="https://assets.thermofisher.com/TFS-Assets/LSG/">https://assets.thermofisher.com/TFS-Assets/LSG/</a> (2008)</li><li>. RNA Integrity Number (RIN)- Standardization of RNA Quality Control Available from: <a target="_blank" href="https://www.agilent.com/cs/library/">https://www.agilent.com/cs/library/</a> (2016)</li><li>Scheyer, A. F., 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=Cannabinoid+Exposure+via+Lactation+in+Rats+Disrupts+Perinatal+Programming+of+the+Gamma-Aminobutyric+Acid+Trajectory+and+Select+Early-Life+Behaviors.">Cannabinoid Exposure via Lactation in Rats Disrupts Perinatal Programming of the Gamma-Aminobutyric Acid Trajectory and Select Early-Life Behaviors.</a> Biological Psychiatry. , (2019).</li><li>Choi, D. W., Rothman, 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+role+of+glutamate+neurotoxicity+in+hypoxic-ischemic+neuronal+death.">The role of glutamate neurotoxicity in hypoxic-ischemic neuronal death.</a> Annual Review of Neuroscience. 13 (1), 171-182 (1990).</li><li>Guhaniyogi, J., Brewer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+mRNA+stability+in+mammalian+cells.">Regulation of mRNA stability in mammalian cells.</a> Gene. 265 (1-2), 11-23 (2001).</li><li>Sampaio-Silva, F., Magalh&#227;es, T., Carvalho, F., Dinis-Oliveira, R. J., Silvestre, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+of+RNA+degradation+for+estimation+of+post+mortem+[corrected]+interval.">Profiling of RNA degradation for estimation of post mortem [corrected] interval.</a> PloS One. 8 (2), 56507 (2013).</li><li>Yamagata, K., 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=Signs+of+biological+activities+of+28,000-year-old+mammoth+nuclei+in+mouse+oocytes+visualized+by+live-cell+imaging.">Signs of biological activities of 28,000-year-old mammoth nuclei in mouse oocytes visualized by live-cell imaging.</a> Scientific Reports. 9 (1), 4050 (2019).</li><li>Allen Brain Atlas: Developing Mouse Brain. Allen Institute Available from: <a target="_blank" href="https://developingmouse.brain-map.org/static/atlas">https://developingmouse.brain-map.org/static/atlas</a> (2008)</li></ol>

Este trabalho descreve uma técnica para isolar pequenas regiões específicas do cérebro, limitando a degradação do ácido nucleico e da proteína. Danos aos tecidos cerebrais acontecem rapidamente quando um organismo morre. Isto deve-se, em parte, ao rápido acúmulo de glutamato extracelular e à excitotoxicidade resultante que ocorre21. O RNA mensageiro é particularmente vulnerável à degradação22,23. A discriminação de prote?...

Este trabalho ilustra a dissecção de regiões cerebrais congeladas para extração de ácido nucleico ou proteína de alta qualidade usando uma referência, como o Allen Mouse Brain Atlas1, como guia. Nesta técnica, os cérebros são congelados e armazenados a -80 °C para secção posterior e dissecção enquanto são mantidos em uma condição congelada. Esse processo permite ao pesquisador colher um grande número de cérebros em uma sessão e depois dissecá-los para uma coleta precisa de múltiplas regiões cerebrais.
A coleta precisa de regiões cerebrais de interesse (ROIs) é frequentemente necessária ao responder perguntas relacionadas à expressão genética e proteica. Enquanto a farmacologia, a eletrofisiologia e a optogenética podem ser usadas em roedores selvagens ou geneticamente modificados para ajudar a elucidar mudanças moleculares que sustentam comportamentos observados2,3,4, a medição de alterações induzidas em transcriptomas e proteomas é frequentemente usada para apoiar esses achados. Técnicas como a reação em cadeia de polimerase de transcrição reversa quantitativa (RT-qPCR), a mancha ocidental, RNAseq5,MAPSeq6 e HPLC7 são robustas e relativamente baixas de custo, permitindo que muitos laboratórios estudem alterações moleculares induzidas em pequenas regiões cerebrais22,4,5,6.
Existem várias maneiras de extrair e purificar o ácido nucleico ou proteína das regiões cerebrais8,,9,,10,,11,12. Muitos laboratórios colhem regiões cerebrais resfriando e cortando cérebros no gelo no momento da colheita9,13. Embora essa abordagem possa resultar em ácido nucleico e proteína de alta qualidade, é um pouco limitada, pois a degradação dentro do microambiente do tecido pode ocorrer a essas temperaturas. Isso pode ser particularmente verdadeiro ao tentar dissecar um grande número de animais ou ROIs em uma sessão. Manter amostras congeladas ajuda a manter moléculas alvo labile, ao mesmo tempo em que fornece ao pesquisador tempo para comparar cuidadosamente marcos em ambos os lados de cada seção no esforço de coletar amostras relativamente puras. A captura a laser é outra forma de coletar tecido para RNA ou análise de proteínas de áreas cerebrais10. Este procedimento é superior à dissecção mecânica em que os ROIs muito pequenos e de forma irregular podem ser identificados e isolados. No entanto, a captura a laser é limitada pelo uso de equipamentos caros e reagentes, é demorada e também pode ser mais suscetível à degradação da amostra.
A dissecção de microperfuração em tecidos congelados não é nova. Os primeiros trabalhos de Miklos Palkovits e outros descrevem as técnicas básicas em detalhes14,15. Esta apresentação segue em grande parte o trabalho original, com algumas melhorias para facilitar a eficiência e diminuir o gasto com os equipamentos necessários. Por exemplo, seções cerebrais são feitas em um bloco cerebral congelado em vez de em um criostato. Isso produz seções mais grossas, o que reduz o número de seções necessárias para coletar amostras de ROI. Este método também disseca amostras em uma placa de vidro congelada que se senta em gelo seco dentro de uma caixa isolada. Isso produz uma fase de subcongelamento no banco em que trabalhar. As seções dissecadas dessa forma são facilmente manipuladas, permitindo ao pesquisador comparar ambos os lados de cada seção com uma referência, a fim de limitar a contaminação de regiões fora do ROI desejado.
As vantagens deste protocolo são que 1) o cérebro é mantido em uma condição congelada durante todo o processo, o que ajuda a preservar proteína e ácido nucleico e dá ao pesquisador tempo para colher cuidadosamente os ROIs, e 2) os reagentes necessários são baratos e são encontrados na maioria dos laboratórios de biologia molecular. Nesse processo, cérebros inteiros são seccionados em 0,5-1,0 mm em uma matriz cerebral e colocados em uma placa de vidro congelada que é continuamente resfriada com gelo seco. Marcos encontrados no Atlas do Cérebro Allen1 ou em outros atlas cerebrais16,17 são usados para identificar regiões de interesse, que são então dissecadas usando um soco frio ou bisturi. Como o tecido nunca é descongelado, as regiões colhidas dessa forma fornecem RNA de alta qualidade e proteína para análises a jusante.

<table>
	<tbody>
		<tr>
			<td>0.5 mm Mouse coronal brain matrice</td>
			<td>Braintree Scientific</td>
			<td>BS-SS 505C</td>
			<td>Cutting block</td>
		</tr>
		<tr>
			<td>0.5 mm Rat coronal brain matrice</td>
			<td>Braintree Scientific</td>
			<td>BS-SS 705C</td>
			<td>Cutting block</td>
		</tr>
		<tr>
			<td>1.0 mm Biopsy Punch with plunger</td>
			<td>Electron Microscopy Sciences</td>
			<td>69031-01</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tubes</td>
			<td>Dot Scientific</td>
			<td>229443</td>
			<td>For storing frozen ROIs</td>
		</tr>
		<tr>
			<td>1.5 mm Biopsy Punch with plunger</td>
			<td>Electron Microscopy Sciences</td>
			<td>69031-02</td>
			<td></td>
		</tr>
		<tr>
			<td>2.0 mm Biopsy Punch with plunger</td>
			<td>Electron Microscopy Sciences</td>
			<td>69031-03</td>
			<td></td>
		</tr>
		<tr>
			<td>4-12% NuPage gel</td>
			<td>Invitrogen</td>
			<td>NPO323BOX</td>
			<td>protein gradient gel</td>
		</tr>
		<tr>
			<td>Bioanalyzer System</td>
			<td>Agilent</td>
			<td>2100</td>
			<td>RNA analysis system</td>
		</tr>
		<tr>
			<td>Dounce tissue grinder</td>
			<td>Millipore Sigma</td>
			<td> 
			D8938</td>
			<td>Glass tissue homogenizer</td>
		</tr>
		<tr>
			<td>Dry Ice</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fiber-Lite</td>
			<td>Dolan-Jenner Industries Inc.</td>
			<td>Model 180</td>
			<td>Cool lamp</td>
		</tr>
		<tr>
			<td>Glass plates</td>
			<td>LabRepCo</td>
			<td>11074010</td>
			<td></td>
		</tr>
		<tr>
			<td>HALT</td>
			<td>ThermoFisher</td>
			<td>78440</td>
			<td>protease inhibitor cocktail</td>
		</tr>
		<tr>
			<td>Low profile blades</td>
			<td>Sakura Finetek USA Inc.</td>
			<td>4689</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse anti-actin antibody</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>JLA20</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>Nanodrop</td>
			<td>Thermo Scientific</td>
			<td>2000C</td>
			<td>Used in initial RNA purity analysis</td>
		</tr>
		<tr>
			<td>No. 15 surgical blade</td>
			<td>Surgical Design Inc</td>
			<td>17467673</td>
			<td></td>
		</tr>
		<tr>
			<td>Odyssey Blocking buffer</td>
			<td>LiCor Biosciences</td>
			<td>927-40000</td>
			<td>Western blocking reagent</td>
		</tr>
		<tr>
			<td>Omni Tissue Master 125</td>
			<td>VWR</td>
			<td>10046-866</td>
			<td>Tissue homogenizer</td>
		</tr>
		<tr>
			<td>rabbit anti-KCC2 antibody</td>
			<td>Cell Signaling Technology</td>
			<td>94725S</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>RNA Plus Micro Kit</td>
			<td>Qiagen</td>
			<td>73034</td>
			<td>Used to extract RNA from small tissue samples</td>
		</tr>
		<tr>
			<td>RNaseZap</td>
			<td>Life Technologies</td>
			<td>AM9780</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel handle</td>
			<td>Excelta Corp.</td>
			<td>16050103</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard razor blades</td>
			<td>American Line</td>
			<td>66-0362</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol Reagent</td>
			<td>ThermoFisher Scientific</td>
			<td>15596026</td>
			<td>Used to extract RNA from tissue</td>
		</tr>
	</tbody>
</table>

collection of frozen rodent brain regions for downstream analyses

À medida que nossa compreensão da neurobiologia progrediu, análises moleculares são frequentemente realizadas em pequenas áreas cerebrais, como o córtex pré-frontal medial (mPFC) ou núcleo accumbens. O desafio neste trabalho é dissecar a área correta, preservando o microambiente a ser examinado. Neste artigo, descrevemos um método simples e de baixo custo usando recursos prontamente disponíveis na maioria dos laboratórios. Este método preserva o ácido nucleico e as proteínas mantendo o tecido congelado durante todo o processo. Os cérebros são cortados em seções de 0,5 a 1,0 mm usando uma matriz cerebral e dispostos em uma placa de vidro congelada. Marcos dentro de cada seção são comparados a uma referência, como o Atlas do Cérebro do Rato Allen, e as regiões são dissecadas usando um bisturi frio ou um soco de biópsia. O tecido é então armazenado a -80 °C até o uso. Através desse processo, o mPFC de ratos e ratos, núcleo accumbens, hipocampo dorsal e ventral e outras regiões foram analisados com sucesso utilizando ensaios qRT-PCR e ocidentais. Este método está limitado a regiões cerebrais que podem ser identificadas por marcos claros.

Para validar esse método, o córtex pré-frontal medial foi coletado de camundongos machos adultos do tipo CD1 e RNA e proteína foram extraídos e caracterizados. O RNA foi analisado por eletroforese capilar. O RNA degradado apresenta uma perda na intensidade das bandas ribossômicas 28S e 18S e também mostra produtos de degradação como uma mancha entre 25 e 200 nucleotídeos(Figura 5A, amostra 1). O RNA de alta qualidade mostra bandas ribossômicas distintas com pouco ou nenhum sinal n...

Watch this Scientific Journal Video about Collection of Frozen Rodent Brain Regions for Downstream Analyses at JoVE.com

Collection of Frozen Rodent Brain Regions for Downstream Analyses

Este procedimento descreve a coleção de regiões cerebrais congeladas discretas para obter proteínas de alta qualidade e RNA usando ferramentas baratas e comumente disponíveis.

Coleção de regiões cerebrais de roedores congelados para análises a jusante

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex ...

Este procedimento descreve a coleção de regiões cerebrais congeladas discretas para obter proteínas de alta qualidade e RNA usando ferramentas baratas e comumente disponíveis. Como as regiões cerebrais são mantidas congeladas da colheita através da dissecção, as moléculas-alvo são preservadas e o pesquisador tem tempo para dissecar cuidadosamente e armazenar regiões de interesse. Identificar regiões de interesse pode inicialmente ser um desafio.O uso de marcos cerebrais comuns à medida que emergem e recuam através das seções em relação às regiões desejadas, deixará a identificação clara. Depois de remover cérebros de camundongos adultos de CD-1, o flash congela o tecido por 60 segundos em nitrogênio líquido ou isopentane. Pré-frio com gelo seco e armazene-o a menos 80 graus Celsius.24 horas antes de dissecar o tecido, coloque uma matriz cerebral limpa em uma pilha de embalagens de congelador descongeladas. E sanduiche as laterais para a matriz entre dois pacotes de congelador certificando-se de deixar aproximadamente meio centímetro entre a parte inferior das ranhuras de barbear e a parte superior das embalagens. Configure uma placa de vidro congelada para a dissecção preenchendo uma caixa isolada com gelo até aproximadamente cinco centímetros do topo.Em seguida, coloque uma camada de 2,5 centímetros de gelo seco em cima. E cubra com lençóis plásticos pretos. Coloque uma placa de vidro em cima do plástico.E gelo seco ao redor das fronteiras da placa. Remova a matriz cerebral congelada do congelador e insira o cérebro, lado do córtex para cima, na matriz. Deixe o tecido equilibrar à temperatura da caixa por 10 minutos.Mantendo a tampa aberta durante este tempo. Use fórceps frios para ajustar a posição do cérebro na matriz para que o seio sagital e o seio transversal se alinhem com as ranhuras perpendiculares do bloco. Uma vez que o cérebro esteja em posição, coloque uma lâmina de barbear gelada perto de seu centro e pressione-a aproximadamente um milímetro no tecido.Em seguida, coloque uma lâmina gelada em cada extremidade do cérebro e pressione todo o caminho até a matriz. Em seguida, comece a adicionar lâminas geladas na extremidade rostral, colocando-as nas ranhuras uma de cada vez e pressionando-as suavemente um milímetro no tecido. Continue adicionando lâminas em intervalos de um milímetro, trabalhando em direção ao final caudal.Quando todas as lâminas estiverem no lugar, pressione para baixo em cima com os dedos, palma ou um objeto contundente e balance-as lentamente de um lado para o outro para movê-las através do tecido. Uma vez que as lâminas tenham atingido a parte inferior das ranhuras, segure cada lado do grupo de lâminas e liberte-as da matriz balançando para frente e para trás. Depois de liberar o grupo de lâminas coloque-os rostral lado para cima na placa de vidro, e coloque gelo seco ao lado ou em cima da pilha para congelar ainda mais as amostras para facilitar a separação.Em seguida, coloque a pilha com bordas afiadas para baixo e separe as lâminas deslocando a pilha entre o polegar e os dedos. Forque a seção das placas de vidro do rostral ao caudal e separe o tecido das lâminas flexionando-os entre os dedos, ou separando-os com uma segunda lâmina gelada. Às vezes, o tecido pode grudar em ambos os lados de uma lâmina e deve-se tomar cuidado para manter rostral à orientação caudal.Antes de iniciar a dissecção, abra o Allen Mouse Brain Atlas ou outra referência e encontre os marcos necessários para identificar regiões de interesse. Use fórceps ou lâminas geladas para virar a seção. E certifique-se de que a região de interesse seja consistente em toda a seção.Corte na seção com um bisturi limpo ou um soco, empurrando o metal suavemente, mas firmemente no tecido. Balançando para frente e para trás para fazer o corte. Depois de colher a região de interesse, coloque-a em tubos de 1,5 milímetros pré-refrigerados e armazene-os a menos 80 graus Celsius.Para processar o tecido, adicione tampão RIPA frio para extração de proteínas ou um guanidinium contendo solvente para extração de RNA e homogeneize-o imediatamente com uma queda de vidro ou homogeneizador mecânico. Para validar esse método, o córtex pré-frontal medial foi coletado de camundongos machos adultos do tipo CD-1, e o RNA e a proteína foram caracterizados. Quando analisado com eletroforese capilar, o RNA degradado apresenta uma perda na intensidade das bandas ribossômicas 28S e 18S, bem como uma mancha entre 25 e 200 nucleotídeos.Enquanto o RNA de alta qualidade mostra bandas ribossômicas distintas pouco ou nenhum sinal na região de menor peso molecular. O RNA obtido utilizando o método de dissecção congelado foi comparado com o RNA preparado a partir de tecido recém-colhido. Ambos os métodos produzem RNA com altos números de integridade e forte banda ribossômica.Para confirmar que este método pode ser usado para preservar o microambiente do tecido dissecado para análise posterior, o RNA foi extraído do córtex pré-frontal medial dissecado que havia sido armazenado ao longo de várias semanas. Todas as amostras produziram RNA de alta qualidade com distintas faixas ribossômicas e números de integridade de RNA acima de oito. Para confirmar a integridade proteica das amostras dissecadas congeladas, a proteína foi coletada, transferida para nitrocelulose e sonda com anticorpo contra o alvo de alto peso molecular KCC2, e a actina de proteína de menor peso molecular.Em todos os casos, as bandas eram nítidas e distintas, sem produtos de decomposição perceptíveis. É importante manter os tecidos congelados durante todo o processo, pois isso preserva o microambiente das seções e manter a morfologia da seção para comparação com as imagens do atlas cerebral. As regiões de interesse coletadas dessa forma podem ser armazenadas por vários meses a 80 graus Celsius negativos e podem ser utilizadas em muitas aplicações a jusante, incluindo RT-qPCR, RNA-Seq, western blot analysis e HPLC.Este método tem sido usado para explorar alterações moleculares dentro dos sistemas límbicos de roedores perinatais expostos ao THC e outros canabinóides para entender melhor como essas drogas podem afetar o desenvolvimento cerebral.

collection-of-frozen-rodent-brain-regions-for-downstream-analyses

Research

JoVE Journal

Neuroscience

Simultaneous fMRI and Electrophysiology in the Rodent Brain

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

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

FMRI simultânea e Eletrofisiologia no cérebro de roedores

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

Neurociência

DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; Gan et al., 2000). Here we provide a detailed description of each step required together with exemplary images of good and bad outcomes that will help when setting up the technique. In our experience, a few steps proved critical for the successful application of DiOLISTICS. These considerations include the quality of the DiI-coated bullets, the extent of fixative exposure, and the concentration of detergent used in the incubation solutions. Tips and solutions for common problems are provided. This is a versatile labeling technique that can be applied to multiple animal species at a wide range of ages. Unlike other fluorescent labeling techniques that are limited to preparations from young animals or restricted to mice because they rely on the expression of a fluorescent transgene, DiOLISTIC labeling can be applied to animals of all ages, species and genotypes and it can be used in combination with immunostaining to identify a specific subpopulation of cells. Here we demonstrate the use of DiOLISTICS to label neurons in brain slices from adult mice and adult non-human primates with the purpose of quantifying dendrite branching and dendritic spine morphology.

We demonstrate the use of the gene gun to introduce fluorescent dyes, such as DiI, into neurons in brain slices from rodents and non-human primates of different ages. In this particular case, we use adult mice (3-6 months old) and adult cynomologus monkeys (9-15 years old). This technique, originally described by the laboratory of Dr. Lichtman (Gan et al., 2000), is well suited for the study of dendritic branching and dendritic spine morphology and can be combined with traditional immunostaining, if detergents are kept at a low concentration.

Labeling DiOLISTIC de Neurônios de roedores e não-humanos fatias do cérebro do primata

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; ...

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of the breadth of molecular regulation. Newer approaches such as high-resolution RNA sequencing (RNA-Seq)1 provides new and expansive information about tissue- or state-specific expression such as relative transcript levels, alternative splicing, and micro RNAs2-4. Prospects for employing the RNA-Seq method in comparative whole transcriptome profiling5 within discrete tissues or between phenotypically distinct groups of individuals affords new avenues for elucidating molecular mechanisms involved in both normal and abnormal physiological states. Recently, whole transcriptome profiling has been performed on human brain tissue, identifying gene expression differences associated with disease progression6. However, the use of next-generation sequencing has yet to be more widely integrated into mammalian studies.
Gene expression studies in mouse models have reported distinct profiles within various brain nuclei using laser capture microscopy (LCM) for sample excision7,8. While LCM affords sample collection with single-cell and discrete brain region precision, the relatively low total RNA yields from the LCM approach can be prohibitive to RNA-Seq and other profiling approaches in mouse brain tissues and may require sub-optimal sample amplification steps. Here, a protocol is presented for microdissection and total RNA extraction from discrete mouse brain regions. Set-diameter tissue corers are used to isolate 13 tissues from 750-&mu;m serial coronal sections of an individual mouse brain. Tissue micropunch samples are immediately frozen and archived. Total RNA is obtained from the samples using magnetic bead-enabled total RNA isolation technology. Resulting RNA samples have adequate yield and quality for use in downstream expression profiling. This microdissection strategy provides a viable option to existing sample collection strategies for obtaining total RNA from discrete brain regions, opening possibilities for new gene expression discoveries.

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling  |

RNA expression profiling of discrete mouse brain regions requires a precise and repeatable tissue collection strategy. A protocol that uses both coronal brain sectioning and tissue corer-assisted microdissection is described here. The yield and quality of total RNA obtained from the resulting samples confirms the utility of the outlined method.

Laser não-Abordagem Microscopia de captura para o Microdissecção de regiões cerebrais discretas Mouse para isolamento de RNA total e Seqüenciamento Next-Generation Downstream e perfil de expressão gênica

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of ...

Morphometric Analyses of Retinal Sections

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the retinal ganglion cell layer (RGCL) in rat models with N-methyl-D-aspartate (NMDA)&#x2013;induced excitotoxicity1, retinal ischemia-reperfusion injury2 and glaucoma3. Reduction of INL and inner plexiform layer (IPL) thicknesses were reversed with citicoline treatment in rats' eyes subjected to kainic acid-mediated glutamate excitotoxicity4. Alteration of RGC density and soma sizes were observed with different drug treatments in eyes with elevated intraocular pressure3,5,6. Therefore, having objective methods of analyzing the retinal morphometries may be of great significance in evaluating retinal pathologies and the effectiveness of therapeutic strategies.
The retinal structure is multi-layers and several different kinds of neurons exist in the retina. The morphometric parameters of retina such as cell number, cell size and thickness of different layers are more complex than the cell culture system. Early on, these parameters can be detected using other commercial imaging software. The values are normally of relative value, and changing to the precise value may need further accurate calculation. Also, the tracing of the cell size and morphology may not be accurate and sensitive enough for statistic analysis, especially in the chronic glaucoma model. The measurements used in this protocol provided a more precise and easy way. And the absolute length of the line and size of the cell can be reported directly and easy to be copied to other files. For example, we traced the margin of the inner and outer most nuclei in the INL and formed a line then using the software to draw a 90 degree angle to measure the thickness. While without the help of the software, the line maybe oblique and the changing of retinal thickness may not be repeatable among individual observers. In addition, the number and density of RGCs can also be quantified. This protocol successfully decreases the variability in quantitating features of the retina, increases the sensitivity in detecting minimal changes.
 	This video will demonstrate three types of morphometric analyses of the retinal sections. They include measuring the INL thickness, quantifying the number of RGCs and measuring the sizes of RGCs in absolute value. These three analyses are carried out with Stereo Investigator (MBF Bioscience &mdash; MicroBrightField, Inc.). The technique can offer a simple but scientific platform for morphometric analyses.

This video demonstrates three types of morphometric analyses of the retina, which include measuring the inner nuclear layer thickness, quantifying the number of retinal ganglion cells (RGCs) and measuring the sizes of RGCs. The technique can offer a simple but scientific platform for morphometric analyses.

Análises morfométricas de secções da retina

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the ...

Growing Neural Stem Cells from Conventional and Nonconventional Regions of the Adult Rodent Brain

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the adult brain. However, the mechanisms these normally quiescent cells employ to ensure proper functioning of neural networks, as well as their role in recovery from injury and mitigation of neurodegenerative processes are little understood. These cells reside in regions referred to as &#34;niches&#34; that provide a sustaining environment involving modulatory signals from both the vascular and immune systems. The isolation, maintenance, and differentiation of CNS SCs under defined culture conditions which exclude unknown factors, makes them accessible to treatment by pharmacological or genetic means, thus providing insight into their in vivo behavior. Here we offer detailed information on the methods for generating cultures of CNS SCs from distinct regions of the adult brain and approaches to assess their differentiation potential into neurons, astrocytes, and oligodendrocytes in vitro. This technique yields a homogeneous cell population as a monolayer culture that can be visualized to study individual SCs and their progeny. Furthermore, it can be applied across different animal model systems and clinical samples, being used previously to predict regenerative responses in the damaged adult nervous system.

Neural stem cells harvested from the adult brain are increasing utilized in applications ranging from the basic research of nervous system development to exploring potential clinical applications in regenerative medicine. This makes rigorous control in the isolation and culturing conditions used to grow these cells critical to sound experimental outcomes.

Crescer a partir de células-tronco neurais convencionais e não convencionais Regiões do cérebro adulto de roedores

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

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

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

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

In vivo Optogenetic estimulação do sistema nervoso central de roedores

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

High Resolution Quantitative Synaptic Proteome Profiling of Mouse Brain Regions After Auditory Discrimination Learning

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory discrimination learning in mice is described. In this learning paradigm, mice are trained in a shuttle box Go/NoGo-task to discriminate between rising and falling frequency-modulated tones in order to avoid a mild electric foot-shock. The protocol involves the enrichment of synaptosomes from four brain areas, namely the auditory cortex, frontal cortex, hippocampus, and striatum, at different stages of training. Synaptic protein expression patterns obtained from trained mice are compared to na&#239;ve controls using a proteomic approach. To achieve sufficient analytical depth, samples are fractionated in three different ways prior to mass spectrometry, namely 1D SDS-PAGE/in-gel digestion, in-solution digestion and phospho-peptide enrichment. 
High-resolution proteomic analysis on a mass spectrometer and label-free quantification are used to examine synaptic protein profiles in phospho-peptide-depleted and phospho-peptide-enriched fractions of synaptosomal protein samples. A commercial software package is utilized to reveal proteins and phospho-peptides with significantly regulated relative synaptic abundance levels (trained/na&#239;ve controls). Common and differential regulation modes for the synaptic proteome in the investigated brain regions of mice after training were observed. Subsequently, meta-analyses utilizing several databases are employed to identify underlying cellular functions and biological pathways.

The identification of molecules and pathways controlling synaptic plasticity and memory is still a major challenge in neuroscience. Here, a workflow is described addressing the relative quantification of synaptic proteins supposedly involved in the molecular reorganization of synapses during learning and memory consolidation in an auditory learning paradigm.

Alta resolução quantitativa Synaptic Proteome Profiling de rato regiões cerebrais Depois Auditivo Discriminação Aprendizagem

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory ...

Personalized Needles for Microinjections in the Rodent Brain

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to deliver gene or cell therapy products. Unfortunately, current microinjection techniques use steel or glass needles that are suboptimal for multiple reasons: in particular, steel needles may cause tissue damage, and glass needles may bend when lowered deeply into the brain, missing the target region. In this article, we describe a protocol to prepare and use quartz needles that combine a number of useful features. These needles do not produce detectable tissue damage and, being very rigid, ensure reliable delivery in the desired brain region even when using deep coordinates. Moreover, it is possible to personalize the design of the needle by making multiple holes of the desired diameter. Multiple holes facilitate the injection of large amounts of solution within a larger area, whereas large holes facilitate the injection of cells. In addition, these quartz needles can be cleaned and re-used, such that the procedure becomes cost-effective.

We describe here a protocol for microinjection in the rodent brain that uses quartz needles. These needles do not produce detectable tissue damage and ensure reliable delivery even in deep regions. Moreover, they can be adapted to research needs by personalized designs and can be re-used.

Agulhas personalizadas para alevinos do cérebro de roedor

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to ...

Stereotactic Atlas-Guided Laser Capture Microdissection of Brain Regions Affected by Traumatic Injury

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial distribution. Such techniques also potentially skew gene expression analysis because the process itself can alter expression patterns in individual cells. Here we describe a laser capture microdissection (LCM) method to selectively collect specific brain regions affected by traumatic brain injury (TBI) by using a modified Nissl (cresyl violet) staining protocol and the guidance of a rat brain atlas. LCM provides access to brain regions in their native positions and the ability to use anatomical landmarks for identification of each specific region. To this end, LCM has been used previously to examine brain region specific gene expression in TBI. This protocol allows examination of TBI-induced alterations in gene and microRNA expression in distinct brain areas within the same animal. The principles of this protocol can be amended and applied to a wide range of studies examining genomic expression in other disease and/or animal models.

We describe the use of laser capture microdissection to obtain samples of distinct cell populations from different brain regions for gene and microRNA analysis. This technique allows the study of differential effects of traumatic brain injury in specific regions of the rat brain.

Estereotáxica guiada por Atlas Laser Microdissection de captura de regiões do cérebro afetadas por lesão traumática

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is tightly regulated during development and neuronal maturation. Importantly, deficits in synapse number can lead to cognitive dysfunction. Therefore, the evaluation of synapse number is an integral part of neurobiology. However, as synapses are small and highly compact in the intact brain, the assessment of absolute number is challenging. This protocol describes a method to easily identify and evaluate synapses in hippocampal rodent slices using immunofluorescence microscopy. It includes a three-step procedure to evaluate synapses in high-quality confocal microscopy images by analyzing the co-localization of pre- and postsynaptic proteins in hippocampal slices. It also explains how the analysis is performed and gives representative examples from both excitatory and inhibitory synapses. This protocol provides a solid foundation for the analysis of synapses and can be applied to any research investigating the structure and function of the brain.

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

Avaliação da densidade de sinapse em fatias Hippocampal cérebro de roedor

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...