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 trabajo fue apoyado por los NIH, DA043982 y DA046196.

Los animales utilizados en este estudio fueron tratados de una manera ética y humana según lo establecido por las directrices del Comité Institucional de Cuidado y Uso de Animales (IACUC) de la Universidad de Indiana y los Institutos Nacionales de Salud (NIH).
NOTA: Todas las herramientas y superficies deben lavarse con un disolvente adecuado para eliminar las nucleasas18 antes de comenzar cualquier trabajo.
1. Almacenar cerebros
<ol><li...

<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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+Clusters,+Hubs,+and+Communities+in+the+Cortical+Microconnectome.">Functional Clusters, Hubs, and Communities in the Cortical Microconnectome.</a> Cerebral cortex. 25 (10), 3743-3757 (2015).</li><li>Wang, Z., Gerstein, M., Snyder, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-Seq:+a+revolutionary+tool+for+transcriptomics.">RNA-Seq: a revolutionary tool for transcriptomics.</a> Nature Reviews Genetics. 10 (1), 57-63 (2009).</li><li>Kebschull, J. M., 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=High-throughput+mapping+of+single+neuron+projections+by+sequencing+of+barcoded+RNA.">High-throughput mapping of single neuron projections by sequencing of barcoded RNA.</a> bioRxiv. , 054312 (2016).</li><li>Mitulovi&#263;, G., Mechtler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HPLC+techniques+for+proteomics+analysis&#8212;a+short+overview+of+latest+developments.">HPLC techniques for proteomics analysis&#8212;a short overview of latest developments.</a> Briefings in Functional Genomics. 5 (4), 249-260 (2006).</li><li>Bettscheider, M., Murgatroyd, C., Spengler, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+DNA+and+RNA+isolation+from+brain+punches+for+epigenetics.">Simultaneous DNA and RNA isolation from brain punches for epigenetics.</a> BMC Research Notes. 4, 314 (2011).</li><li>Chiu, K., Lau, W. 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 trabajo describe una técnica para aislar pequeñas regiones específicas del cerebro mientras limita la degradación del ácido nucleico y la proteína. El daño a los tejidos cerebrales ocurre rápidamente una vez que un organismo muere. Esto se debe en parte a una rápida acumulación de glutamato extracelular y la excitotoxicidad resultante que se produce21. El ARN mensajero es particularmente vulnerable a la degradación22,23. La...

Este trabajo ilustra la disección de regiones cerebrales congeladas para la extracción de ácido nucleico o proteína de alta calidad utilizando una referencia, como el Allen Mouse Brain Atlas1, como guía. En esta técnica, los cerebros se congelan con flash y se almacenan a -80 oC para su posterior seccionamiento y disección mientras se mantienen en una condición congelada. Este proceso permite al investigador cosechar un gran número de cerebros en una sesión y luego diseccionarlos para una colección precisa de múltiples regiones cerebrales.
La recopilación precisa de regiones cerebrales de interés (ROI) a menudo es necesaria al responder preguntas relacionadas con la expresión de genes y proteínas. Mientras que la farmacología, electrofisiología y optogenética se pueden utilizar en roedores de tipo salvaje o modificados genéticamente para ayudar a dilucidar los cambios moleculares que sustentan los comportamientos observados2,3,4, la medición de los cambios inducidos en transcriptomes y proteomes se utiliza a menudo para apoyar estos hallazgos. Técnicas como la reacción en cadena cuantitativa de la polimerasa de transcripción inversa (RT-qPCR), la hincha occidental, el RNAseq5,el MAPSeq6 y el HPLC7 son robustos y relativamente bajos en costo, lo que permite a muchos laboratorios estudiar los cambios moleculares inducidos dentro de las regiones cerebrales pequeñas2,,4,5,6.
Hay varias maneras de extraer y purificar el ácido nucleico o proteína de las regiones cerebrales8,9,10,11,12. Muchos laboratorios cosechan regiones cerebrales enfriando y cortando cerebros en hielo en el momento de la cosecha9,13. Si bien este enfoque puede dar lugar a ácido nucleico y proteína de alta calidad, es algo limitado en el tiempo, ya que la degradación dentro del microambiente del tejido puede tener lugar a estas temperaturas. Esto puede ser particularmente cierto cuando se intenta diseccionar un gran número de animales o IRO en una sola sesión. Mantener las muestras congeladas ayuda a mantener las moléculas diana lábiles mientras que proporciona al investigador tiempo para comparar cuidadosamente puntos de referencia en ambos lados de cada sección en el esfuerzo por recoger muestras relativamente puras. La captura láser es otra forma de recolectar tejido para el análisis de ARN o proteínas de las áreas cerebrales10. Este procedimiento es superior a la disección mecánica en el que se pueden identificar y aislar ROI muy pequeños y de forma irregular. Sin embargo, la captura láser está limitada por el uso de costosos equipos y reactivos, consume mucho tiempo y también puede ser más susceptible a la degradación de la muestra.
La disección de micropundado en tejidos congelados no es nueva. Los primeros trabajos de Miklos Palkovits y otros describen las técnicas básicas en detalle14,15. Esta presentación sigue en gran medida el trabajo original, con algunas mejoras para facilitar la eficiencia y disminuir el gasto del equipo necesario. Por ejemplo, las secciones cerebrales se hacen en un bloqueo cerebral congelado en lugar de en un criostato. Esto produce secciones más gruesas que reduce el número de secciones necesarias para recoger muestras de ROI. Este método también disecciona muestras en una placa de vidrio congelado que se sienta en hielo seco dentro de una caja aislada. Esto produce una etapa de subcongelación en el banco en el que trabajar. Las secciones diseccionadas de esta manera son fácilmente manipulables, lo que permite al investigador comparar ambos lados de cada sección con una referencia con el fin de limitar la contaminación de regiones fuera del ROI deseado.
Las ventajas de este protocolo son que 1) el cerebro se mantiene en una condición congelada durante todo el proceso, lo que ayuda a preservar la proteína y el ácido nucleico y da al investigador tiempo para cosechar cuidadosamente los IRO, y 2) los reactivos requeridos son baratos y se encuentran en la mayoría de los laboratorios de biología molecular. En este proceso, los cerebros enteros se seccionan a 0.5–1.0 mm en una matriz cerebral y se colocan en una placa de vidrio congelado que se enfría continuamente con hielo seco. Los lugares de interés que se encuentran en el Allen Brain Atlas1 u otros atlas cerebrales16,17 se utilizan para identificar las regiones de interés, que luego se diseccionan usando un puñetazo frío o un bisturí. Debido a que el tejido nunca se descongela, las regiones cosechadas de esta manera proporcionan ARN y proteínades de alta calidad para análisis posteriores.

<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

A medida que nuestra comprensión de la neurobiología ha progresado, los análisis moleculares a menudo se realizan en pequeñas áreas cerebrales como la corteza prefrontal medial (mPFC) o núcleo accumbens. El reto de este trabajo es diseccionar el área correcta preservando al mismo tiempo el microambiente a examinar. En este artículo, describimos un método simple y de bajo costo utilizando recursos disponibles en la mayoría de los laboratorios. Este método preserva el ácido nucleico y las proteínas manteniendo el tejido congelado durante todo el proceso. Los cerebros se cortan en secciones de 0,5-1,0 mm usando una matriz cerebral y dispuestos en una placa de vidrio congelado. Las marcas de interés dentro de cada sección se comparan con una referencia, como el Allen Mouse Brain Atlas, y las regiones se diseccionan usando un bisturí frío o un punzón de biopsia. A continuación, el tejido se almacena a -80 oC hasta su uso. A través de este proceso mPFC de rata y ratón, núcleo accumbens, hipocampo dorsal y ventral y otras regiones se han analizado con éxito utilizando qRT-PCR y ensayos occidentales. Este método se limita a las regiones cerebrales que se pueden identificar por puntos de referencia claros.

Para validar este método, la corteza prefrontal medial se recogió de ratones machos de tipo salvaje CD1 adultos y ARN y proteínas fueron extraídos y caracterizados. El ARN fue analizado por electroforesis capilar. El ARN degradado muestra una pérdida en la intensidad de las bandas ribosomales 28S y 18S y también muestra los productos de degradación como un frotis entre 25 y 200 nucleótidos(Figura 5A,muestra 1). ARN de alta calidad muestra bandas ribosomales distintas con poca o ningu...

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 procedimiento describe la recolección de regiones cerebrales congeladas discretas para obtener proteínas y ARN de alta calidad utilizando herramientas baratas y comúnmente disponibles.

Colección de regiones cerebrales de roedores congelados para análisis posteriores

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

Este procedimiento describe la recolección de regiones cerebrales congeladas discretas para obtener proteínas y ARN de alta calidad utilizando herramientas baratas y comúnmente disponibles. Debido a que las regiones cerebrales se mantienen congeladas de la cosecha a través de la disección, las moléculas diana se conservan y el investigador tiene tiempo para diseccionar y almacenar cuidadosamente las regiones de interés. Identificar regiones de interés puede ser inicialmente difícil.El uso de puntos de referencia cerebral comunes a medida que surgen y retroceden a través de las secciones en relación con las regiones deseadas hará que la identificación sea clara. Después de eliminar los cerebros de ratones adultos eutanizados de tipo salvaje CD-1, congelar el tejido durante 60 segundos en nitrógeno líquido o isopentano. Pre-enfriar con hielo seco y almacenarlo en menos 80 grados Celsius.24 horas antes de diseccionar el tejido, coloque una matriz cerebral limpia en una pila de paquetes de congelador descongelados. Y empareje los lados a la matriz entre dos paquetes de congelador asegurándose de dejar aproximadamente medio centímetro entre la parte inferior de las ranuras de afeitar y la parte superior de los paquetes. Configure una placa de vidrio congelada para la disección llenando una caja aislada con hielo hasta aproximadamente cinco centímetros desde la parte superior.A continuación, coloque una capa de 2,5 centímetros de hielo seco en la parte superior. Y cúbrelo con láminas de plástico negro. Coloque una placa de vidrio encima del plástico.Y hielo seco alrededor de los bordes de la placa. Retire la matriz cerebral congelada del congelador e inserte el cerebro, del lado de la corteza hacia arriba, en la matriz. Deje que el tejido se equilibre a la temperatura de la caja durante 10 minutos.Mantener la tapa abierta durante este tiempo. Utilice fórceps fríos para ajustar la posición del cerebro en la matriz de modo que el seno sagital y el seno transversal se alineen con las ranuras perpendiculares del bloque. Una vez que el cerebro esté en posición, coloque una cuchilla de afeitar fría cerca de su centro y presione aproximadamente un milímetro en el tejido.A continuación, coloca una hoja fría en cada extremo del cerebro y presiona hasta abajo en la matriz. Luego comience a agregar cuchillas refrigeradas en el extremo rostral, colocándolas en las ranuras de una en una y presionando suavemente un milímetro en el tejido. Continúe agregando cuchillas a intervalos de un milímetro, trabajando hacia el extremo caudal.Cuando todas las cuchillas estén en su lugar, presione hacia abajo en la parte superior con los dedos, la palma de la mano o un objeto contundente y mece lentamente de lado a lado para moverlas a través del tejido. Una vez que las cuchillas hayan llegado a la parte inferior de las ranuras, agarre cada lado del grupo de cuchillas y libéntelas de la matriz balanceándose hacia adelante y hacia atrás. Después de liberar el grupo de cuchillas colocarlos lado rostral hacia arriba en la placa de vidrio, y poner hielo seco al lado o en la parte superior de la pila para congelar aún más las muestras para una separación más fácil.A continuación, coloque la pila con bordes afilados hacia abajo y separe las cuchillas desplazando la pila entre el pulgar y los dedos. Alinee la sección en las placas de vidrio de rostral a caudal y separe el tejido de las cuchillas flexionándolas entre los dedos, o separándolas con una segunda hoja refrigerada. A veces, el tejido puede adherirse a ambos lados de una cuchilla y se debe tener cuidado para mantener la orientación rostral a caudal.Antes de iniciar la disección, abra Allen Mouse Brain Atlas u otra referencia y encuentre los puntos de referencia necesarios para identificar las regiones de interés. Utilice fórceps o cuchillas refrigeradas para voltear la sección. Y asegúrese de que la región de interés sea coherente en toda la sección.Cortar en la sección con un bisturí limpio o un puñetazo, empujando el metal suavemente pero firmemente en el tejido. Mecándolo de un lado a otro para hacer el corte. Después de cosechar la región de interés, colóquela en tubos pre-refrigerados de 1,5 milímetros etiquetados y guárdelos a menos 80 grados Centígrados.Para procesar el tejido, agregue el tampón RIPA frío para la extracción de proteínas o un disolvente que contenga guanidinio para la extracción de ARN y homogeneice inmediatamente con un desgarro de vidrio o un homogeneizador mecánico. Para validar este método, la corteza prefrontal medial se recogió de ratones macho adultos de tipo salvaje CD-1, y se caracterizaron arneses y proteínas. Cuando se analiza con electroforesis capilar, el ARN degradado muestra una pérdida en la intensidad de las bandas ribosomales 28S y 18S, así como un frotis entre 25 y 200 nucleótidos.Mientras que el ARN de alta calidad muestra bandas ribosomales distintas poca o ninguna señal en la región de menor peso molecular. El ARN obtenido utilizando el método de disección congelada se comparó con ARN preparado a partir de tejido recién cosechado. Ambos métodos producen ARN con altos números de integridad y bandas ribosomales fuertes.Para confirmar que este método se puede utilizar para preservar el microambiente del tejido diseccionado para su posterior análisis, el ARN se extrajo de la corteza prefrontal medial diseccionada que se había almacenado durante varias semanas. Todas las muestras produjeron ARN de alta calidad con bandas ribosomales distintas y números de integridad del ARN por encima de ocho. Para confirmar la integridad proteica de las muestras diseccionadas congeladas, se recogió la proteína, se transfirió a la nitrocelulosa y la sonda con anticuerpos contra el objetivo de alto peso molecular KCC2, y la actina de proteína de menor peso molecular.En todos los casos las bandas eran afiladas y distintas, sin productos de descomposición discernibles. Es importante mantener los tejidos congelados durante todo el proceso, ya que esto preserva el microambiente de las secciones y mantiene la morfología de la sección para la comparación con las imágenes del atlas cerebral. Las regiones de interés recogidas de esta manera se pueden almacenar durante varios meses a 80 grados Celsius negativos y se pueden utilizar en muchas aplicaciones posteriores, incluyendo RT-qPCR, RNA-Seq, análisis de manchas occidentales y HPLC.Este método se ha utilizado para explorar los cambios moleculares dentro de los sistemas límbicos de roedores perinatales expuestos a THC y otros cannabinoides para entender mejor cómo estos fármacos pueden afectar el desarrollo 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 simultánea y electrofisiología en el cerebro 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 ...

Investigación

Neurociencias

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.

Etiquetado DiOLISTIC de las neuronas de los roedores y no humanos rodajas de cerebro de los primates

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.

Sin láser y de captura de enfoque de Microscopía de la microdisección de regiones discretas del cerebro del ratón para el aislamiento de ARN total y Downstream secuenciación de próxima generación y de perfiles de expresión génica

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.

Los análisis morfométricos de las Secciones de 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.

Cultivo de células madre neurales de Regiones convencionales y no convencionales del cerebro 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 optogenético la estimulación del sistema nervioso 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 Resolución cuantitativa Synaptic proteoma perfiles de cerebro de ratón Regiones Después de discriminación auditiva de aprendizaje

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.

Agujas personalizadas para microinyecciones en el cerebro 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.

Microdisección de captura láser Atlas estereotáctico de regiones del cerebro afectadas por la lesión 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.

Evaluación de la densidad de sinapsis en rebanadas de hipocampo cerebral de roedor

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