Este trabajo fue financiado por el Consejo Sueco de Investigación Médica (K2009-75SX-21.028-01-3, K2008-61X-20700-01-3); FONCICYT 000000000091984, los cimientos de Jeansson, Söderström Königska sjukhemmet, Lundqvist Marta y Sigurd och Elsa Goljes Minne, y la Sociedad Sueca de Medicina SLS-95151. Profesor Gene D. Block, UCLA, se agradece por sus valiosos comentarios sobre el manuscrito. Se agradece al Dr. Michael Andang y la Dra. Helena Johard para el bloqueador del canal de HCN, y el Prof. Abdel El Manira para proporcionar video microscopio. Consideraciones éticas: Todos los experimentos con animales se realizaron de acuerdo con las directrices y normas establecidas por el Instituto Karolinska y &quot;Estocolmo Norra Djurförsöksetiska Nämnd&quot;. Todos los experimentos con animales se llevan a cabo con la intención de reducir al mínimo cualquier posible estrés o incomodidad para el animal.

<ol>
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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biological+clock+nucleus:+a+multiphasic+oscillator+network+regulated+by+light.">The biological clock nucleus: a multiphasic oscillator network regulated by light.</a> J Neurosci. 23, 8070-8076 (2003).</li><li>LeSauter, J., Yan, L., Vishnubhotla, B., Quintero, J. E., Kuhlman, S. J., McMahon, D. G., Silver, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+short+half-life+GFP+mouse+model+for+analysis+of+suprachiasmatic+nucleus+organization.">A short half-life GFP mouse model for analysis of suprachiasmatic nucleus organization.</a> Brain Res. 964, 279-287 (2003).</li><li>Nakamura, W., Yamazaki, S., Takasu, N. N., Mishima, K., Block, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+response+of+Period+1+expression+within+the+suprachiasmatic+nucleus.">Differential response of Period 1 expression within the suprachiasmatic nucleus.</a> J Neurosci. 25, 5481-5487 (2005).</li><li>Davidson, A. J., Yamazaki, S., Arble, D. M., Menaker, M., Block, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resetting+of+central+and+peripheral+circadian+oscillators+in+aged+rats.">Resetting of central and peripheral circadian oscillators in aged rats.</a> Neurobiol Aging. 29, 471-477 (2008).</li><li>Croning, M. D., Haddad, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+brain+slice+chamber+designs+for+investigations+of+oxygen+deprivation+in+vitro.">Comparison of brain slice chamber designs for investigations of oxygen deprivation in vitro.</a> J Neurosci Methods. 81, 103-111 (1998).</li><li>Burgoon, P. W., Boulant, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-sensitive+properties+of+rat+suprachiasmatic+nucleus+neurons.">Temperature-sensitive properties of rat suprachiasmatic nucleus neurons.</a> Am J Physiol Regul Integr Comp Physiol. 281, 706-715 (2001).</li><li>Nishide, S. Y., Honma, S., Honma, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+circadian+pacemaker+in+the+cultured+suprachiasmatic+nucleus+from+pup+mice+is+highly+sensitive+to+external+perturbation.">The circadian pacemaker in the cultured suprachiasmatic nucleus from pup mice is highly sensitive to external perturbation.</a> Eur J Neurosci. 27, 2686-2690 (2008).</li><li>Yoshikawa, T., Sellix, M., Pezuk, P., Menaker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Timing+of+the+ovarian+circadian+clock+is+regulated+by+gonadotropins.">Timing of the ovarian circadian clock is regulated by gonadotropins.</a> Endocrinology. 150, 4338-4347 (2009).</li><li>Kohn, R. A., Dunlap, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calculation+of+the+buffering+capacity+of+bicarbonate+in+the+rumen+and+in+vitro.">Calculation of the buffering capacity of bicarbonate in the rumen and in vitro.</a> J Anim Sci. 76, 1702-1709 (1998).</li><li>Wilsbacher, L. D., Yamazaki, S., Herzog, E. D., Song, E. J., Radcliffe, L. A., Abe, M., Block, G., Spitznagel, E., Menaker, M., Takahashi, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photic+and+circadian+expression+of+luciferase+in+mPeriod1-luc+transgenic+mice+in+vivo.">Photic and circadian expression of luciferase in mPeriod1-luc transgenic mice in vivo.</a> Proc Natl Acad Sci U S A. 99, 489-494 (2002).</li><li>Sellix, M. 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Ventajas y desventajas de la tecnología de luciferasa En contraste con los métodos ex vivo, tales como RT-PCR, hibridación in situ y Western blot que requieren muestras de tejido en muchos diferentes puntos de tiempo (dando un tiempo de resolución de baja normalmente 2-4 horas dependiendo de la frecuencia de muestreo) para estudiar diurna variaciones en la expresión de genes y proteínas, la tecnología de luciferasa permite alta resolución (1-10 min) los estudios de las oscilaciones circadiano durante muchos días en la misma preparación. Estudios por lo tanto, el número de animales utilizados se reduce al mínimo y detallado de los efectos de la fase y el período del ritmo son factibles, que normalmente no es posible utilizando técnicas convencionales de muestreo con el tiempo de resolución de baja. Sin embargo, a pesar de las mediciones relativas amplitud del ritmo es posible, hay que destacar que la tecnología de periodista no es cuantitativo y no puede ser utilizado para medir las cantidades de los genes transcritos o traducidos proteínas. Las grabaciones de los tejidos se pueden iniciar y analizadas inmediatamente después de un cultivo, una gran ventaja para estudiar directamente los efectos moleculares de estrés en vivo, en vivo los cambios de fase inducido por la luz, etc La cultura SCN organotípicos permite a largo plazo (semanas) la manipulación farmacológica del cerebro adulto tejido que contiene una intacta red madura sináptica y la tecnología permite grabaciones de luciferasa estable durante varias semanas. Por lo tanto, el tejido no tiene por qué ser postnatal neonatal o principios con el fin de obtener cultivos sanos, y en contraste con el agudo corte tratamientos farmacológicos crónicos se puede realizar. En contraste con transfections plásmido reportero en cultivos de células, que no hacer lugar a la incorporación de ADN en el genoma, la luciferasa transgénicos modelos animales (así como transfecciones lenti-virus) son favorables si las alteraciones epigenéticas son objeto de estudio. Se debe en este contexto también se mencionó que las imágenes de luminiscencia es hoy en día es posible con cámaras CCD de alta sensibilidad 11, lo que permite grabaciones, incluso en las neuronas SCN sola (~ 6.10 m) y otros tipos de células, utilizada por los principales laboratorios estudian los ritmos circadianos. Finalmente, varios investigadores circadiano comúnmente seguimiento del uso de la expresión molecular mediante la prensa, como la proteína verde fluorescente, con el fin de estudiar genes reloj / oscilaciones de proteínas 16-19. Los aspectos de grosor de corte Los espesores aquí se recomienda el corte de SCN (200-300 micras) podrían reducirse o aumentarse si así lo desea. Sin embargo, aunque el tejido se aplana en la membrana después de unos días en la cultura, no se recomienda para más de 500 micras de espesor de la rebanada cortada inicialmente con el fin de preservar la viabilidad de los tejidos 7. Una rebanada de menos de 100 m es de razones mecánicas y prácticos difíciles de manejar. Debido a que el tejido SCN es heterogéneo el grosor del corte puede afectar a la fase de la señal de salida de la luciferasa, ya que las diferentes regiones del SNC (por ejemplo, el &quot;núcleo&quot; versus la &quot;cáscara&quot;, y en comparación con las regiones dorsal ventral) oscilan con las diferentes fases 20 y diferencialmente volver a sincronizar los cambios después de la fase 21. El grosor de la rebanada también afecta a la línea de base del recuento de fotones, ya que más tejido emite un mayor número de fotones. La amplitud de las oscilaciones moleculares pueden de esta manera indirectamente verse afectada por el tamaño de los explantes. Por estas razones, el control y cultivos tratados deben ser siempre del mismo tamaño, grosor, y que contiene la misma región del SCN, para oscilar en la misma fase y con la misma amplitud. Los dos núcleos unilateral, por su parte, oscilan en fase con los demás y con amplitudes similares, siempre y cuando el corte es horizontal y no vibratome y en ángulo (que a su vez depende de que el corte de separación entre el cerebelo y los dos hemisferios es perpendicular a la superficie de la mesa). Un investigador que realiza el cultivo de SCN pueden experimentar las culturas SCN no oscilan en fase con la otra. La práctica y la estandarización de la técnica de corte, es decir, los cortes siempre se cortan en el mismo rostro-caudal a nivel del SNC, mejorará el resultado. Los pasos críticos <ol><li> Suministro de oxígeno es crítico. Dado que el cerebro requiere una gran cantidad de oxígeno 22, el procedimiento de corte tiene que ser rápido (cuanto más cerca 3,1-3,10 es ~ 6.5 minutos, el mejor es el fin de mantener el tejido sano). </li><li> El SCN es sensible a los cambios de temperatura y el intercambio de mediano plazo. El SCN es sensible a la temperatura y los cambios de temperatura grandes pueden desplazamiento de fase del marcapasos y causar artefactos experimentales 23. Si el medio o las soluciones que se intercambian o añadidos durante un experimento en curso, por ejemplo, al aplicar un fármaco después de un par de días en la cultura, el medio o solution tiene que ser pre-calentado a 36-37 ° C (la misma temperatura en la cámara) antes de la adición. En experimentos farmacológicos que implican intercambios medio, es importante trabajar siempre con pre-condicionada medio de cultivo celular. Además / lavado con agua dulce, medio condicionado potencialmente cambio de fase del ritmo molecular SCN 24, así como otros cultivos de células de tipo 25. </li><li> Composición del medio. Es importante a la cultura del tejido en el pH y la osmolalidad correcta. El medio de aire buffer contiene una gran cantidad de HEPES, que tiene una capacidad de amortiguación óptima en el rango de pH 6.8 a 8.2 y también es apropiado para amortiguar los cambios de pH que pueden ocurrir como resultado de la respiración celular. HEPES, por el contrario, es más sensible a los cambios de temperatura en comparación con bicarbonato de sodio, que se incluye en pequeñas cantidades en el medio de cultivo. El bicarbonato de sodio tiene una mayor capacidad de amortiguación en el pH bajo (5,1-7,1) entre 26 y, por lo tanto apropiado en una atmósfera de CO 2. Sin embargo, el aumento de cantidades de bicarbonato de sodio en el medio del aire-buffering (DMEM 2902) aumenta la osmolaridad de manera significativa y no se puede añadir al mismo tiempo, sin diluir el medio, lo que a su vez se traduce en la composición incorrecta de aminoácidos, vitaminas e iones. Consejos para preparar el medio: <ol><li> Sea preciso y cuidado al mezclar el medio. </li><li> Utilice las soluciones madre sólo fresco o descongelado recientemente. </li><li> En el caso de osmolalidad muy alta que no vale la pena diluyendo. Más de 6.5% diluido en medio no lo más probable es el trabajo. El uso de D-glucosa para aumentar la osmolaridad si es demasiado baja. </li></ol></li><li> Cambios de fase debido al tiempo de preparación El tiempo de preparación rebanada es fundamental. Efecto nulo o mínimo en la fase se obtiene si el procedimiento de corte se realiza entre 6 a 12 dic. ZT. Todas las disecciones corte debe realizarse en la misma fase del ciclo diurno o circadiano con el fin de minimizar el error debido a la fase de variación entre los preparativos. </li></ol> Posibles modificaciones <ol><li> De electrofisiología SCN aguda, el corte debe realizarse con un timbre (líquido cerebro espinal artificial; ACSF) de amortiguación, saturada de oxígeno (95% O 2, 5% CO 2) antes de que comience la corte. Además, el oxígeno es necesario agregar continuamente al búfer durante todo el procedimiento corte, como la actividad eléctrica y sináptica depende directamente del suministro de oxígeno 22. </li><li> Cortes horizontales y sagitales también puede ser preparado con éxito y culto de la SCN. Los autores tienen experiencia con las grabaciones de la luciferasa en cortes horizontales, que de acuerdo a nuestra experiencia dan oscilaciones circadiano de menor amplitud, en comparación con el corte coronal. </li><li> El SCN es de aproximadamente 1 mm de longitud rostro-caudal y el método de los cortes se describe aquí se dirige hacia la adquisición de una sección de SCN en la región central. Sin embargo, más de una sección SCN se pueden obtener de ratón y los cerebros de ratas, lo que permite la investigación de la circadiano de genes / proteínas de expresión en los niveles más rostrales versus caudal del núcleo SCN. </li><li> Culturas sector a partir de los animales más jóvenes, las crías después del parto o de embriones, también se pueden preparar. Tenga en cuenta que el cerebro y el cráneo en cachorros muy jóvenes son muy suaves y sensibles. Además, el nervio óptico es delgado y no ha desarrollado plenamente. Tenga cuidado cuando la disección del cerebro de las crías para que la región SCN no está dañado. Por ejemplo, micro herramientas gubia no puede ser utilizada para abrir el cráneo de crías de ratón, ya que son demasiado grandes. Tijeras finas son suficientes para cortar los tejidos blandos de los cachorros. </li><li> La técnica del cristal de la cubierta cultura basada descritos aquí también se puede utilizar para la luminiscencia de la imagen utilizando una cámara CCD / EM-CCD, también en las neuronas SCN sola. </li><li> Otras regiones del sistema nervioso central y los tejidos periféricos de órganos de transgénicos PER2:: Los LUC se puede obtener fácilmente, cultivadas y analizadas en términos de las oscilaciones moleculares, como el PER2:: expresión LUC sigue oscilando durante muchos días, también en una serie de otros tejidos y tipos de células 10. La mayoría de estos tejidos periféricos, por ejemplo, el hígado, no requieren de una membrana para la supervivencia, sino que son cultivadas directamente bañada por el medio 11. Tenga en cuenta que el desplazamiento de fase debido a la corte, el cultivo y el intercambio de medio no puede seguir los mismos principios que para el SCN. </li></ol> Significado En ratones transgénicos y las cepas de ratas (mPER2:: LUC; mPer1-Luc 8, 10, 27), actividad de la enzima luciferasa refleja las proteínas o los ritmos de la expresión génica y pueden evaluarse mediante el registro de la bioluminiscencia. La bioluminiscencia luciferasa generado da una señal débil, pero la luminiscencia de fondo es cercano a cero, por lo que este método ventajoso. Por otra parte, porque la molécula de luciferasa es inestable y se degrada rápidamente que no hay phototoxicidad, que puede aparecer durante la iluminación de larga duración de excitación 28. En conjunto, estas propiedades permiten a los experimentos de larga duración haciendo que la tecnología de luciferasa muy ventajosa en la investigación circadiano.

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

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

Un centro circadiano (~ 24 h) la coordinación de los ritmos diarios del reloj en la fisiología y el comportamiento reside en el núcleo supraquiasmático (NSQ), ubicado en el hipotálamo anterior. El reloj se sincroniza directamente con la luz a través de la retina y el nervio óptico. Oscilaciones circadiano son generados por la interacción bucles de retroalimentación negativa de un número de los llamados &quot;genes reloj&quot; y sus productos proteicos, incluyendo el período de (por) los genes. El reloj de núcleo también depende de la despolarización de la membrana, el calcio y el campamento 1. El SCN presenta oscilaciones diarias en la expresión de genes reloj, la actividad metabólica y la actividad eléctrica espontánea. Cabe destacar que esta actividad cíclica endógena persiste en cortes de tejido adulto de la SCN 2-4. De esta manera, el reloj biológico puede ser estudiada in vitro, lo que permite las investigaciones moleculares, electrofisiológicas y metabólicas del funcionamiento del marcapasos. El SCN es una pequeña y bien definida estructura bilateral situada justo por encima del quiasma óptico 5. En la rata que contiene ~ 8.000 neuronas en cada núcleo y tiene unas dimensiones de aproximadamente 947 m (largo, eje rostrocaudal) x 424 m (ancho) x 390 m (altura) 6. Para diseccionar el SCN, es necesario cortar una rodaja del cerebro en el nivel específico del cerebro donde se encuentra el SCN identificadas. Aquí se describe el procedimiento de disección y el corte de la SCN, que es similar para el ratón y el cerebro de las ratas. Además, nos muestran cómo la cultura de los tejidos disecados organotypically en una membrana de 7, una técnica desarrollada para el cultivo de tejidos SCN por Yamazaki et al. 8. Por último, demostrar cómo el tejido transgénico puede ser utilizado para medir la expresión de los genes del reloj / proteínas de uso de la tecnología dinámica de luciferasa, un método que fue utilizado originalmente para la medición circadiano por Geusz et al. 9. Estamos aquí, usar pañuelos SCN de los transgénicos knock-in período2:: ratones luciferasa producida por Yoo et al 10.. Los ratones que contienen una proteína de fusión del período (PER) 2 y la enzima luciferasa de luciérnaga. Cuando PER2 se traduce en la presencia del sustrato para la luciferasa, luciferina, es decir, la expresión PER2 se puede controlar con bioluminiscencia cuando luciferasa cataliza la oxidación de la luciferina. El número de fotones emitidos se correlaciona positivamente con la cantidad de proteína producida PER2, y los ritmos de la bioluminiscencia coincidir con el ritmo de proteínas PER2 in vivo 10. De esta manera, la variación cíclica en la expresión PER2 puede ser de un seguimiento continuo en tiempo real durante muchos días. El protocolo que siguen para el cultivo de tejidos y la grabación en tiempo real bioluminiscencia ha sido completamente descrito por Yamazaki y Takahashi 11.

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

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

El procedimiento de preparación de cortes que contiene el ratón adulto núcleo supraquiasmático del hipotálamo (NSQ), y de una manera rápida a la cultura del tejido SCN en condiciones de cultivo organotípico, se informó. Además, la medición de la expresión de la proteína del gen del reloj oscilatorio utilizando la tecnología dinámica de luciferasa se describe.

Preparación Cortar, cultivo de tejidos organotípicos y grabación de la actividad luciferasa de reloj de genes en el núcleo supraquiasmático

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

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

Research

JoVE Journal

Neuroscience

34.9K vistas.  Karolinska Institutet. El procedimiento de preparación de cortes que contiene el ratón adulto núcleo supraquiasmático del hipotálamo (NSQ), y de una manera rápida a la cultura del tejido SCN en condiciones de cultivo organotípico, se informó. Además, la medición de la expresión de la proteína del gen del reloj oscilatorio utilizando la tecnología dinámica de luciferasa se describe.

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

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

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

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

El organotípicos rebanada hipocampo modelo de cultivo para el examen de la lesión neuronal

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

Investigación

Neurociencias

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

Organotípicos culturas rebanada hipocampo

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

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

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

Grabación de células enteras de una rebanada preparación organotípicos del neocórtex

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

Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation

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

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

Imágenes de detección de feromonas en una preparación de ratón Vomeronasal aguda lámina de tejido

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

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11 are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.

El análisis de la morfología de células dendríticas Purkinje en cultivos de corte organotípicos

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

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

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

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

Registro y análisis de los ritmos circadianos en funcionamiento las ruedas Actividad en roedores

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

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

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

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

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

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

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

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

Culturas Organotípicos Slice para Estudiar Oligodendrocyte Dinámica y mielinización

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

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

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

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

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

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

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

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