La investigación financiada por el Departamento de Defensa (Defense Advanced Research Projects Agency, Premio Facultad Young, número de concesión N66001-09-1-2117) y NINDS (R15NS070734).

1. Preparación quirúrgica de Animales 1. Los sujetos experimentales Utilice ratones de la B6.Cg-Tg (Thy1-COP4/eYFP) 18Gfng / J línea transgénica 9; tensión JAX # 7612) o los otros ratones que expresan el azul crepuscular canal catiónico, canalrodopsina-2, en las neuronas corticales cerebrales. La aplicación de luz azul a la corteza cerebral de la B6.Cg-Tg (Thy1-COP4/eYFP) 18Gfng / J línea transgénica provoca que las neuronas piramidales que expresan canalrodopsina-2 para despolarizar y se someten a los potenciales de acción 9,10. Como consecuencia de la activación de células piramidales, interneuronas locales se activan, y el cambio en el potencial se propaga a las neuronas más allá del sitio de estimulación 10. Realizar cirugía en condiciones estériles en la adhesión a las directrices reguladoras aplicables: autoclave herramientas quirúrgicas y softpacks (campo quirúrgico, gasas); caliente cordón esterilizar todos tolerantes al calor implantados dispositivos metálicos (cánulas, tornillos y cables)por 30-segundos; Desinfectar calor intolerantes a los productos implantables (cable de fibra óptica conector de plástico) con un dip de 10 segundos en etanol; golpe de cruce de etanol artículos seca mediante la aplicación de aire comprimido. 2. Anestesia, colocación estereotáxica y Liquidación de cráneo Utilice el IMPAC 6 Integrada Paciente Multi (Vet Equip Inc) sistema para anestesiar el ratón. Utilice un 5% Isoflurane/95% de oxígeno para la inducción y el oxígeno del 3% Isoflurane/97% para el mantenimiento durante la cirugía. Colocar el animal sobre una manta de circulación de agua ajustado a 37 º C. No es necesario controlar la temperatura del cuerpo si la manta se mantiene a esta temperatura. 3. Preparación de la Calavera de Implantación Prepare el sitio de la incisión por el afeitado todo piel de la parte superior del cráneo. El área de afeitado debe extenderse lateralmente de oreja a oreja y antero-posterior de los ojos con el extremo posterior del cráneo. Desinfecte la piel expuesta con tres CONSECUTIVOSe hisopos de Betadine seguido por etanol. Hacer una incisión medial en la parte superior del cráneo, de entre los ojos a la parte posterior del cráneo. Limpiar el cráneo con peróxido de hidrógeno y solución salina estéril (0,9% NaCl). Controlar la hemorragia con una herramienta de cauterización. Localizar y marcar bregma y lambda para determinar las coordenadas estereotáxica. Las coordenadas estereotáxicas para las configuraciones de los electrodos de estímulo / optogenético que hemos utilizados se presentan en la Tabla 1 y se muestra esquemáticamente en la Figura 1A. 4. Preparación de los lugares de implantación en el cráneo Use un taladro dental de alta velocidad con una bola de 0,5 mm bit fresa para establecer cada sitio de implantación en el cráneo. Bisele el borde externo de cada hoyo con una bola de 0,7 mm poco fresa para facilitar la inserción del tornillo. 5. Inserción y fijación de cánulas, EMG conduce y conduce EEG Inserte los tornillos de EEG (0,8 mm de longitud, con 2 cm de 3 sin recubrimiento0-medidor de cobre estañado autobús alambre pre-soldado a la cabeza del tornillo) en los orificios y los llevan en una mano con un destornillador ranurado revoluciones aproximadamente 4-5 para obtener la profundidad deseada. Mantenga cánulas guía en su lugar con un soporte de cánula estereotáxico y luego péguelas en el cráneo y anclajes con cemento acrílico. Cada cánula guía debe contener un maniquí de cánula / estilete (Plastics One, parte # C312 DC \ 1) desde el momento de la cirugía para el tiempo de experimentación (10-14 días) para mantener la permeabilidad. 6. Cierre del sitio quirúrgico Una vez que los EEG y cánulas de guía se coloca en el cráneo, unirlos juntos con cemento acrílico dental (Dental Lang - Ortho-Jet cemento). Después de fraguar el cemento, colocar el conector de plástico (parte Pinnacle Tecnología # 8402) por encima del montículo de cemento seco. Soldar los extremos de los cables procedentes de derivaciones de EEG para los contactos del conector de plástico. Encajar los conectores del cable en el cemento. A continuación, dibuje los cables de EMG a través del músculo nucals deslizándolas en el barril de una aguja 21gA perforado a través del músculo. Ate un nudo de cirujano doble sutura de nylon 5-0 alrededor de estos cables justo distal a donde sale el músculo. Sutura de nuevo juntos de la piel que se retrae para acceder al tejido muscular con un solo cirujano nudos interrumpidas, con un retroceso de corte P-3 y 5-0 aguja de sutura de nylon. 7. Analgesia postoperatoria y recuperación Administrar buprenorfina como analgésico (0,1 mg / kg, subcutánea) y flunixina meglumina (0,1 mg / kg, subcutánea) como un anti-inflamatorio inmediatamente después de la cirugía, y diariamente en los días 1-2 después de la cirugía. Deje que los animales puedan recuperarse en condiciones estándar de colonias de vivienda durante al menos siete días antes de la experimentación. Con ninguna atención adicional más allá de la vivienda de colonias estándar, las cánulas se puede esperar que permanecen patente durante al menos dos semanas, con los estiletes permanentes asegurado en su lugar. Sensores EEG y EMG también se puede esperar que retenerfuncionalidad de esta duración. 2. Generación de Pulsos de luz temporizado 1. Programación de la Unidad de Estímulo MC Conecte la unidad MC-estímulo (Multi-Channel Systems) a través de una conexión USB a una computadora de escritorio. Utilice la hoja de cálculo similar a la interfaz de usuario MC-Estímulo para programar la unidad de Estímulo MC. Introduce el deseado TTL (lógica transistor-transistor) de tensión para el periodo en adelante, las duraciones de estímulo y fuera de períodos, el número de ciclos por segundo deseados, y el número total de ciclos repetidos para la sesión de estímulo entero. Para más detalles, véase el MC-Estímulo manual técnico: 3. Fabricación / Montaje de Fibra Óptica Probes para la prestación de Luz a la corteza cerebral <ol><li> Suministros y equipos necesarios se encuentran en: <a href="http://www.thorlabs.com/Thorcat/1100/1166-D02.pdf" target="_blank">http://www.thorlabs.com/Thorcat/1100/1166-D02.pdf</a> </li><li> Corte y pulido de los cables de fibra óptica. <a href="http://www.thorlabs.com/Thorcat/1100/1166-D02.pdf" target="_blank">http://www.thorlabs.com/Thorcat/1100/1166-D02.pdf</a> </li></ol> 4. Asamblea del Sistema de Estimulación roedores optogenético 1. Interfaz de unidad MC-Estímulo y Laser Una vez que la unidad MC-estímulo está programado, correr como un generador de señal independiente de un sistema binario de 5 voltios ON / OFF de la señal. Conecte la unidad MC-Estímulo a la unidad de potencia TTL enabled del láser con conectores BNC. 2. Producción de la salida láser a través de cable de fibra óptica Utilice la pantalla digital y el dial variable de la potencia de salida de la unidad de potencia del láser en el manualLy ajustar la potencia de salida del láser. Conectar la unidad de potencia del láser en el láser a través de un cable de cinta. Conectar el conector hembra FC (férula de conector) adaptador de fibra del láser para el conector macho de FC en el cable de conexión de fibra óptica (una longitud de 3 m, 200 m de diámetro de fibra de vidrio recubierto de conducción en la luz que refleja acrilato de revestimiento de polímero). 3. Transmitir el láser para el animal: Conmutador giratorio Conectar el cable de fibra óptica originario (3 m de longitud) y el cable de fibra óptica de entrega (45 cm de longitud) a los extremos opuestos de la junta óptica giratoria que encierra dos lentes (lentes de colimación dóricas). La fibra óptica de la junta rotativa sirve como un conmutador: como el animal se mueve alrededor de su jaula, la junta giratoria gira para evitar la rotura de los cables de fibra óptica. Use lazos de cable de plástico para fijar el conmutador, a un soporte metálico colocado por encima de la jaula cilíndrica en la que se aloja el animal. 4. Colocación de Entrega de fibra ópticaCable de Enfrentamientos Prohibir al ratón con una sola mano a la clavija del ratón debajo de una palma ahuecada. Oriente la cabeza entre medio del experimentador y el dedo índice. Extraer la cánula de maniquí de la cánula guía colocada por la cánula de fibra óptica. Desactive la cánula guía de escombros con una aguja estéril 25GA. Inserte el cable de fibra óptica a mano y fijarlo a la cánula de fibra óptica de guía con una tapa roscada rescatado de una cánula maniquí. Controlar la profundidad de inserción del cable de fibra óptica en el cerebro mediante un nudo de sutura atada en el cable de fibra óptica a una distancia fija desde el extremo plano-troceados. El aparato experimental entera se muestra en la Figura 1B. 5. Medición del EEG del sueño definido y la concentración de lactato durante la estimulación optogenético 1. Precalibración del sensor de lactato Pre-calibración del sensor de lactato se realiza con un in vitrokit (parte Pinnacle Tecnología # 7000-K1-T). El sensor se equilibra en solución salina tamponada con fosfato (pH 7,4), luego se expuso a tres concentraciones de L-lactato de manera escalonada por los protocolos del fabricante. 2. La inserción del sensor de lactato y el cable EEG Insertar el sensor de pre-calibrada en el cráneo montado en la cánula guía de lactato de una manera idéntica a la fibra óptica procedimiento de inserción de cable (sección 4,4). Conecte el sensor de lactato al preamplificador del Pinnacle 8.400 biosensor con conectores bipolares. Conecte este preamplificador en el conector de 8-pin en el headmount implantado quirúrgicamente. 3. El establecimiento de la intensidad del estímulo optogenético Antes de recoger los datos, usar la intensidad del láser mando de control para ajustar la intensidad del estímulo optogenético. La amplitud de la respuesta del EEG varía en los animales, debido a factores que no se han estudiado de forma sistemática, una<html> d debe ser verificada mediante inspección visual en el inicio de la estimulación. En la práctica, una intensidad de 80-120 μWatts típicamente producirá un incremento en la amplitud EEG de aproximadamente 50% en la frecuencia de estimulación. Post-hoc rápido análisis de Fourier de transformación (ver sección 5.5) es necesario cuantificar la magnitud absoluta de la respuesta. 4. Recopilación de datos Recopilar datos utilizando el sistema Pinnacle 8400: <a href="http://pinnaclet.com/pal-8400.html" target="_blank">http://pinnaclet.com/pal-8400.html</a> . 5. Procesamiento de Datos con Neuroscore Clasificar los estados de suspensión por inspección visual de los datos de EEG y EMG con la interfaz Neuroscore (DataSciences, International: <a href="http://www.datasci.com/products/software/NeuroScore_CNS_Software.asp" target="_blank">http://www.datasci.com/products/software/NeuroScore_CNS_Software.asp</a> ) o la interfaz de Sirenia (Pinnacle Technology:f = &quot;http://www.pinnaclet.com/sirenia.html&quot; target = &quot;_blank&quot;&gt; http://www.pinnaclet.com/sirenia.html). Los datos de proceso en diez segundos épocas como consecuencia, no rápido movimiento de los ojos de sueño, o sueño de movimientos oculares rápidos basados ​​en el EEG y EMG. Guardar datos en hojas de cálculo Microsoft Excel para análisis adicional y pruebas estadísticas.

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No hay conflictos de interés declarado.

Los métodos aquí presentados permiten medir la relación entre el sueño y los cambios en la concentración cerebral de la lactato intermediario glicolítico en una escala de tiempo no era posible anteriormente. Los animales experimentan transiciones espontáneas entre vigilia, NREMS y REMS. Además, estamos en condiciones de aplicar estímulos optogenético mientras que los animales se someten a estas transiciones. Los datos recogidos hasta la fecha demuestran que las ondas de impacto tanto espontánea e inducida en ...

<table border="1"><tbody><tr><td> Componente </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios (opcional) </td></tr><tr><td> Basi Ratón cánula guía </td><td> Pinnacle Tecnología / Inc Basi </td><td> 7032 </td><td></td></tr><tr><td> Biosensor de lactato </td><td> Pinnacle Tecnología </td><td> 7004 </td><td></td></tr><tr><td> Montado en el cabezal </td><td> Pinnacle Tecnología </td><td> 8402 </td><td></td></tr><tr><td> Sleep / Sistema de grabación Biosensor </td><td> Pinnacle Tecnología </td><td> 8400-K1-SL </td><td> 2 canales de EEG, EMG canal 1, y biosensor 1 </td></tr><tr><td> Ratón Tethered in vitro kit de calibración </td><td> Pinnacle Tecnología </td><td> 7000-K1-T </td><td></td></tr><tr><td> Fibra óptica cánula guía </td><td> Plastics One </td><td> C312G </td><td> 21 Medidor de guía para cánula </td></tr><tr><td> Cánula Maniquí </td><td> Plastics One </td><td> C312DC </td><td> 21 Gauge Maniquí </td></tr><tr><td> Diamond Scribe fibra </td><td> Thorlabs </td><td> S90W </td><td></td></tr><tr><td> Conector de fibra Crimp Tool </td><td> Thorlabs </td><td> CT042 </td><td></td></tr><tr><td> Bifurcación tubo </td><td> Thorlabs </td><td> FT030 </td><td> 03,0 mm </td></tr><tr><td></td><td> Thorlabs </td><td> T10S13 </td><td> Max Dia. 0,012 </td></tr><tr><td> Bifurcación Stripper Tube </td><td> Thorlabs </td><td> FTS3 </td><td></td></tr><tr><td> La fibra multimodo Bare Revestimiento Duro </td><td> Thorlabs </td><td> BFL37-200 </td><td> 200 micras Core, 0,37 NA </td></tr><tr><td> Alambre Tijeras / Tijeras de Kevlar </td><td> Thorlabs </td><td> T865 </td><td></td></tr><tr><td> Epoxy de fibra óptica </td><td> Thorlabs </td><td> F112 </td><td></td></tr&gt; <tr><td> Fibra de herramientas Stripper </td><td> Thorlabs </td><td></td><td></td></tr><tr><td> Placa de cristal pulido </td><td> Thorlabs </td><td> CTG913 </td><td></td></tr><tr><td> Pulido Pad Rubber </td><td> Thorlabs </td><td> NRS913 </td><td></td></tr><tr><td> Ojo de lupa </td><td> Thorlabs </td><td> JEL10 </td><td></td></tr><tr><td> Kim Wipes </td><td> Thorlabs </td><td> KW32 </td><td></td></tr><tr><td> Aire comprimido </td><td> Thorlabs </td><td> CA3 </td><td></td></tr><tr><td> Pulido Puck </td><td> Thorlabs </td><td> D50-xx </td><td></td></tr><tr><td> Fibra alcance de Inspección </td><td> Thorlabs </td><td> CL-200 </td><td></td></tr><tr><td> Películas de pulir </td><td> Thorlabs </td><td> LFG5P, LFG3P, LFG1P, LFG03P </td><td></td></tr><tr><td> FC / PC conector del extremo </td><td> Thorlabs </td&gt; <td> 30126G2-240 </td><td> Diámetro 240 m, SS virola </td></tr><tr><td> MC Unidad Estímulo </td><td> Sistemas multicanal </td><td> STG-4002 </td><td></td></tr><tr><td> MC Software Estímulo </td><td> Sistemas multicanal </td><td> MC-Estímulo V 2.1.5 </td><td></td></tr><tr><td> Láser azul </td><td> CrystaLaser </td><td> CL473-050-0 </td><td></td></tr><tr><td> Laser Fuente de alimentación </td><td> CrystaLaser </td><td> CL2005 </td><td></td></tr><tr><td> Fiber Optic junta rotativa </td><td> Lentes dóricas </td><td> FRJ-v4 </td><td></td></tr><tr><td></td><td></td><td></td><td> Tabla 2. Suministros y equipo. </td></tr></tbody></table>

simultaneous electroencephalography, real-time measurement of lactate concentration and optogenetic manipulation of neuronal activity in the rodent cerebral cortex

Aunque el cerebro representa menos del 5% del cuerpo en masa, utiliza aproximadamente un cuarto de la glucosa utilizada por el cuerpo en reposo 1. La función del sueño no REM (NREMS), la mayor parte de sueño por el tiempo, es incierto. Sin embargo, una característica sobresaliente de NREMS es una reducción significativa en la tasa de utilización de la glucosa cerebral relativo a la vigilia 2-4. Esta y otras conclusiones han dado lugar a la creencia generalizada de que el sueño tiene una función relacionada con el metabolismo cerebral. Sin embargo, los mecanismos subyacentes a la reducción del metabolismo cerebral de la glucosa durante NREMS aún no se han dilucidado. Un fenómeno asociado con NREMS que podrían afectar la tasa metabólica cerebral es la aparición de ondas lentas, oscilaciones en frecuencias de menos de 4 Hz, en el electroencefalograma 5,6. Estas ondas lentas detectadas en el nivel de la superficie del cráneo o cortical cerebral reflejar laoscilaciones de las neuronas subyacentes entre un estado despolariza / arriba y un estado hiperpolarizado / abajo 7. Durante el estado de abajo, las células no se someten a los potenciales de acción para los intervalos de hasta varios milisegundos cien. Restauración de los gradientes de concentración iónica posteriores a los potenciales de acción representa una carga significativa metabólico de la célula 8; ausencia de potenciales de acción durante los estados asociados a abajo NREMS pueden contribuir al metabolismo reducido en relación a despertar. Dos retos técnicos que había que abordar para que esta relación hipotética a probar. En primer lugar, era necesario para medir el metabolismo glicolítico cerebral con una resolución temporal reflectante de la dinámica de la cerebral EEG (es decir, más de segundos en lugar de minutos). Para ello, se midió la concentración de lactato, el producto de la glicolisis aerobia, y por lo tanto, una lectura de la tasa de metabolismo de la glucosa en el cerebro de los ratones. El lactato fuemedido usando un sensor basado en lactato oxidasa tiempo real incorporado en la corteza frontal. El mecanismo de detección consta de un electrodo de platino-iridio, rodeado por una capa de moléculas de lactato oxidasa. Metabolismo de lactato por la lactato oxidasa produce peróxido de hidrógeno, que produce una corriente en el electrodo de platino-iridio. Así, un aumento gradual de la glucólisis cerebral proporciona un aumento en la concentración de sustrato para la lactato oxidasa, que a continuación se refleja en corriente aumentó en el electrodo sensor. Era necesario adicionalmente para medir estas variables, mientras que la manipulación de la excitabilidad de la corteza cerebral, a fin de aislar esta variable a partir de otras facetas de NREMS. Hemos ideado un sistema experimental para la medición simultánea de la actividad neuronal a través de la elecetroencephalogram, medición de flujo glucolítico a través de un biosensor de lactato, y la manipulación de la actividad cerebral neuronal cortical través de la activación de optogenético PyraMIDAL neuronas. Hemos utilizado este sistema para documentar la relación entre el sueño relacionado con formas de onda electroencefalográfica y la dinámica de momento a momento de la concentración de lactato en la corteza cerebral. El protocolo puede ser útil para cualquier individuo interesado en estudiar, comportarse libremente en roedores, la relación entre la actividad neuronal medida a nivel electroencefalográfico y la energética celular en el cerebro.

<html> Como se muestra en la Figura 2, un ratón que disponga para la estimulación optogenético y lactato / EEG / EMG colección de datos se sometieron espontáneas de sueño / vigilia transiciones de estado mientras EEG, EMG y la concentración de lactato cerebral fueron monitoreados continuamente. Actual en el sensor de lactato se incrementó durante los períodos de baja amplitud EEG y disminuyó durante los períodos de alta amplitud EEG. Como se muestra en la Figura 3,</...

Watch this Scientific Journal Video about Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral C

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Se describe un procedimiento para la manipulación de la actividad de las neuronas piramidales corticales cerebrales optogenetically mientras que el electroencefalograma, electromiograma, y ​​la concentración de lactato cerebral son monitoreados. Grabaciones experimentales se realizaron en ratones de cable-atados mientras se someten espontáneas de sueño / vigilia ciclos. Optogenético equipo es ensamblado en nuestro laboratorio; aparato de control está disponible comercialmente.

Electroencefalografía simultánea, en tiempo real Medición de la concentración de lactato y Manipulación optogenético de la actividad neuronal en la corteza cerebral de roedores

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

simultaneous-electroencephalography-real-time-measurement-lactate

Research

JoVE Journal

Neuroscience

20.1K vistas.  Washington State University. Se describe un procedimiento para la manipulación de la actividad de las neuronas piramidales corticales cerebrales optogenetically mientras que el electroencefalograma, electromiograma, y ​​la concentración de lactato cerebral son monitoreados. Grabaciones experimentales se realizaron en ratones de cable-atados mientras se someten espontáneas de sueño / vigilia ciclos. Optogenético equipo es ensamblado en nuestro laboratorio; aparato de control está disponible ...

Video: Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

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

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the first born neurons remain closer to the ventricle while the last born neurons migrate past the first born neurons towards the surface of the brain1. In addition to neuronal migration2, a key process for normal cortical function is the regulation of neuronal morphogenesis3. While neuronal morphogenesis can be studied in vitro in primary cultures, there is much to be learned from how these processes are regulated in tissue environments.
We describe techniques to analyze neuronal migration and/or morphogenesis in organotypic slices of the cerebral cortex4,6. A pSilencer modified vector is used which contains both a U6 promoter that drives the double stranded hairpin RNA and a separate expression cassette that encodes GFP protein driven by a CMV promoter7-9. Our approach allows for the rapid assessment of defects in neurite outgrowth upon specific knockdown of candidate genes and has been successfully used in a screen for regulators of neurite outgrowth8. Because only a subset of cells will express the RNAi constructs, the organotypic slices allow for a mosaic analysis of the potential phenotypes. Moreover, because this analysis is done in a near approximation of the in vivo environment, it provides a low cost and rapid alternative to the generation of transgenic or knockout animals for genes of unknown cortical function. Finally, in comparison with in vivo electroporation technology, the success of ex vivo electroporation experiments is not dependant upon proficient surgery skill development and can be performed with a shorter training time and skill.

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth.

Métodos de estudio de la morfogénesis neuronal:<em&gt; Ex vivo</em&gt; RNAi electroporación en la corteza cerebral embrionario murino

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

La visualización y la manipulación genética de las dendritas y espinas en la corteza cerebral y el hipocampo del ratón usando<em&gt; En el útero</em&gt; La electroporación

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Dos registros electrofisiológicos de sinápticamente respuestas evocadas neuronal y astroglial en agudas rodajas de hipocampo

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

La manipulación genética de las neuronas cerebelosa gránulo<em&gt; In Vitro</em&gt; Y<em&gt; En Vivo</em&gt; Estudiar Neuronal Morfología y Migración

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

La inducción de la plasticidad de astrocítico Receptores por manipulación de Neuronales cocción Precios

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

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 ...

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools to selectively manipulate brain rhythms. Here we describe an approach combining projection-specific optogenetics with extracellular electrophysiology for high-fidelity control of hippocampal theta oscillations (5-10 Hz) in behaving mice. The specificity of the optogenetic entrainment is achieved by targeting channelrhodopsin-2 (ChR2) to the GABAergic population of medial septal cells, crucially involved in the generation of hippocampal theta oscillations, and a local synchronized activation of a subset of inhibitory septal afferents in the hippocampus. The efficacy of the optogenetic rhythm control is verified by a simultaneous monitoring of the local field potential (LFP) across lamina of the CA1 area and/or of neuronal discharge. Using this readily implementable preparation we show efficacy of various optogenetic stimulation protocols for induction of theta oscillations and for the manipulation of their frequency and regularity. Finally, a combination of the theta rhythm control with projection-specific inhibition addresses the readout of particular aspects of the hippocampal synchronization by efferent regions.

We describe the use of optogenetics and electrophysiological recordings for selective manipulations of hippocampal theta oscillations (5-10 Hz) in behaving mice. The efficacy of the rhythm entrainment is monitored using local field potential. A combination of opto- and pharmacogenetic inhibition addresses the efferent readout of hippocampal synchronization.

Optogenetic de arrastre de las oscilaciones Theta Hippocampal en comportarse ratones

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools ...

Combined Transcranial Magnetic Stimulation and Electroencephalography of the Dorsolateral Prefrontal Cortex

Transcranial magnetic stimulation (TMS) is a non-invasive method that produces neural excitation in the cortex by means of brief, time-varying magnetic field pulses. The initiation of cortical activation or its modulation depends on the background activation of the neurons of the cortical region activated, the characteristics of the coil, its position and its orientation with respect to the head. TMS combined with simultaneous electrocephalography (EEG) and neuronavigation (nTMS-EEG) allows for the assessment of cortico-cortical excitability and connectivity in almost all cortical areas in a reproducible manner. This advance makes nTMS-EEG a powerful tool that can accurately assess brain dynamics and neurophysiology in test-retest paradigms that are required for clinical trials. Limitations of this method include artifacts that cover the initial brain reactivity to stimulation. Thus, the process of removing artifacts may also extract valuable information. Moreover, the optimal parameters for dorsolateral prefrontal (DLPFC) stimulation are not fully known and current protocols utilize variations from the motor cortex (M1) stimulation paradigms. However, evolving nTMS-EEG designs hope to address these issues. The protocol presented here introduces some standard practices for assessing neurophysiological functioning from stimulation to the DLPFC that can be applied in patients with treatment resistant psychiatric disorders that receive treatment such as transcranial direct current stimulation (tDCS), repetitive transcranial magnetic stimulation (rTMS), magnetic seizure therapy (MST) or electroconvulsive therapy (ECT).

The protocol presented here is for TMS-EEG studies utilizing intracortical excitability test-retest design paradigms. The intent of the protocol is to produce reliable and reproducible cortical excitability measures for assessing neurophysiological functioning related to therapeutic interventions in the treatment of neuropsychiatric diseases such as major depression.

Estímulo magnético de Transcranial combinado y electroencefalograma (EEG) de la corteza Prefrontal Dorsolateral

Transcranial magnetic stimulation (TMS) is a non-invasive method that produces neural excitation in the cortex by means of brief, time-varying magnetic ...

Simultaneous Cryosectioning of Multiple Rodent Brains

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In neuroscience, as well as a plethora of other scientific fields, these techniques are used. Immunohistochemistry can be done on slide mounted tissue or free-floating sections. Preparing slide-mounted samples is a time intensive process. The following protocol for a technique, called the Megabrain, reduced the time taken to cryosection and mount brain tissue by up to 90% by combining multiple brains into a single frozen block. Furthermore, this technique reduced variability seen between staining rounds, in a large histochemical study. The current technique has been optimized for using rodent brain tissue in downstream immunohistochemical analyses; however, it can be applied to different scientific fields that use cryosectioning.

Here, we present a protocol to freeze and section brain tissue from multiple animals as a timesaving alternative to processing single brains. This reduces staining variability during immunohistochemistry and reduces time cryosectioning and imaging.

Cryosectioning simultánea de varios cerebros de roedores

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In ...