<p class="jove_content"&gt; Agradecemos al Dr. Yuval Gottlieb-Dror para la ilustración de la electroporación. Este trabajo fue apoyado por becas a DSD de El Instituto Nacional de Psicobiología en Israel y en el programa de cooperación Niedersachsen-Israel Investigación y de subvenciones a AK de la Fundación Ciencias de Israel, el Ministerio de Salud de Israel, y el Centro del Patrimonio Excelencia-Legacy asociación Ciencias Biomédicas.</p&gt;

1. La electroporación Rombencéfalo 1.1 Manejo de Huevos <ol><li> Coloque los huevos horizontalmente en un incubador humidificado (37-38.5 ° C). Los embriones son electroporación después de 65-70 horas de incubación, cuando alcanzan 16-17 (HH) etapa (25-30 somitas). </li><li> Eliminar los huevos de la incubadora, sino que permanecen en la posición horizontal. </li></ol> 1.2 Preparativos <ol><li> Tire de capilares de vidrio (0,5 mm de diámetro). </li><li> Conectar dobladas en forma de L de oro Genetrodes electrodos (3 mm de diámetro) con un soporte del adaptador para el generador de impulsos. Electroporación parámetros incluyen 25 voltios, 5 números de impulsos, 45 ms de longitud de pulso, los intervalos de 300 ms de pulso. </li><li> Preparar una pipeta de boca, 10 ml de aguja de la jeringa, tiras de parafilm, un cepillo suave, unas tijeras de disección afilados y 25 ml de solución de PBS estéril. Esta cantidad es por lo general suficiente para manejar una bandeja de huevos (18 huevos). </li><li> Preparar una mezcla adecuada de los plásmidos de ADN (5 g / l cada uno) yañadir alrededor de 0,025% de colorante verde rápido para visualizar la inyección de ADN. Por lo general, un volumen de 4 l es suficiente para la inyección de una bandeja. </li></ol> 1.3 de ventanas, de inyección y electroporación <ol><li> Limpie las cáscaras de huevo con etanol al 70%. </li><li> Manejar un huevo a la vez para minimizar el tiempo de exposición al aire embrionaria. </li><li> Hacer un agujero en el polo de huevo con los extremos tijeras y retire 3-4 ml de albúmina con la jeringa. No extraiga la mayor cantidad de albúmina, ya que reduce la viabilidad. </li><li> Abra una ventana oval pequeño (2,5 x 2 cm de grosor) en el centro superior de la cáscara de huevo con las tijeras de disección. </li><li> Por goteo unas gotas de PBS en la parte superior del embrión para evitar la sequedad durante todo el proceso. </li><li> Cargar una pequeña cantidad de la mezcla de ADN en el capilar de vidrio usando una pipeta de boca. </li><li> Coloque el embrión con su extremo posterior hacia usted. No se requiere la inyección de tinta en esta etapa el embrión es claramente visible. Evitar el aumento de inyección de tintasupervivencia embrionaria. Penetrar en el cerebro posterior caudal mediante la celebración de los capilares en el ~ 45 °. No retire cualquier membrana de todo el embrión. Inyectar solución de ADN con cuidado en el lumen cerebro posterior y exhalar sin que se formen burbujas de aire. Solución de ADN se extiende hacia delante. </li><li> Inmediatamente después de la inyección de ADN, coloque los electrodos paralelos a la AP precisa y DV posición rombencéfalo tu objetivo es orientar la campaña. Empuje los electrodos ligeramente ventralmente sin tocar el corazón, ya que puede reducir la viabilidad. Pulso del electroporador. Los electrodos deben estar cubiertos con burbujas para indicar la conductividad eléctrica (Figura 1A). </li><li> Retire con cuidado los electrodos y gotear unas gotas de PBS para enfriar el embrión y para eliminar las burbujas. Selle el orificio correctamente con una tira de parafina para evitar que se sequen durante la incubación de los huevos. Lave los electrodos en PBS con un cepillo suave. </li><li> Coloque huevo en la incubadora durante el período de tiempo deseado. La supervivencia de los embriones es de aproximadamente 90% después de 2-3 díaselectroporación, y reduce hasta el 50% 12 días después de la electroporación. </li></ol> 2. Análisis de embriones 2.1 La elaboración del plano de montaje e inmunofluorescencia <ol><li> Preparaciones plana de montaje de la parte posterior del cerebro se pueden realizar hasta E6.5 para visualizar fácilmente los axones bajo un microscopio o un estereomicroscopio. </li><li> Diseccionar el embrión cuidadosamente por lo separa de membranas que rodean y los vasos sanguíneos. Coloque el embrión en una pequeña silicona - placa de Petri recubierto que contiene PBS. Conecte el embrión al plato con alfileres de tungsteno enfrenta dorsal. Realice una hendidura dorsal en el cerebro posterior en el techo placa con fuerte capilares de vidrio. </li><li> Con ayuda de pinzas cortantes y micro-tijeras sujetar la parte superior de la cabeza y tirar del tejido de cerebro posterior muy cuidadosamente de rostral a caudal. Después de la separación de la parte posterior del cerebro eliminar los tejidos ventrales restantes para alcanzar una preparación limpia. </li><li> Incubar el cerebro posterior con 4% De solución de paraformaldehído (PFA) para 1-2 hr a temperatura ambiente (RT). Lavar el cerebro posterior con PBT (PBS/0.1% de Triton X-100) para eliminar PFA. </li><li> Por inmunofluorescencia en hindbrains-montados plana añadir 500 l de solución de bloqueo (5% de suero de cabra en PBT y se incuba durante 2 horas a RT Incubar con 1 pt de anticuerpo ON a 4 ° C. Lavar con PBT (15 min x 3). Incubar con 2 ª anticuerpo durante 2 horas a TA. Lavar con PBT (15 min x 3). </li><li> Coloque el cerebro posterior frente dorsalmente en un portaobjetos de vidrio. Añadir medio de montaje fluorescentes en la parte posterior del cerebro. Usa capilares de vidrio o agujas de tungsteno para aplanar la parte posterior del cerebro en la diapositiva. </li><li> Añadir pequeña cantidad de grasa en cada esquina para evitar exprimir del tejido por la hoja de la cubierta. Con cuidado, coloque una hoja de cubierta en la parte superior de la parte posterior del cerebro y evitar burbujas de aire. Para añadir la adhesión esmalte de uñas para cubrir los bordes del vidrio. </li></ol> 2.2 Cryo-secciones e inmunofluorescencia <ol><li> Cryo-secciones del tronco cerebral can llevarse a cabo en cualquier etapa de visualizar las trayectorias axonales. Sin embargo, después de E6.5 este debe ser el método preferido debido a la dificultad para visualizar los axones en el embrión de montaje plana, que se convierte en demasiado grueso. Por otra parte, la demostración de las sinapsis requiere la sección del tejido y una vista de gran aumento bajo un microscopio. </li><li> Disección del embrión y eliminar todos los tejidos circundantes. Lavar con PBS. Cortar el embrión en la parte caudal de la médula mediante micro-tijeras y pinzas cortantes. Hacer una amplia incisión entre el cerebelo y el cerebro medio y retirar todo el tronco cerebral. El tejido debe contener el bulbo raquídeo, la protuberancia y el cerebelo. </li><li> Fijar el tronco cerebral en PFA al 4% durante toda la noche (ON) a 4 ° C, enjuague con PBS (3 x 10 min), y se incuba con 30% de sacarosa para ON a 4 ° C hasta que se hunde. </li><li> Insertar el cerebro posterior en un molde con la crio-compuesto OCT (temperatura óptima de corte), y colocarlo a -20 ° C hasta su congelación, tal como se describe en otro lugar 11. </li><li&gt; Coloque el bloque de octubre en el criostato en el eje transversal deseada. Ancho de sección es de 12 micras. </li><li> Secar los portaobjetos. Añadir 100 l de solución de bloqueo en cada portaobjetos durante 2 h a TA. Añadir 100 l de anticuerpos (diluido en la solución de bloqueo a la concentración deseada). El período de incubación de 1 ª y 2 ª anticuerpos está encendido a 4 ° C o 2 horas a temperatura ambiente, respectivamente. Para cada tiempo de incubación, añadir con cuidado tira de parafina en la parte superior de la corredera para evitar la sequedad. Lavar con PBS (10 min x 3) entre los anticuerpos. Si es necesario, añadir 1:1000 DAPI (4&#39;-6-diamidino-2-fenilindol), diluido en PBS durante 20 min a TA. Lavar con PBS. </li><li> Coloque el portaobjetos con medio de montaje fluorescentes. Deje que se seque durante 1 hora antes de su análisis. </li></ol>

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<p class="jove_content"&gt; No hay conflictos de interés declarado.</p

En electroporación in ovo es una herramienta viable, fiable y eficaz para examinar especificación de las células y la guía axonal durante el desarrollo del sistema nervioso polluelo 20. En este protocolo se describe un modo de electroporación en el cerebro posterior chica en E2.75 utilizando elementos potenciadores que permiten el etiquetado condicional de interneuronas específicos. Esta estrategia se combina con el sistema de transposición mediada por PiggyBac para insertar genes ext...

El cerebro posterior representa un concentrador relé clave del sistema nervioso mediante la comunicación entre los sistemas nerviosos central y periférico por medio de ascendente y descendente redes neuronales. Regula las funciones básicas como la respiración, la conciencia, el oído y la coordinación motora 1-3. Durante el desarrollo embrionario temprano, la parte posterior del cerebro de vertebrados se subdivide transitoriamente a lo largo de su eje antero-posterior (AP) en rhombomeres repetitivas, en las que se forman distintos tipos de células neuronales y generan múltiples centros de los núcleos del tronco cerebral 4. El cerebro posterior también se divide a lo largo de su eje dorsal-ventral (DV) en una placa basal y alar, en la que los progenitores neuronales discretas se especifican y se diferencian en lugares distintos DV 3,5,6. Como a principios del AP y DV-específicos patrones neuronales se rige el establecimiento de circuitería tronco cerebral funcional es en gran parte desconocida. Para adquirir conocimientos sobre este fundamentalcuestión, se requieren herramientas para etiquetar subconjuntos específicos de neuronas en el cerebro posterior y principios para trazar su trayectoria axonal y la conectividad en las etapas más avanzadas. Hemos utilizado previamente elementos específicos potenciador, y un sistema de expresión condicional Cre / loxP basada para el seguimiento de la trayectoria axonal de interneuronas espinales dorsales en el embrión de pollo temprano 7-9. En el manuscrito actual se ha centrado en el cerebro posterior y mejorado el paradigma experimental para etiquetar finales interneuronas rombencéfalo embrionarias, axones y sus objetivos sinápticas, mediante una estrategia de electroporación modificado y el PiggyBac - Adaptación de ADN mediada. Nuestra nueva estrategia permite que el etiquetado de los subtipos neuronales distintas en un lado del cerebro posterior y el seguimiento de sus proyecciones axonales y sitios sinápticos en las diversas etapas embrionarias, desde 2 hasta 12 días después de la electroporación. En base a este método, se calificó el dorsal más subgrupo de interneuronas del cerebro posterior (dA1/Atoh1 + </sa&gt; las células) y reveló dos patrones de proyección axonal ascendentes contralaterales, cada uno deriva de una ubicación AP diferente y se alarga en un funiculus distinta. DA1 axones se encontraron para formar sinapsis en los núcleos auditivos, cerebro medio y en múltiples capas del cerebelo 10 proyecto. La combinación de polluelo de la electroporación, el rastreo genético de las neuronas y el análisis de los sitios de proyección en una fase mucho más avanzadas de desarrollo proporciona una plataforma única para estudiar la formación de redes neuronales en el cerebro y para dilucidar los mecanismos moleculares que gobiernan la formación de circuito.

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Reagent/Equipment</td>
			<td>Company</td>
			<td>Catalogue Number</td>
		 </tr>
		 <tr>
			<td>L-shaped gold Genetrodes 3 mm electrodes</td>
			<td>BTX, Harvard Apparatus</td>
			<td>45-0162 </td>
		 </tr>
		 <tr>
			<td>pulse generator, ECM 830</td>
			<td>BTX, Harvard Apparatus</td>
			<td>45-0002</td>
		 </tr>
		 <tr>
			<td>OCT (Optimal Cutting Temperature) Compound</td>
			<td>Tissue-Tek
				Sakura</td>
			<td>4583 O.C.T. Compound </td>
		 </tr>
		 <tr>
			<td>Nail Polish</td>
			<td>From Any Commercial Supplier </td>
			<td></td>
		 </tr>
		</tbody>
 </table>

electroporation of the hindbrain to trace axonal trajectories and synaptic targets in the chick embryo

La electroporación del tubo neural de embriones de pollo tiene muchas ventajas tales como ser rápida y eficiente para la expresión de genes extraños en células neuronales. En este manuscrito se proporciona un método que demuestra cómo única para electroporar ADN en el cerebro posterior aviar en E2.75 con el fin de etiquetar específicamente un subconjunto de células progenitoras neuronales, y cómo seguir sus proyecciones axonales y metas sinápticas en una fase mucho más avanzada de desarrollo, hasta E14.5. Hemos utilizado herramientas genéticas nuevas que incluyen elementos específicos potenciadoras, Cre / Lox - plásmidos basados ​​y el sistema de transposición de ADN PiggyBac mediada para dirigir la expresión de GFP en un subtipo de células del cerebro posterior (dorsal más subgrupo de interneuronas, DA1). Trayectorias y objetivos de DA1 axonal axones se siguen en las etapas embrionarias tempranas y tardías en varias regiones del tronco cerebral. Esta estrategia contribuye con técnicas avanzadas para atacar a las células de interés en la parte posterior del cerebro embrionario y de tracing formación circuito en múltiples etapas de desarrollo.

Este protocolo fue utilizado recientemente para descubrir los patrones axonal y sitios de proyección de DA1 subgrupo de interneuronas en el cerebro posterior chica 10. Para etiquetar específicamente estos axones, se confirmó un elemento potenciador (Atoh1), que anteriormente ha sido caracterizado como específico para las neuronas espinales ED1 8,12,13, que se expresa en las células del cerebro posterior 10 DA1. El elemento fue clonado aguas arriba de la recombinasa Cre y co-electrop...

Watch this Scientific Journal Video about Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo at JoVE.com

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

¿Cómo las redes neuronales se establecen en el cerebro embrionario es una cuestión fundamental en la neurobiología del desarrollo. Aquí hemos combinado una técnica de electroporación con herramientas genéticas novedosas, tales como Cre / Lox-plásmidos y el sistema de transposición de ADN PiggyBac mediada por el cerebro posterior aviar para etiquetar interneuronas dorsal y seguimiento de sus proyecciones axonales y metas sinápticas en las diversas etapas de desarrollo.

La electroporación del Rombencéfalo trazar trayectorias axonal y metas sinápticas en el embrión de pollo

Electroporation of  the chick embryonic neural tube has many advantages such as being quick and efficient  for the expression of foreign genes into ...

electroporation-hindbrain-to-trace-axonal-trajectories-synaptic

Research

JoVE Journal

Neuroscience

10.8K vistas.  The Hebrew University of Jerusalem. ¿Cómo las redes neuronales se establecen en el cerebro embrionario es una cuestión fundamental en la neurobiología del desarrollo. Aquí hemos combinado una técnica de electroporación con herramientas genéticas novedosas, tales como Cre / Lox-plásmidos y el sistema de transposición de ADN PiggyBac mediada por el cerebro posterior aviar para etiquetar interneuronas dorsal y seguimiento de sus proyecciones axonales y metas sinápticas en las diversas etapas de desa...

Video: Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Labeling and Imaging Cells in the Zebrafish Hindbrain

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish embryos, injection of plasmid DNA results in mosaic expression, allowing for the visualization of single cells or small clusters of cells 1 . We describe how injection of plasmid DNA encoding membrane-targeted Green Fluorescent Protein (mGFP) under the control of a ubiquitous promoter can be used for imaging cells undergoing neurulation. Central to this protocol is the methodology for imaging labeled cells at high resolution in sections and also in real time. This protocol entails the injection of mGFP DNA into young zebrafish embryos. Embryos are then processed for vibratome sectioning, antibody labeling and imaging with a confocal microscope. Alternatively, live embryos expressing mGFP can be imaged using time-lapse confocal microscopy. We have previously used this straightforward approach to analyze the cellular behaviors that drive neural tube formation in the hindbrain region of zebrafish embryos 2. The fixed preparations allowed for unprecedented visualization of cell shapes and organization in the neural tube while live imaging complemented this approach enabling a better understanding of the cellular dynamics that take place during neurulation.

Key to understanding the morphogenetic processes that shape the early embryo is the ability to image cells at high resolution. We describe here a technique for labeling single cells or small clusters of cells in whole zebrafish embryos with membrane-targeted Green Fluorescent Protein.

Etiquetado y las Pilas de imágenes en la parte posterior del cerebro de pez cebra

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish ...

Investigación

Neurociencias

Production of Chick Embryo Extract for the Cultivation of Murine Neural Crest Stem Cells

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent progenitor cells to be an integral part of the neural crest1. Phenotypically, neural crest stem cells (NCSC) are defined by simultaneously expressing p75 (low-affine nerve growth factor receptor, LNGFR) and SOX10 during their migration from the neural crest2,3,4,5. These progenitor cells can differentiate into smooth muscle cells, chromaffin cells, neurons and glial cells, as well as melanocytes, cartilage and bone6,7,8,9. To cultivate NCSC in vitro, a special neural crest stem cell medium (NCSCM) is required10. The most complex part of the NCSCM is the preparation of chick embryo extract (CEE) representing an essential source of growth factors for the NCSC as well as for other types of neural explants. Other NCSCM ingredients beside CEE are commercially available. Producing CCE using laboratory standard equipment it is of high importance to know about the challenging details as the isolation, maceration, centrifugation, and filtration processes. In this protocol we describe accurate techniques to produce a maximized amount of pure and high quality CEE.

To cultivate neural crest stem cells (NCSC) in vitro, a special medium (NCSCM) is required. Essential part of NCSCM is chick embryo extract (CEE). We here describe accurate techniques to produce a maximized amount of pure and high quality CEE, including details as the isolation, maceration, centrifugation, and filtration processes.

La producción de extracto de embrión de pollo para el Cultivo de murino crestas de células neurales madre

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

Fast Micro-iontoforesis de glutamato y GABA: una herramienta útil para investigar la integración sináptica

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation of the guidance receptors is the first step of the signaling mechanisms allowing axon tips, the growth cones, to respond to the ligands. As such, the modulation of their availability at the cell surface is one of the mechanisms that participate in setting the growth cone sensitivity. We describe here a method to precisely visualize the spatio-temporal cell surface dynamics of an axon guidance receptor both in vitro and in vivo in the developing chick spinal cord. We took advantage of the pH-dependent fluorescence property of a green fluorescent protein (GFP) variant to specifically detect the fraction of the axon guidance receptor that is addressed to the plasma membrane. We first describe the in vitro validation of such pH-dependent constructs and we further detail their use in vivo, in the chick spinal chord, to assess the spatio-temporal dynamics of the axon guidance receptor of interest.

We describe here the use of a pH-sensitive green fluorescent protein variant, pHluorin, to study the spatio-temporal dynamics of axon guidance receptors trafficking at the cell surface.&#160;The pHluorin-tagged receptor is expressed both in cell culture and in vivo, using electroporation of the chick embryo.

Uso De pHluorin Para Evaluar La Dinámica De Los Receptores De Orientación Axón En El Cultivo Celular Y En El Embrión De Polluelo

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

El sistema olfativo como un modelo para estudiar los patrones de crecimiento axonal y Morfología<em&gt; En Vivo</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

Simultánea de dos fotones<em&gt; En Vivo</em&gt; Imágenes de entradas sinápticas y Objetivos postsinápticos en la corteza del ratón retroesplenial

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.

Trace eyeblink classical conditioning (ECC) was used to assess hippocampal-dependent associative learning in adult rats that were administered a high concentration (11.9% v/v) of alcohol during early neonatal brain development. In general, ECC procedures are sound diagnostic tools for detecting brain dysfunction across many psychological and biomedical settings.

El uso de condicionamiento de clásico de Eyeblink rastro para evaluar disfunción hipocampal en un modelo de rata de trastornos del espectro alcohólico Fetal

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

Grabación de la plasticidad sináptica en rebanadas Hippocampal agudas mantenidas en un pequeño volumen reciclado - perfusión- y sistema de cámara de tipo inmersión

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Use of Synaptic Zinc Histochemistry to Reveal Different Regions and Laminae in the Developing and Adult Brain

Characterization of anatomical and functional brain organization and development requires accurate identification of distinct neural circuits and regions in the immature and adult brain. Here we describe a zinc histochemical staining procedure that reveals differences in staining patterns among different layers and brain regions. Others have utilized this procedure not only to reveal the distribution of zinc-containing neurons and circuits in the brain, but also to successfully delineate areal and laminar boundaries in the developing and adult brain in several species. Here we illustrate this staining procedure with images from developing and adult ferret brains. We reveal a zinc-staining pattern that serves as an anatomical marker of areas and layers, and can be reliably used to distinguish visual cortical areas in the developing and adult visual cortex. The main goal of this protocol is to present a histochemical method that allows the accurate identification of layers and regions in the developing and adult brain where other methods fail to do so. Secondarily, in conjunction with densitometric image analysis, this method allows one to assess the distribution of synaptic zinc to reveal potential changes throughout development. This protocol describes in detail the reagents, tools, and steps necessary to successively stain frozen brain sections. Although this protocol is described using ferret brain tissue, it can easily be adapted for use in rodents, cats, or monkeys as well as in other brain regions.

We describe a histochemical procedure that reveals characteristic laminar and areal zinc staining patterns in different brain regions. The zinc-staining pattern may be used in conjunction with other anatomical markers to reliably distinguish layers and regions in the developing and adult brain.

Uso de Zinc sináptica histoquímica para revelar las diferentes regiones y láminas en el cerebro en desarrollo y adulto

Characterization of anatomical and functional brain organization and development requires accurate identification of distinct neural circuits and regions ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

Combinando Optogenetics con microRNAs artificiales para caracterizar los efectos de Gene caída en la función presináptica en circuitos neuronales intactas

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...