Nos gustaría dar las gracias a todos los miembros de nuestro laboratorio para sus consejos, las discusiones y comentarios útiles sobre el manuscrito. También nos gusta especialmente dar las gracias a Marta Koch y Hassan Bassem para la excelente sugerencia que esperar hasta 1,5 horas antes de la disección de los cerebros de la APF para el cultivo en los experimentos de remodelación. Agradecemos también a la instalación de imágenes de NHGRI para el uso de su microscopio confocal. Este trabajo fue apoyado por el Programa de Neurociencia Básica del Programa de Investigación Intramural del NINDS, NIH (Z01 NS003106).

A. Preparación de los medios <ol><li> Preparar medios completos por filtración esterilizante Drosophila Schneider medio suplementado con suero fetal bovino al 10% y 1% de penicilina / estreptomicina. </li><li> Medios de comunicación deben estar preparados frescos cada vez. Alternativamente, una parte alícuota de la pre-congelados medios de comunicación pueden ser descongelado antes de cada uso. </li><li> Antes de cultivo, añadir 10 g / ml de insulina y 2 g / ml ecdisona a los medios. Los fármacos se preparó como poblaciones concentradas y se congelaron en alícuotas pequeñas. Alícuotas descongeladas no volver a congelar. </li><li> Todas las soluciones de trabajo debe mantenerse a 25 ° C. </li></ol> El cultivo de los cerebros de larvas 1. La selección y la disección de los cerebros de larvas <ol><li> Añadir 500 l de medios de comunicación para una inserción de 12 mm de la cultura Millicell celular. Los cerebros disecados se coloca en estos insertos para facilitar inmunotinción fácil. </li><li> Añadir una pequeña cantidad de medios preparados para un vidrio de reloj disección. </li><li> Ucantar una pequeña espátula, recoger las larvas de tercer estadio vagando y colocarlos en el vidrio de reloj. (Suponemos que el método in vivo la cultura franco se aplicará igualmente a las larvas primero o segundo, pero no he probado esto.) </li><li> Manteniendo el extremo posterior de la larva con un par de pinzas, cuidadosamente abrir el extremo anterior con un segundo par de pinzas # 5. </li><li> Rápidamente diseccionar el cerebro, manteniendo el cordón nervioso ventral intacto. </li></ol> 2. Ex cultivo in vivo de los cerebros disecados larvarias <ol><li> Uso de pre-húmedas puntas de pipeta, transferir el cerebro con cuidado en los insertos de cultivo celular, pre-llenado con los medios de comunicación. </li><li> Cerebros de transferencia, en los medios, a una incubadora a 25 ° C hasta el deseado punto de tiempo que se alcanza. Este método funciona de manera eficiente para un máximo de 48 horas en la cultura. </li><li> Vuelva a colocar los medios de comunicación a las 8 horas. Más allá de eso, reemplazar la mitad de los medios de comunicación cada 5-6 horas. </li></ol> 3. La manipulación farmacológica deLos cerebros de larvas Este método de cultivo ex vivo puede ser utilizado para corroborar farmacológicamente interacciones genéticas demostrado previamente en nuestro laboratorio 1. <ol><li> Añadir 100 roscovitina mM y 100 mM olomoucina a los medios preparados. </li><li> Transferencia de cerebros disecados a este medio. </li><li> Los cerebros de Cultura a 25 ° C durante 24 horas. </li><li> Vuelva a colocar los medios de comunicación a las 8 horas. Más allá de eso, reemplazar la mitad de los medios de comunicación (con medicamentos) cada 5-6 horas. </li></ol> El cultivo de los cerebros de pupas 1. Selección de las pupas de Disección <ol><li> Marcar pupas blancas en las botellas y frascos. </li><li> Sólo elija pupas que son completamente blancos. Las pupas con cualquier tipo de color no deben ser incluidos. </li><li> Esta etapa es de 0 horas después de la formación Pupario (APF). </li><li> Estas pupas estará listo para la disección de 1,5 horas después de marcar. Este paso es crucial para garantizar que la poda de desarrollo se produce en ex vivo </em&gt; cultura. </li></ol> 2. La disección de las pupas <ol><li> Añadir 500 l de medios de comunicación para una inserción de 12 mm de la cultura Millicell celular. (Los cerebros disecados se coloca en estos insertos para facilitar inmunotinción fácil). </li><li> Una vez que el deseado punto de tiempo se ha alcanzado, añadir una pequeña cantidad de medios preparados para un vidrio de reloj disección. </li><li> Usando una espátula pequeña, húmeda, recoger las pupas previamente marcada para la disección y colocarlos en el vidrio de reloj. </li><li> Manteniendo el extremo posterior de las pupas con un par de pinzas, abra cuidadosamente el extremo anterior del caso pupa con un segundo par de pinzas # 5. </li><li> Rápidamente diseccionar el cerebro, manteniendo el cordón nervioso ventral intacto y unido. </li></ol> 3. Ex cultivo in vivo de los cerebros disecados pupas <ol><li> Transferir el cerebro con cuidado en los insertos de cultivo celular, pre-llenado con los medios de comunicación. </li><li> Cerebros de transferencia, en los medios, a una incubadora a 25 ° C hasta el deseado tiempo point se alcanza. Se utilizó este método para hasta 10 horas en cultivo para el experimento se muestra, pero puede extenderse a más largos períodos de observación y tratamiento, si es necesario. </li><li> Para los puntos de tiempo de más de 8 horas APF, sustituir los medios de comunicación cada 5-6 horas. </li></ol> Partir de este paso, el protocolo se mantiene sin cambios, tanto para las larvas y pupas. B. Fijación de cerebros disecados <ol><li> Una vez que el deseado punto de tiempo se alcanza, separa el medio y añadir 500 l de formaldehído al 4% en PBS 1x con 0,5% de Triton X-100 (PBST) durante 30 minutos, reemplazando con formaldehído fresco después de 15 minutos. </li><li> Asegúrese de no permitir que se seque el cerebro al reemplazar los medios de comunicación con el fijador. </li><li> Lavar formaldehído con cuatro lavados de 10 minutos de 1x PBS con 0,5% TritonX-100 (PBST). </li></ol> C. La tinción cerebros fijos <ol><li> Después de formaldehído ha sido lavada, bloquear cerebros durante 1 hora en una solución de bloqueo de 1x PBS con un 1% normalSuero de cabra y 1% de seroalbúmina bovina. </li><li> Añadir anticuerpos primarios formados en solución de bloqueo. </li><li> Los cerebros de incubar durante la noche a 4 ° C. </li><li> A la mañana siguiente, lave los anticuerpos primarios con cuatro lavados de 10 minutos de PBST. </li><li> Añadir secundaria de anticuerpos formados en solución de bloqueo. </li><li> Dejar a temperatura ambiente durante 4 horas. </li><li> Lavar la zona secundaria de anticuerpos con cuatro lavados de 10 minutos de PBST. </li></ol> D. Montaje de cerebros fijados y teñidos de Microscopía <ol><li> Equilibre los cerebros de los medios de comunicación Vectashield montaje durante 1 hora. </li><li> Monte cerebro en portaobjetos de vidrio que utilizan los medios de comunicación Vectashield montaje. </li><li> Coloque los chips de vidrio a cada lado de los cerebros antes de montar el cubreobjetos. Esto evita que los cerebros de ser aplastado, mientras se mantiene la muestra lo suficientemente delgada como para analizar todos los planos focales con un microscopio confocal. Los cerebros fueron más o menos orientada al montaje para facilitar su lectura las características de interés. Cuando sea necesario,imágenes del cerebro fueron rotados con el software de Imaris para proporcionar una visión óptima de la documentación y la medición. </li><li> Selle cubreobjetos con esmalte de uñas transparente. </li><li> Tienda desliza lejos de la luz. </li></ol> E. Representante Resultados La Figura 1 muestra un panel de tercer estadio larval de cerebros (A) de tipo salvaje moscas y (B) las moscas que expresan un derivado dominante negativo de Cdk5 (Cdk5DN) (Connell-Crowley et al., 2000). Ambos expresan la actina-GFP (verde) en las neuronas gamma MB. Como se ha descrito previamente (Trunova et al., 2011), hay una región en los axones proximales de las neuronas gamma de tipo salvaje que falla para acumular actina-GFP, mostrando así como una &quot;zona clara&quot; (A, soporte), y fasciclin dos proteínas (rojo) se acumula en los axones sólo hasta el borde ventral de la zona (línea horizontal blanca). La línea de puntos en el tercer panel de la Figura 1 (A) indica la posición de la MB, destacando el actoen la &quot;zona libre&quot; y su borde ventral. En las moscas que expresan Cdk5DN, la zona de actina claro está ausente (B) y fasciclin 2 (FasII) inmunoreactividad extiende dorsalmente a través de esa región. (Tenga en cuenta que un número variable de FasII positivo del proyecto axones horizontal a través de la MB, estos no son relevantes para el fenotipo). Barra de escala representa a 20 micras. Utilizando el método de cultivo ex vivo, que tratan de tipo salvaje moscas con una combinación de roscovitina y olomoucina, inhibidores farmacológicos de Cdk5 actividad. Figura 2 muestra un panel de tercer estadio larval cerebros de control (A) y tratados con el fármaco (B ) de tipo salvaje moscas. Al igual que las moscas que expresan una dominante negativa Cdk5 construcción, los cerebros tratados con inhibidores de la Cdk5 durante 24 horas, muestran la pérdida de característica de la zona de actina clara y cambio en los límites FasII (entre corchetes). Las líneas blancas muestran la posición de tipo salvaje frontera FasII. Barra de escala representa a 20 micras. FiguRe 3 muestra una serie representativa de los organismos de hongos Drosophila de 0-10 horas de la APF, marcado con la membrana objetivo mCD8-GFP (verde). Los paréntesis indican el cáliz, es decir, las proyecciones dendríticas de la MB. Antes de la metamorfosis, se puede ver axones MB cursan dorsoventral en haces gruesos, así como una &quot;neblina &#39;de señal fluorescente resultante de la dendrítica denso eje (soporte). Panel A muestra cerebros disecados en el momento indicado. Tenga en cuenta la poda de las dendritas en la región indicada por los soportes, de tal manera que por 6-8 hrs APF, la bruma de la señal dendríticas se ha ido (aunque la señal no se ve afectada axonal). Cerebros en el panel B fueron disecados a las 0 horas APF, antes de que el alza interna de la ecdisona (Truman y Riddiford, 2002), y cultivadas ex vivo como se indica. Estos no muestran la poda en el desarrollo de las dendritas, sino la señal dendríticas persiste durante 10 horas APF. Para evitar esto, las pupas se marcan a 0 APF horas, pero disecado de 1,5 horas APF y culta. Panell C muestra una serie representativa de estos cuerpos seta de Drosophila APF 0-10 horas. Al igual que en las muestras no cultivadas, la señal dendrítica es indetectable por 8 horas APF. Barra de escala representa a 20 micras.

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No hay conflictos de interés declarado.

<html> Crónicas manipulaciones genéticas de la estructura del tejido y la función, incluyendo las mutaciones y la expresión de los transgenes, por lo general producen una combinación de efectos inmediatos y finales de las consecuencias indirectas que pueden ser muy difíciles de separar. Como complemento de los métodos genéticos con la farmacología puede facilitar el interrogatorio de la evolución en el tiempo detrás de un fenotipo. Por ejemplo, este ha sido un enfoque de gran alcance para la disección de las ...

<table border="1"><tbody><tr><td> Nombre de Reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> La concentración final </td></tr><tr><td> Schneider Drosophila medios de comunicación </td><td> Invitrogen </td><td> 21720-024 </td><td></td></tr><tr><td> Suero bovino fetal (inactivado por calor) </td><td> Invitrogen </td><td> 10082-147 </td><td> 10% </td></tr><tr><td> La penicilina / estreptomicina </td><td> Invitrogen </td><td> 15140-122 </td><td> 1% </td></tr><tr><td> Insulina </td><td> Invitrogen </td><td> 12585-014 </td><td> 10μg/ml </td></tr><tr><td> Ecdisona </td><td> Sigma-Aldrich </td><td> H5142 </td><td> 2μg/ml </td></tr><tr><td> Roscovitina </td><td> Sigma-Aldrich </td><td> R7772 </td><td> 100 micras </td></tr><tr><td> Olomoucina </td><td> Sigma-Aldrich </td><td> O0886 </td><td> 100 micras </td></tr><tr><td> Suero normal de cabra </td><td> Invitrogen </td><td> 50062Z </td><td> 1% </td></tr><tr><td> Albúmina de suero bovino </td><td> Fisher Scientific </td><td> M9501 </td><td> 1% </td></tr><tr><td> Formaldehído </td><td> Sigma-Aldrich </td><td> D4551 </td><td> 4% </td></tr><tr><td> Triton X-100 </td><td> Fisher Scientific </td><td> BP151 </td><td> 0,5% </td></tr><tr><td> Tampón fosfato salino </td><td> Invitrogen </td><td> 70011-044 </td><td> 1X </td></tr><tr><td> Vectashield </td><td> Vector Laboratories </td><td> H1000 </td&gt; <td></td></tr><tr><td> Millicell Inserta cultivo celular </td><td> Millipore </td><td> PI8P01250 </td><td></td></tr><tr><td> Multidish, 4 y </td><td> Thermo Scientific </td><td> 176740 </td><td></td></tr><tr><td> Dumont # 5 Pinzas </td><td> Herramientas de Bellas Ciencia </td><td> 11251-20 </td><td></td></tr><tr><td> Preparaciones para microscopio </td><td> Fisher Scientific </td><td> 12-544-7 </td><td></td></tr><tr><td> Cierre de vidrio </td><td> Fisher Scientific </td><td> 12-548-5M </td><td></td></tr></tbody></table>

ex vivo culturing of whole, developing drosophila brains

Se describe un método para el cultivo ex vivo de cerebros completos de Drosophila. Esto puede ser usado como un contrapunto de crónicas manipulaciones genéticas para investigar la biología celular y el desarrollo de estructuras cerebrales centrales al permitir agudas intervenciones farmacológicas y formación de imágenes en vivo de procesos celulares. Como un ejemplo de la técnica, antes de trabajar de nuestro laboratorio 1 ha demostrado que un compartimiento subcelular no reconocido previamente se encuentra entre los compartimentos axonales y somatodendrítica de los axones del cerebro de Drosophila central. El desarrollo de este compartimiento, referido como el segmento inicial del axón (AIS) 2, se muestra genéticamente a depender de la quinasa neuronal específica dependiente de ciclina, Cdk5. Mostramos aquí que el tratamiento ex vivo de tipo salvaje cerebro de Drosophila larvas con la roscovitina Cdk5 específico de los inhibidores farmacológicos y olomoucina 3 se producen cambios agudos en la organización de la actina, yen la localización de la proteína de la superficie celular fasciclin 2, que imitan los cambios observados en los mutantes que carecen de actividad de Cdk5 genéticamente. Un segundo ejemplo de la técnica in vivo la cultura ex está prevista la remodelación de las conexiones del cuerpo de setas embrionario (MB), las neuronas gamma durante la metamorfosis de larva a adulto. El cuerpo de hongo es el centro de aprendizaje y la memoria olfativa en la mosca de 4, y estas neuronas gamma podar sus ramas dendrítica y axonal durante el desarrollo de la pupa y luego volver a extender las ramas en un punto de tiempo después para establecer el patrón de inervación de adultos 5. La poda de estas neuronas de la MB se ha demostrado que se producen a través de la degeneración local de ramas neuritas 6, por un mecanismo que se activa por la ecdisona, una hormona esteroide, que actúa en el receptor de ecdisona B1 7, y que es dependiente de la actividad del ubiquitina-proteasoma sistema 6. Nuestro método de cultivo ex vivo puede be utilizan para interrogar mejor el mecanismo de la remodelación de desarrollo. Se encontró que en la configuración de la cultura vivo ex, las neuronas gamma de la MB recapituló el proceso de la poda de desarrollo con un tiempo similar al que en vivo. Es esencial, sin embargo, que esperar hasta 1,5 horas después de la formación pupario antes explantar el tejido a fin de que las células de cometer irreversiblemente a la metamorfosis; disección de animales en el inicio de pupariation llevó a metamorfosis poca o ninguna en cultivo. Así, con las modificaciones apropiadas, el método in vivo cultivo ex puede ser aplicado para estudiar dinámica así como los aspectos de estado estacionario de la biología cerebro central.

Watch this Scientific Journal Video about Ex vivo Culturing of Whole, Developing Drosophila Brains at JoVE.com

Ex vivo Culturing of Whole, Developing Drosophila Brains

Este artículo describe un método por el cual se puede imitar<em&gt; En vivo</em&gt; Desarrollo de la<em&gt; Drosophila</em&gt; Cuerpo de hongo en un<em&gt; Ex vivo</em&gt; Sistema de cultivo.

Ex cultivo in vivo de la Plenaria, los cerebros en desarrollo de Drosophila

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for ...

ex-vivo-culturing-of-whole-developing-drosophila-brains

Research

JoVE Journal

Neuroscience

Ex vivo Culturing of Whole, Developing Drosophila Brains

13.0K vistas.  National Institute of Neurological Disorders and Stroke. Este artículo describe un método por el cual se puede imitar En vivo Desarrollo de la Drosophila Cuerpo de hongo en un Ex vivo Sistema de cultivo.

Video: Ex vivo Culturing of Whole, Developing Drosophila Brains

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the procedure for the visualization of Drosophila embryonic nervous system using antibodies to neuronal proteins. Since the entire embryonic peripheral nervous and central nervous systems are well characterized at the level of individual cells (Dambly-Chaudi&egrave;re et al., 1986; Bodmer et al., 1987; Bodmer et al., 1989), any aberrations to these systems can be easily identified using antibodies to different neuronal proteins. The developing embryos are collected at certain times to ensure that the embryos are in the proper developmental stages for visualization. After collection, the outer layers of the embryo, the chorion membrane and the vitelline envelope that surrounds the embryo, are removed before fixation. Embryos are then incubated with neuronal antibodies and visualized using fluorescently labeled secondary antibodies. Embryos at stages 12-17 are visualized to access the embryonic nervous system. At stage 12 the CNS germ band starts shortening and by stage 15 the definitive pattern of the commissure has been achieved. By stage 17 the CNS contracts and the PNS is fully developed (Campos-Ortega et al. 1985). Thus changes in the pattern of the PNS and CNS can be easily observed during these developmental stages.

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

We describe the procedure to prepare staged Drosophila embryos for the visualization of the embryonic nervous system during embryogenesis.

Visualización del sistema nervioso embrionario en Todo el montaje Drosophila Los embriones

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the ...

Investigación

Neurociencias

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

En electroporación in vivo de la retina del ratón en desarrollo

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of existing mouse lines for genetic cell lineage tracing: a tamoxifen-inducible Cre line and a Cre reporter line expressing dsRed upon Cre-mediated recombination. By using a relatively low level of tamoxifen, we were able to induce recombination in a small number of cells, permitting us to follow individual cell divisions. Additionally, we observed the transcriptional response to Sonic Hedgehog (Shh) signaling using an Olig2-eGFP transgenic line 1-3 and we monitored formation of cilia by infecting the cultured slice with virus expressing the cilia marker, Sstr3-GFP 4. In order to image the neuroepithelium, we harvested embryos at E8.5, isolated the neural tube, mounted the neural slice in proper culturing conditions into the imaging chamber and performed time-lapse confocal imaging. Our ex vivo live imaging method enables us to trace single cell divisions to assess the relative timing of primary cilia formation and Shh response in a physiologically relevant manner. This method can be easily adapted using distinct fluorescent markers and provides the field the tools with which to monitor cell behavior in situ and in real time.

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Here we develop the tools necessary for ex vivo live imaging to trace single cell divisions in the mouse E8.5 neuroepithelium

Ex vivo de imágenes en vivo de las divisiones sola celda en ratón neuroepitelio

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Todo el montaje de Immunolabeling olfativa del receptor de las neuronas en el<em&gt; Drosophila</em&gt; Antena

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

Simultáneo<em&gt; Ex vivo</em&gt; Pruebas funcionales de dos retinas por<em&gt; In vivo</em&gt; Sistema Electrorretinograma

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

Ex Vivo Optogenética Disección de circuitos del miedo en rodajas de cerebro

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

Un simple de un solo paso para la disección Protocolo Todo el montaje Preparación de adulto<em&gt; Drosophila</emLos cerebros&gt;

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Here, we provide a low-cost and reliable method to generate electroporated brain organotypic slice cultures from mouse embryos suitable for confocal microscopy and live-imaging techniques.

Ex útero Electroporación y culturas Organotypic rebanada de cerebros embrionarios de ratón para vivir-la proyección de imagen de interneuronas GABAérgicas de migrando

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model animal for endocrine research, Drosophila melanogaster, which has sophisticated genetic tools and genome information, is being increasingly used. This article describes a method for the calcium imaging of Drosophila brain explants. This method enables the detection of the direct signaling of a hormone to the brain. It is well known that many peptide hormones act through G-protein-coupled receptors (GPCRs), whose activation causes an increase in the intracellular Ca2+concentration. Neural activation also elevates intracellular Ca2+ levels, from both Ca2+ influx and the release of Ca2+ stored in the endoplasmic reticulum (ER). A calcium sensor, GCaMP, can monitor these Ca2+ changes. In this method, GCaMP is expressed in the neurons of interest, and the GCaMP-expressing larval brain is dissected and cultured ex vivo. The test peptide is then applied to the brain explant, and the fluorescent changes in GCaMP are detected using a spinning disc confocal microscope equipped with a CCD camera. Using this method, any water-soluble molecule can be tested, and various cellular events associated with neural activation can be imaged using the appropriate fluorescent indicators. Moreover, by modifying the imaging chamber, this method can be used to image other Drosophila organs or the organs of other animals.

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

This paper describes a protocol for ex vivo calcium imaging of the Drosophila brain. In this method, natural or synthetic compounds can be applied to the buffer to test their ability to activate particular neurons in the brain.

Ex Vivo Proyección de imagen de calcio para la visualización de las respuestas cerebrales a la señalización endocrina en Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model ...

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs provides a tissue-level understanding of energy utilization that cannot be captured when analyzing primary cells and cell lines. While this analysis is ex vivo, it allows for the measurement from a number of specialized cells working together to perform a function in one tissue and more closely models the in vivo organ. Metabolic reprogramming has been observed in many neurological diseases, including neoplasia, and neurodegenerative diseases. This protocol was developed to assist the D. melanogaster community&#39;s investigation of metabolism in neurological disease models using a commercially available metabolic analyzer. Measuring metabolism of whole brains in the metabolic analyzer is challenging due to the geometry of the brain. This analyzer requires samples to remain at the bottom of a 96-well plate. Cell samples and tissue punches can adhere to the surface of the cell plate or utilize spheroid plates, respectively. However, the spherical, three-dimensional shape of D. melanogaster brains prevents the tissue from adhering to the plate. This protocol requires a specially designed and manufactured micro-tissue restraint that circumvents this problem by preventing any movement of the brain while still allowing metabolic measurements from the analyzer&#39;s two solid-state sensor probes. Oxygen consumption and extracellular acidification rates are reproducible and sensitive to a treatment with metabolic inhibitors. With a minor optimization, this protocol can be adapted for use with any whole tissue and/or model system, provided that the sample size does not exceed the chamber generated by the restraint. While basal metabolic measurements and an analysis after a treatment with mitochondrial inhibitors are described within this protocol, countless experimental conditions, such as energy source preference and rearing environment, could be interrogated.

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

We present a protocol for measuring oxygen consumption and extracellular acidification in Drosophila melanogaster larval and adult brains. A metabolic analyzer is utilized with an adapted and optimized protocol. Micro-tissue restraints are a critical component of this protocol and were designed and created specifically for their use in this analysis.

Análisis metabólico del cerebro adulto y Larval de Drosophila melanogaster

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...

Ex cultivo in vivo de la Plenaria, los cerebros en desarrollo de Drosophila

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