Damos las gracias a RIKEN para su financiación. P10-Gal4 fue una especie de regalo de Alain Vicente (Université Paul Sabatier, Toulouse, Francia).

1. Preparación de los reactivos Notas antes de comenzar: la disección y la tinción inmunohistoquímica se llevan a cabo en una cámara magnética y la larva se fija por el uso de clavos de insectos de forma especial. Las instrucciones detalladas para la construcción de una cámara magnética, y la preparación de estos pines se pueden encontrar en las referencias relacionadas con 14,15. En resumen, un agujero cuadrado 1x1cm se corta en una hoja magnética y un cubreobjetos colocado en la parte posterior de la hoja para hacer una pequeña cámara. Los lados de la cámara está sellada con pegamento epóxico, después de esta cola se ha fijado la cámara se lava varias veces con etanol al 70% antes de su uso. Pines disección de insectos son preparados por la flexión a la forma deseada y luego pegado a una lengüeta metálica 14,15. Como alternativa a una ficha de metal, se ha utilizado una invertida de cabeza plana chincheta de acero con un mango de una punta amarilla de corte. Uso de esta cámara magnética permite un estrecho control over posicionamiento pin y el tejido se extiende durante la disección. Para conducir la expresión del gen en diferentes subgrupos de neuronas DA investigadores pueden utilizar varias líneas diferentes Gal4 (resumidos por Shimono y sus colegas 16). Muchas de estas líneas están disponibles de los centros públicos de acciones. En este protocolo representante, llevamos a cabo la inmunotinción de una línea en la que dos clases opuestas de neuronas DA son co-etiquetado: el más simple de clase I y la mayoría de los complejos de clase IV (P10-Gal4 17,18, UAS-mCD8:: Kusabira-Orange (KO)). <ol><li> Prepare Ca + + libre HL3.1 solución salina <ol><li> En mM: 70 NaCl, 5 KCl, 20 de MgCl 2, 10 NaHCO 3, 5 HEPES, sacarosa 115, y trehalosa 5, pH 7,2 19. Filtro-esterilizar y almacenar a 4 ° C. Nota: Ca + + sin solución impide la contracción muscular durante la disección. </li></ol></li><li> Prepare 2x tampón PHEM <ol><li> En mM: 130 TUBOS, 60 HEPES, 20 EGTA, 4 MgSO4, pH 7,0. Filtro-esterilizar y almacenar a 4 ° C. Nota: Estos materiales no se disuelven hasta que se acerque el pH 7,0. </li></ol></li><li> Preparar el fijador recién inmediatamente antes de la fijación. <ol><li> Con el fin de preparar una solución de 25 ml, paraformaldehído primera mezcla de 2 g, 100μl 1M NaOH y 10 ml de agua en un tubo de 50 ml Falcon. </li><li> Agite el fijador en un baño de agua a 55 ° C con agitación hasta que la solución es clara. (Volumen total será de unos 11,5 ml.) </li><li> Fijador de enfriar en hielo. </li><li> Añadir buffer 12.5ml 2x PHEM. </li><li> Ajustar el pH a 7.0 con HCl 1M. </li><li> Llene la solución con agua hasta que el volumen es de 25 ml. </li><li> Filtrar la solución con papel Whatman. </li></ol></li></ol> 2. Disección de las larvas Tenga en cuenta antes de empezar: las redes de microtúbulos, y en particular los de las dendritas sensorial, se descompondrá rápidamente después del inicio de la disección. Corriente alternahieving disección rápida en menos de cinco minutos, seguido de la fijación inmediata son factores clave en el éxito de este protocolo. <ol><li> Lave la larva en el agua y rápidamente pasar a esta caída con un bucle de cabello. </li><li> Orientar la larva hasta la parte dorsal y la parte ventral en el cristal. Nota: Esta orientación es el de examinar las neuronas grupo ventral. Para examinar las neuronas dorsales cluster, invertir la orientación. </li><li> Use el centro de insectos-pin a pin del extremo anterior cerca de los ganchos de la boca. Coloque el pasador cerca del final para obtener mejores resultados. </li><li> Ponga una gota de solución salina HL3.1 en la cámara de disección. </li><li> Cortar la punta posterior de la larva de con micro tijeras. Nota: Este paso se abre una abertura en el extremo posterior de la larva que va a permitir el acceso de la micro tijeras (paso 2.7). </li><li> Coge con las pinzas de la región del intestino que está asomando de la abertura en el extremo posterior de la larva. Retire con cuidado todo el intestino. </li><li> Coloque elpunta de una hoja de la micro tijeras a través de la apertura y corte a lo largo de la línea media ventral hacia la parte anterior. </li><li> Utilizando los pines esquina, las esquinas del ahora libre, posterior, luego anterior. Al mismo tiempo estira suavemente abrir el filete de larvas. </li></ol> 3. Fijación, bloqueo, tinción y montaje de los filetes de larvas Notas antes de empezar: Todas las medidas de fijación y tinción se llevó a cabo en la sala de disección. Durante este proceso, tenga cuidado de no tocar los pines de la celebración de la larva del insecto, ya que puede conducir a daños en los tejidos. Para evitar que el experimento de la desecación, se todos los pasos de tinción en un pequeño recipiente Tupperware rodeado por tejidos húmedos. <ol><li> Aspirar el Ca + + libre HL3.1 buffer usando una punta amarilla. Inmediatamente añadir el fijador con otro pipetteman. </li><li> Pipeta suavemente arriba y abajo para mezclar el fijador con las trazas restantes de Ca + + libre HL3.1 bufferen la cámara. Inmediatamente aspirado y luego añadir el fijador fresco en la cámara. </li><li> Incubar a temperatura ambiente durante 20 minutos. </li><li> Lavado de 10 minutos 6 veces en PBST (0,1% Triton X-100 en PBS). </li><li> Bloquear con suero de cabra al 5% en PBST durante 20 minutos a temperatura ambiente. </li><li> Vuelva a colocar la solución de bloqueo con anticuerpos primarios diluidos en suero de cabra al 5% en PBST. Los anticuerpos primarios utilizados son el ratón anti-α-tubulina (DM1A) y la rata anti-CD8 (5H10), tanto diluido 1 / 1000. Nota: El investigador puede desear a sustituir el ratón anti-α-tubulina (DM1A) con el ratón anti-Futsch (22C10) diluido 20,21 1 / 1000 en algunas circunstancias (véase la discusión). </li><li> Incubar toda la noche (por lo menos a las 16h) a 4 ° C. </li><li> Lavado de 10 minutos 6 veces en PBST. </li><li> Añadir la solución de anticuerpo secundario diluido en suero de cabra al 5% en PBST. Los anticuerpos secundarios utilizados son Alexa Fluor 488 cabra anti-ratón IgG y Cy3 burro IgG anti-rata. Mantener la muestra cubierta para evitar fluoróforofoto-blanqueo 22. </li><li> Incubar ya sea a temperatura ambiente durante dos horas, o durante la noche a 4 ° C, seguida de una hora a temperatura ambiente. </li><li> Lavado de 10 minutos 6 veces en PBST. </li><li> Coloque los filetes de larvas en la diapositiva de la cutícula hacia abajo, monte en el 80% de glicerol, y el sello de los lados del cubreobjeto con esmalte de uñas para un &quot;rápido&quot; de montaje. Nota: Para limpiar el tejido mejora, y una muestra de estabilidad permanente, montar en DPX como se describió previamente 23. </li></ol> 4. Los resultados representativos: Tinción fluorescente se examinan bajo un microscopio confocal. En las Figuras 1-2 ramas, diferentes dentro de un jardín de dendritas tienen la organización del citoesqueleto diferentes. La figura 1 muestra una región de la glorieta de la clase IV DA neuronas en la 1 ª fase estadio larval. La pérgola entera está marcada con mCD8:: KO y detectado usando un anticuerpo anti-CD8 y fluorescentes secundaria (Cy3). Tubulina se detecta el uso de anti α-tubulina de anticuerpos y fluorescent secundario (Alexa Fluor 488). Las ramas principales son positivos para la tubulina, algunas ramas laterales delgadas son tubulina-negativo. Movie 1 es un conjunto de secciones en serie a través de una coloración similar de una clase I DA neuronas. La figura 2 muestra una región de la glorieta de la clase IV DA neuronas teñidas con anticuerpos contra Futsch y CD8 en la 1 ª fase estadio larval. Las ramas principales son Futsch-positivas, algunas ramas laterales delgadas son Futsch negativo. Figura 3. Tráqueas en la pared del cuerpo de larvas muestran una organización compleja de microtúbulos. Películas 2 y 3 muestran las secciones de serie de manchas a través de los músculos de la pared del cuerpo y la tráquea. <img alt="Figura 1" src="/files/ftp_upload/3662/3662fig1.jpg" /> Figura 1. Fig. 1 muestra una región de la glorieta de la clase IV DA neuronas en la 1 ª fase estadio larval. La pérgola entera está marcada con mCD8:: KO y detectado usando anticuerpo anti-CD8 y fluorescentes secundaria (Cy3). Tubulina se detecta el uso de anti-&amp;-túbulosn de anticuerpos fluorescentes y secundario (Alexa 488). Paneles AC secuencial confocal z-secciones (0.5μm), C &#39;-C &quot;, la tinción de anticuerpos única desde el panel de C. Ejemplo de ramas con (punta de flecha amarilla) o sin (púrpura punta de flecha) se destacan los microtúbulos. Puntas de flecha roja microtúbulos destacar en el subyacente las células epiteliales. <img alt="Figura 2" src="/files/ftp_upload/3662/3662fig2.jpg" /> Figura 2. Fig. La figura 2 muestra una región similar de la glorieta de la clase IV da neuronas teñidas con anticuerpos contra Futsch y CD8 en la 1 ª fase estadio larval. La pérgola entera está marcada con mCD8:: KO y detectado usando anticuerpo anti-CD8 y fluorescentes secundaria (Cy3). Futsch se detecta el uso de anti-anticuerpos fluorescentes y Futsch secundario (Alexa 488). Las ramas principales son Futsch-positivas, algunas ramas laterales delgadas son Futsch negativo. Ejemplo de las ramas con (punta de flecha amarilla) o sin (púrpura punta de flecha) Futsch se destacan. Figura 3. Tráquea teñidas utilizando el protocolo anti-tubulina se ha descrito anteriormente. Esta larva es el tercer estadio y diseccionado como se describió previamente 23,24. Movie 3 muestra ampliada de serie de secciones en el campo marcado por un cuadrado. Secciones de cine 1. Serie trazado tinción tubulina a través de la glorieta dendríticas de una clase que las neuronas. La pérgola entera está marcada con mCD8:: KO (Magenta) y detectado usando anticuerpo anti-CD8 y fluorescentes secundaria (Cy3). Tubulina (verde) se detecta mediante un anticuerpo anti-α-tubulina y fluorescentes secundario (Alexa Fluor 488). Escala: de un lado de la imagen de vídeo se corresponde con 46.88μm en la sección. <a href="http://www.jove.com/files/ftp_upload/3662/YalginMooreMovie1.avi">Haga clic aquí para ver la película.</a> Movie 2. Serie secciones trazado tinción tubulina en cuerposy la pared muscular de un tercer instar de la larva. Tubulina se detecta el uso de anti-α-tubulina de anticuerpos fluorescentes y secundario (Alexa Fluor 488). Escala: de un lado de la imagen de vídeo se corresponde con 46.88μm en la sección. <a href="http://www.jove.com/files/ftp_upload/3662/YalginMooreMovie2.avi">Haga clic aquí para ver la película.</a> Movie 3. Las secciones seriadas búsqueda tinción tubulina en un cuerpo de la pared de la tráquea tercera instar de la larva, marcado con un cuadrado en Figure.3. Tubulina se detecta el uso de anti-α-tubulina de anticuerpos fluorescentes y secundario (Alexa Fluor 488). Escala: de un lado de la imagen de vídeo se corresponde con 46.88μm en la sección. <a href="http://www.jove.com/files/ftp_upload/3662/YalginMooreMovie3.avi">Haga clic aquí para ver la película.</a>

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Para entender la complejidad de las formas celulares se logró es importante ser capaz de precisión organización de microtúbulos ensayo. Aquí se describe un método robusto de etiquetado inmunohistoquímico para la organización de los microtúbulos ensayo dendríticas dendritas arborización neurona sensorial. Además de la tinción de las neuronas sensoriales, este método logra tinción inmunohistoquímica robusta de la tráquea, músculos y otros tejidos de la pared del cuerpo. Nosot...

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Catálogo número </th><th> Comentarios (Opcional) </th></tr><tr><td> Fórceps </td><td> Dumont </td><td> 11251-20 </td><td></td></tr><tr><td> Microtijeras </td><td> FST </td><td> 15000-08 </td><td></td></tr><tr><td> Mouse anti-α-tubulina (Clon: DM1A) </td><td> Sigma </td><td> T9026 </td><td> Dilución 1 / 1000 </td></tr><tr><td> Anti-ratón Futsch (Clon: 22C10), sobrenadante </td><td> De desarrollo Estudios Banco hibridoma </td><td> 22C10 </td><td> Dilución 1 / 1000 </td></tr><tr><td> Rata anti-CD8 (clon: 5H10) </td><td> Caltag </td><td> MCD0800 </td><td> Dilución 1 / 1000 </td></tr><tr><td> Alexa Fluor 488 IgG anti-ratón </td><td> Invitrogen </td><td> A-11.001 </td><td> Dilución 1 / 500 </td></tr><tr><td> Cy3 anti-IgG de rata </td><td> Jackson Immunoresearch </td><td> 712-166-150 </td><td> Dilución 1 / 200 </td></tr></tbody></table>

immunohistological labeling of microtubules in sensory neuron dendrites, tracheae, and muscles in the drosophila larva body wall

Para entender cómo las diferencias en las formas de células complejas se consiguen, es importante seguir con precisión la organización de los microtúbulos. El muro de las larvas de Drosophila cuerpo contiene varios tipos de células que son modelos para el estudio celular y morfogénesis de tejidos. Por ejemplo tráqueas se utilizan para examinar la morfogénesis del tubo 1, y la arborización dendrítica (DA), las neuronas sensoriales de la larva de Drosophila se han convertido en un sistema principal para el esclarecimiento de los generales y las neuronas específicas de la clase de mecanismos de diferenciación 5.2 dendríticas y degeneración 6 . La forma de dendritas pueden variar significativamente entre las clases de neuronas, e incluso entre las diferentes ramas de una sola neurona 7,8. Los estudios genéticos en las neuronas DA sugieren que la organización del citoesqueleto diferencial puede subyacen a las diferencias morfológicas en forma de ramas dendríticas 4,9-11. Ofrecemos un método inmunológico etiquetado robusta a unssay en la organización de los microtúbulos en vivo DA sensorial cenador neurona dendrita (Figuras 1, 2, Movie 1). Este protocolo se muestra la disección y la inmunotinción de larva de primer estadio, una etapa en la actividad sensorial consecuencia dendritas neuronales y la organización de ramificación se está produciendo 12,13. Además de la tinción de las neuronas sensoriales, este método logra el etiquetado robusto de organización de microtúbulos en los músculos (Películas 2, 3), la tráquea (Figura 3, Movie 3), y otros tejidos de la pared del cuerpo. Es valioso para los investigadores que deseen analizar la organización de microtúbulos in situ en la pared del cuerpo en la investigación de los mecanismos de control que el tejido y la forma celular.

Watch this Scientific Journal Video about Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall at JoVE.com

Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall

Para entender cómo las formas complejas de células, como las dendritas neuronales, se obtienen durante el desarrollo, es importante ser capaz de precisión organización de microtúbulos ensayo. Aquí se describe un método robusto de etiquetado inmunohistoquímico para examinar la organización de microtúbulos de las dendritas arborización dendrítica neurona sensorial, la tráquea, músculos y otros<em&gt; Drosophila</em&gt; Larva tejidos de la pared del cuerpo.

Etiquetado inmunohistológica de microtúbulos en las dendritas neurona sensorial, tráqueas y los músculos en el Drosophila Larva pared del cuerpo

To understand how differences in complex cell shapes are achieved, it is important to accurately follow microtubule organization. The Drosophila larval ...

immunohistological-labeling-microtubules-sensory-neuron-dendrites

Research

JoVE Journal

Neuroscience

Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall

12.3K vistas.  RIKEN Brain Science Institute. Para entender cómo las formas complejas de células, como las dendritas neuronales, se obtienen durante el desarrollo, es importante ser capaz de precisión organización de microtúbulos ensayo. Aquí se describe un método robusto de etiquetado inmunohistoquímico para examinar la organización de microtúbulos de las dendritas arborización dendrítica neurona sensorial, la tráquea, músculos y otros Drosophila Larva tejidos de la pared del cuerpo.

Video: Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Células vivas imágenes de células de los órganos sensoriales Precursor en Intacto Drosophila Las pupas

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Investigación

Neurociencias

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic development and axonal wiring. Recently the dendritic arborization (DA) sensory neurons of the Drosophila larval peripheral nervous system (PNS) have become powerful genetic models in which to elucidate both general and class-specific mechanisms of neuron differentiation.
There are four main DA neuron classes (I-IV)1. They are named in order of increasing dendrite arbor complexity, and have class-specific differences in the genetic control of their differentiation2-10. The DA sensory system is a practical model to investigate the molecular mechanisms behind the control of dendritic morphology11-13 because: 1) it can take advantage of the powerful genetic tools available in the fruit fly, 2) the DA neuron dendrite arbor spreads out in only 2 dimensions beneath an optically clear larval cuticle making it easy to visualize with high resolution in vivo, 3) the class-specific diversity in dendritic morphology facilitates a comparative analysis to find key elements controlling the formation of simple vs. highly branched dendritic trees, and 4) dendritic arbor stereotypical shapes of different DA neurons facilitate morphometric statistical analyses.
DA neuron activity modifies the output of a larval locomotion central pattern generator14-16. The different DA neuron classes have distinct sensory modalities, and their activation elicits different behavioral responses14,16-20. Furthermore different classes send axonal projections stereotypically into the Drosophila larval central nervous system in the ventral nerve cord (VNC)21. These projections terminate with topographic representations of both DA neuron sensory modality and the position in the body wall of the dendritic field7,22,23. Hence examination of DA axonal projections can be used to elucidate mechanisms underlying topographic mapping7,22,23, as well as the wiring of a simple circuit modulating larval locomotion14-17.
We present here a practical guide to generate and analyze genetic mosaics24 marking DA neurons via MARCM (Mosaic Analysis with a Repressible Cell Marker)1,10,25 and Flp-out22,26,27 techniques (summarized in Fig. 1).

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

The dendritic arborization sensory neurons of the Drosophila larval peripheral nervous system are useful models to elucidate both general and neuron class-specific mechanisms of neuron differentiation. We present a practical guide to generate and analyze dendritic arborization neuron genetic mosaics.

Análisis morfológico de Drosophila Larvas dendritas neurona sensorial periférica y axones Con mosaicos genéticos

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

Visualización de los propioceptores<em&gt; Drosophila</em&gt; Las larvas y pupas

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

El marcaje de células individuales en el Sistema Nervioso Central de<em&gt; Drosophila melanogaster</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

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 ...

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an excellent model system for such investigations due to its complex behaviors, powerful genetic techniques, and compact nervous system. Laboratory behavioral assays have long been used with Drosophila to simulate properties of the natural environment and study the neural mechanisms underlying the corresponding behaviors (e.g. phototaxis, chemotaxis, sensory learning and memory)1-3. With the recent availability of large collections of transgenic Drosophila lines that label specific neural subsets, behavioral assays have taken on a prominent role to link neurons with behaviors4-11. Versatile and reproducible paradigms, together with the underlying computational routines for data analysis, are indispensable for rapid tests of candidate fly lines with various genotypes. Particularly useful are setups that are flexible in the number of animals tested, duration of experiments and nature of presented stimuli. The assay of choice should also generate reproducible data that is easy to acquire and analyze. Here, we present a detailed description of a system and protocol for assaying behavioral responses of Drosophila flies in a large four-field arena. The setup is used here to assay responses of flies to a single olfactory stimulus; however, the same setup may be modified to test multiple olfactory, visual or optogenetic stimuli, or a combination of these. The olfactometer setup records the activity of fly populations responding to odors, and computational analytical methods are applied to quantify fly behaviors. The collected data are analyzed to get a quick read-out of an experimental run, which is essential for efficient data collection and the optimization of experimental conditions.

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

We describe here a behavioral setup and data analysis method for assaying olfactory responses of up to 100 vinegar flies (Drosophila melanogaster). This system may be used with single or multiple olfactory stimuli, and adaptable for optogenetic activation or silencing of neuronal subsets.

Comportamientos olfativas ensayó mediante seguimiento informático de<em&gt; Drosophila</em&gt; En un olfatómetro de cuatro cuadrantes

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

Etiquetado balístico de las neuronas piramidales en las rebanadas cerebrales y en el cultivo celular primario

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...

Semi-Quantitative Determination of Dopaminergic Neuron Density in the Substantia Nigra of Rodent Models using Automated Image Analysis

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased stereological counting is the standard for quantification of these cells, but it remains a laborious and time-consuming process, which may not be feasible for all projects. Here, we describe the use of an image analysis platform, which can accurately estimate the quantity of labeled cells in a pre-defined region of interest. We describe a step-by-step protocol for this method of analysis in rat brain and demonstrate it can identify a significant reduction in tyrosine hydroxylase positive neurons due to expression of mutant &#945;-synuclein in the substantia nigra. We validated this methodology by comparing with results obtained by unbiased stereology. Taken together, this method provides a time-efficient and accurate process for detecting changes in dopaminergic neuron number, and thus is suitable for efficient determination of the effect of interventions on cell survival.

Here we present an automated method for semi-quantitative determination of dopaminergic neuron number in the rat substantia nigra pars compacta.

Determinación Semicuantita cuantitativa de la densidad de la neurona dopaminérgica en la sustancia negra de modelos de roedores utilizando el análisis automatizado de imágenes

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased ...

Visualización de redes de microtúbulos en uniones neuromusculares y células musculares de Drosophila

Using Drosophila Larval Neuromuscular Junction and Muscle Cells to Visualize Microtubule Network

The microtubule network is an essential component of the nervous system. Mutations in many microtubules regulatory proteins are associated with neurodevelopmental disorders and neurological diseases, such as microtubule-associated protein Tau to neurodegenerative diseases, microtubule severing protein Spastin and Katanin 60 cause hereditary spastic paraplegia and neurodevelopmental abnormalities, respectively. Detection of microtubule networks in neurons is advantageous for elucidating the pathogenesis of neurological disorders. However, the small size of neurons and the dense arrangement of axonal microtubule bundles make visualizing the microtubule networks challenging. In this study, we describe a method for dissection of the larval neuromuscular junction and muscle cells, as well as immunostaining of &#945;-tubulin and microtubule-associated protein Futsch to visualize microtubule networks in Drosophila melanogaster. The neuromuscular junction permits us to observe both pre-and post-synaptic microtubules, and the large size of muscle cells in Drosophila larva allows for clear visualization of the microtubule network. Here, by mutating and overexpressing Katanin 60 in Drosophila melanogaster,&#160;and then examining the microtubule networks in the neuromuscular junction and muscle cells, we&#160;accurately reveal the regulatory role of Katanin 60 in neurodevelopment. Therefore, combined with the powerful genetic tools of Drosophila&#160;melanogaster, this protocol greatly facilitates genetic screening and microtubule dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

Autor destacado: Comprensión de la red de microtúbulos en las uniones neuromusculares de Drosophila 

Here, we present a detailed protocol to visualize the microtubule networks in neuromuscular junctions and muscle cells. Combined with the powerful genetic tools of Drosophila melanogaster, this protocol greatly facilitates genetic screening and microtubule-dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.