Los autores agradecen a RIKEN para su financiación. También agradecemos a Cagri Yalgin, Delandre Caroline, y Jay Parrish para las discusiones sobre los protocolos de genética e inmunohistoquímica.

1.Preparation de reactivos <ol><li> Prepare Ca + + libre HL3.1 solución salina 28. <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. Filtro de esterilizar y almacenar a 4 ° C. Nota: Ca + + sin solución impide la contracción muscular durante la disección. </li></ol></li><li> Hacer de poli-L-lisina (PLL) cubreobjetos. <ol><li> Disolver 100 mg de PLL en el agua 4.2ml y hacer alícuotas 300μl en tubos Eppendorf y se congela a -20 ° C. </li><li> Antes de cubreobjetos de recubrimiento, descongelar primero una parte alícuota, traerlo hasta 10 ml en ddH2O y añadir 20μl de Kodak Photo-Flo, la concentración de PLL final es de 0,7 mg / ml. </li><li> Cubreobjetos sumergir por 30 minutos en una solución de PLL, eliminar y seco, luego enjuague a fondo con agua y seca. Repita dos veces. Cubreobjetos tratados con una duración aproximada de un mes. </li></ol></li></ol> 2. Cruces genéticos <ol><li> To generar clones MARCM. Aquí está una cruz de ejemplo utilizando un controlador de pan-DA 11,27: FRT2A x hsFLP; Gal4 109 (2) 80, UAS-mCD8:: GFP, bañera-GAL80 FRT2A/SM5-TM6B </li><li> Para generar Flp-out clones. Aquí está una cruz de ejemplo utilizando una clase IV controlador específico 8: ppk-Gal4 x yw, hsFLP; UAS-FRT-CD2, y +-stop-FRT-mCD8:: GFP </li></ol> 3. Recolección de los embriones <ol><li> Mantenga cruza Drosophila en una botella de colección a 25 ° C y recoger los embriones en una placa de agar jugo de manzana 29 se extendió con una fina capa de pasta de levadura 30,31. </li></ol> 4. Tratamiento de choque de calor de los embriones <ol><li> Coloque un plato vacío en la oposición a la placa de agar de jugo de manzana en la que los embriones se han establecido. Selle alrededor de estas dos placas con Parafilm (Fig. 2). </li><li> Durante el choque térmico, se sumergen ªplaca e en el baño de agua y se mantiene presionado con un peso de metal. </li><li> Para los embriones MARCM <ol><li> Recoger los embriones durante 2 e incubar en un plato de petri de 10 cm rodeado por tejidos humedecido a 25 ° C durante 2 horas una adicional. Nota: Ajuste del protocolo de choque térmico altera la frecuencia con que se generan clones. </li><li> Para un número menor de clones, con el objetivo de una sola neuronas aisladas, sumergirse y golpe de calor en el baño de agua durante 1 hora a 38 ° C. </li><li> Para un mayor número de clones, choque térmico de 45 minutos a 38 ° C, a temperatura ambiente 30 minutos recuperarse, luego de choque térmico de nuevo durante otros 30 minutos. </li></ol></li><li> Recoger Flp-los embriones durante 24 horas, y el choque térmico de 1 hora a 38 ° C. </li></ol> 5. La detección de los clones <ol><li> Después del choque térmico, retire la tapa de la disposición hermético y coloque el jugo de manzana placa de agar en una placa de Petri de 10 cm rodeado por tejidos húmedos. La cultura los embriones y larvas posteriores a 25 ° C hasta que vaganING 3 º estadio. Nota: las condiciones de cultivo, en especial la alimentación, se han mostrado alteraciones en la morfología DA dendríticas cenador 32,33. Asegúrese de que las larvas en crecimiento tienen el acceso a la pasta de levadura en todo momento, controlar y reponer la pasta de levadura según sea necesario. </li><li> Desde este punto en adelante, en el protocolo, manipular con cuidado las larvas de insecto con unas pinzas. </li><li> En pocas palabras y enjuague suavemente las larvas en el agua del grifo, y luego colocar en una placa de agar. Nota: Este paso reduce el fondo de autofluorescencia de los alimentos o la suciedad en la superficie de las larvas. </li><li> Examinar las larvas bajo un microscopio de fluorescencia de gran alcance de disección. Identificar las larvas con GFP-positivas las neuronas / células en la pared del cuerpo. </li></ol> 6. Imágenes in vivo de las dendritas <ol><li> Lugar de la larva en un portaobjetos de vidrio depresión con una pequeña gota de un 80% de glicerol. Coloque un cubreobjetos sobre el portaobjetos para inmovilizar la larva. Asegúrese de que no quede aire entre la larva y el coverslip. </li><li> Empuje suavemente el cubreobjetos para rodar la larva para permitir la visualización de la neurona de interés. La imagen de la mCD8:: GFP-etiquetados cenador dendríticas a través de microscopía confocal. </li></ol> 7. Disección de las larvas Tenga en cuenta antes de empezar: las dendritas de neuronas DA se degradan rápidamente después del inicio de la disección. Diseccionar cada larva individual en menos de 5 minutos para asegurar una buena morfología dendrítica. <ol><li> Diseccionar vagando 3 de larvas en un plato Sylgard como se describió previamente 34 con las siguientes modificaciones: <ol><li> Para la obtención de imágenes de las neuronas DA axón termini, asegúrese de que el SNC está intacta y los nervios segmentarios no están rotos. Después de la apertura de las larvas, primero corte cuidadosamente las conexiones de la tráquea a la pared del cuerpo y luego retire con cuidado el intestino completo. </li><li> Si las dendritas imágenes solo, eliminar el sistema nervioso central para permitir a un plano de montaje. </li></ol></li></ol> 8. Fixation y el bloqueo de los filetes de larvas <ol><li> Con la larva sigue clavado en la placa Sylgard, fijarlo en el 4% PFA en PBS durante 20 minutos a temperatura ambiente en un agitador girar suavemente. </li><li> Eliminar los restos de tejidos larvarios (grasa corporal, la tráquea, etc) después de la fijación. </li><li> Lave en PBST (PBS con 0,1% Triton X-100) a 10 minutos 3 veces en la placa de Sylgard y agitador. Si el examen de terminales del axón, mantenga el filete en el plato Sylgard. (Si las dendritas tinción es posible la transferencia de los filetes de las larvas a un tubo de 0,5 ml en esta etapa 34) </li><li> Bloquear las larvas por 20 minutos a temperatura ambiente en el 5% de suero normal de burro (NDS) en PBST en un agitador. </li></ol> 9. Tinción de los filetes de larvas <ol><li> Retire la solución de bloqueo y se incuban en anticuerpo primario (en el 5% NDS / PBST) durante la noche a 4 ° C en un pequeño recipiente Tupperware rodeado por tejidos humedecido. </li><li> Retire los tubos de 4 ° C e incubar una hora más a temperatura ambiente. </li><li> Lavado de 10 minutos 6 veces en PBST. Agregue el antib secundariaody (en el 5% NDS / PBST), y la cubierta para evitar la foto-blanqueo fluoróforo 34. </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> Lave el 10 minutos larvas 6x en PBST y proceder al montaje. </li></ol> 10. Montaje de los filetes de larvas para el examen de la glorieta dendrita <ol><li> Monte cada filete de larvas lo más plana posible utilizar unas tijeras de disección o un bisturí para cortar la cabeza (incluyendo los ganchos de la boca), y la posterior (incluyendo los espiráculos) 34. </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 34. </li><li> Para un montaje permanente y una imagen más clara <ol><li> Coloque el filete de músculo diseccionado larvas hacia abajo en una gota de PBS en un cubreobjetos PLL, rápidamente se adhieren a la cubreobjetos. </li><li> Eliminar tanto líquido como sea posible después del montaje, however No deje que se seque por completo. </li><li> Tomar el cubreobjetos (con filete de larvas adjunta) a través de una serie de etanol: 35% de etanol, seguido de 50%, 70%, 95% y el 100% de etanol, finalmente 2x cada uno durante 10 minutos (debe ser el segundo al 100% la solución EtOH cambiar con frecuencia ) y, por último, lavar 2-3x 10 minutos en xileno. </li><li> Ponga una gota de DPX medio de montaje (xileno Distyrene plastificante) en un portaobjetos limpio y coloque cuidadosamente el cubreobjetos filete hacia abajo en la parte superior. Mantener en la oscuridad, y esperar un día para el DPX para establecer antes de exponer. </li></ol></li></ol> 11. Montaje de los filetes de larvas para el examen de las terminales del axón <ol><li> Mantener los nervios segmentarios que corre entre el SNP y el SNC intacto durante la disección, fijación y tinción para permitir la localización de los axones de las células sensoriales periféricas del cuerpo de la neurona VNC. </li><li> Monte cada filete de cutícula larval hacia abajo en glicerol al 80% en una diapositiva de la depresión. Rastro de los axones del cuerpo celular de la DAVNC en el microscopio confocal. Nota: las neuronas SNP en cada proyecto el cuerpo segmento de la pared en el segmento de cognado de la VNC. </li></ol> 12. Los resultados representativos: Los resultados representativos se muestran en las figuras 3-5. Fig. 3 muestra la glorieta completa de una clase IV da la neurona, capturado en vivo bajo el microscopio confocal. Fig. 4 muestra un primer plano de parte de la glorieta dendrita de una neurona inmunohistoquímica etiqueta de clase III que se ha fijado correctamente para preservar la morfología. Los asociados recuadro muestra la degradación que puede ocurrir después de una disección y fijación sin éxito (ambos se tiñeron con anticuerpos anti-GFP). Fig. 5 muestra una sola clase IV terminal axonal en el SNC (anti-GFP, verde), todos los terminales de clase IV son co-teñidas con anti-CD2 (magenta). <img alt="Figura 1" src="/files/ftp_upload/3111/3111fig1.jpg" /> Figura 1 Descripción general de Protocolo. a) Recoger y embriones de choque de calor. b)<html> Seleccione una larva con una GFP-positivas las neuronas DA clon, a continuación, la imagen de la GFP-etiquetados dendrita cenador en la larva en vivo. c) Disección de la larva, y luego inmunohistoquímica mancha el filete. d) Monte el filete de colores, y luego la imagen de la glorieta dendríticas y proyecciones axonal de las neuronas DA de los clones. <img alt="Figura 2" src="/files/ftp_upload/3111/3111fig2.jpg" /> Figura 2: Preparación de la placa de agar de jugo de manzana para el choque térmico. Tome la placa (flecha blanca, ab), añadir una segunda placa de Petri en la parte superior (rojo flecha B) y el sello alrededor con Parafilm (flecha azul, b). <img alt="Figura 3" src="/files/ftp_upload/3111/3111fig3.jpg" /> Figura 3 en la imagen in vivo de la glorieta de una dendrita mCD8:: GFP-etiquetados clon MARCM (genotipo como en 2.1) que representa una clase IV (v&#39;ada) las neuronas de una larva de 3 º estadio. Barra de escala es 50 micras. d/3111/3111fig4.jpg &quot;/&gt; Figura 4 mCD8:: GFP-etiquetados MARCM clon de una neurona de clase III (ddaA) teñidas con anticuerpos anti-GFP, mostrando la morfología buen éxito después de la disección y fijación (verde). El recuadro muestra la degradación de las dendritas (flechas blancas) que ocurren después de la disección y fijación sin éxito (magenta). Barra de escala es 25μm. <img alt="Figura 5" src="/files/ftp_upload/3111/3111fig5.jpg" /> Figura 5 El 3 de estadio larval VNC. Un clon Flp-de una sola clase axón terminal VI (VDAB) (genotipo como en el punto 2.2) se detecta el uso de anticuerpos anti-GFP (verde). Anti-CD2 etiquetas de toda clase IV termini (magenta). Barra de escala es 25μm.

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J., Gunawardena, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+the+Embryonic+Nervous+System+in+Whole-mount+Drosophila+Embryos.">Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos.</a> J. Vis. Exp. (46), e2150-e2150 (2010).</li><li>Featherstone, D. E., Chen, K., Broadie, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harvesting+and+preparing+Drosophila+embryos+for+electrophysiological+recording+and+other+procedures.">Harvesting and preparing Drosophila embryos for electrophysiological recording and other procedures.</a> J Vis Exp. , (2009).</li><li>Medina, P. M., Swick, L. L., Andersen, R., Blalock, Z., Brenman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+forward+genetic+screen+for+identifying+mutations+affecting+larval+neuronal+dendrite+development+in+Drosophila+melanogaster.">A novel forward genetic screen for identifying mutations affecting larval neuronal dendrite development in Drosophila melanogaster.</a> Genetics. 172, 2325-2335 (2006).</li><li>Mirouse, V., Swick, L. L., Kazgan, N., St Johnston, D., Brenman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LKB1+and+AMPK+maintain+epithelial+cell+polarity+under+energetic+stress.">LKB1 and AMPK maintain epithelial cell polarity under energetic stress.</a> J Cell Biol. 177, 387-392 (2007).</li><li>Brent, J., Werner, K., McCabe, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+Larval+NMJ+Immunohistochemistry.">Drosophila Larval NMJ Immunohistochemistry.</a> J. Vis. Exp. 25, e1108-e1108 (2009).</li><li>Brand, A. H., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes.">Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.</a> Development. 118, 401-415 (1993).</li><li>Sugimura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+developmental+modes+and+lesion-induced+reactions+of+dendrites+of+two+classes+of+Drosophila+sensory+neurons.">Distinct developmental modes and lesion-induced reactions of dendrites of two classes of Drosophila sensory neurons.</a> J Neurosci. 23, 3752-3760 (2003).</li><li>Zito, K., Parnas, D., Fetter, R. D., Isacoff, E. Y., Goodman, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Watching+a+synapse+grow:+noninvasive+confocal+imaging+of+synaptic+growth+in+Drosophila.">Watching a synapse grow: noninvasive confocal imaging of synaptic growth in Drosophila.</a> Neuron. 22, 719-729 (1999).</li><li>Landgraf, M., Sanchez-Soriano, N., Technau, G. M., Urban, J., Prokop, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Charting+the+Drosophila+neuropile:+a+strategy+for+the+standardised+characterisation+of+genetically+amenable+neurites.">Charting the Drosophila neuropile: a strategy for the standardised characterisation of genetically amenable neurites.</a> Dev Biol. 260, 207-225 (2003).</li></ol>

No hay conflictos de interés declarado.

La Drosophila larvas DA modelo de neurona ofrece un excelente sistema genético para investigar los mecanismos que controlan la morfología neuronal y la formación de circuitos. MARCM se utiliza generalmente para el etiquetado y para la generación de mutantes DA clones neurona. Para MARCM que utilizar un pan-neural (por ejemplo, Gal4 C155) o DA neuronal específica del controlador. Utilizando un controlador de pan-neuronal, es posible utilizar directamente varias poblaciones amplia...

<table border="1"><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios (opcional) </th></tr><tr><td> SZX16 fluorescencia microscopio de disección (con GFPHQ filtro) </td><td> Olimpo </td><td> SZX16 </td><td></td></tr><tr><td> Pinzas de insectos en vivo </td><td> FST </td><td> 26030-10 </td><td></td></tr><tr><td> 26mm x 76mm depresión de diapositivas de vidrio </td><td> Toshinriko Co. </td><td> T8-R004 </td><td></td></tr><tr><td> Sylgard 184 (o Silpot 184) </td><td> Dow Corning </td><td> 3097358-1004 </td><td></td></tr><tr><td> Poli-L-lisina </td><td> Sigma </td><td> P-1524 </td><td> Este producto ha demostrado ser más eficaz </td></tr><tr><td> DPX medio de montaje </td><td> Sigma </td><td> 44581 </td><td></td></tr><tr><td> De conejo anti-GFP </td><td> Invitrogen </td><td> A-11122 </td><td> Dilución 1:500 </td></tr><tr><td> Rata anti-CD8 </td><td> Caltag </td><td> 5H10 </td><td> Dilución de 1:200 </td></tr><tr><td> Mouse anti-CD2 </td><td> AbD serotec </td><td> MCA443R </td><td> Dilución 1:700 </td></tr><tr><td> Anti-ratón Fasciclin2 </td><td> DSHB </td><td> 1D4 </td><td> Dilución 1:10 </td></tr></table>

morphological analysis of drosophila larval peripheral sensory neuron dendrites and axons using genetic mosaics

Desarrollo del sistema nervioso requiere de la especificación correcta de la posición de la neurona y de la identidad, seguido por el desarrollo neuronal dendríticas precisa específicos de la clase y el cableado axonal. Recientemente, la arborización dendrítica (DA), las neuronas sensoriales del sistema Drosophila larvas nervioso periférico (SNP) se han convertido en poderosos modelos de genética en la que para dilucidar los mecanismos generales y específicos de la clase de diferenciación de las neuronas. Hay cuatro clases principales de neuronas DA (I-IV) 1. Se nombran en orden creciente de complejidad dendrita cenador, y que clase de diferencias específicas en el control genético de la diferenciación de 2.10. El sistema de asimilación sensorial es un modelo práctico para investigar los mecanismos moleculares de control de la morfología dendrítica 11-13 debido a que: 1) se pueden aprovechar las poderosas herramientas genéticas disponibles en la mosca de la fruta, 2) la DA neuronal dendrita cenador se extiende en sólo dos dimensiones por debajo de un culo ópticamentear cutícula larval lo que es fácil de visualizar con alta resolución en vivo, 3) la diversidad específica de la clase en la morfología dendrítica facilita un análisis comparativo para encontrar los elementos clave que controla la formación de los árboles dendríticos muy simple vs ramificada, y 4) dendríticas cenador estereotipadas formas diferentes de neuronas DA facilitar morfométricos análisis estadísticos. DA actividad neuronal modifica la salida de un generador de patrones de locomoción larvas centrales 14-16. Las diferentes clases de neuronas DA tienen distintas modalidades sensoriales, y su activación provoca diferentes respuestas conductuales 14,16-20. Además diferentes clases enviar proyecciones axonales estereotipada en el sistema nervioso central larvas de Drosophila en el cordón nervioso ventral (VNC) 21. Estas proyecciones terminar con representaciones topográficas de los dos DA modalidad de neurona sensorial y la posición en la pared del cuerpo del campo dendríticas 7,22, De 23 años. Por lo tanto, el examen de las proyecciones de DA axonal puede ser utilizado para dilucidar los mecanismos subyacentes de mapas topográficos 7,22,23, así como el cableado de un circuito simple locomoción de larvas de modulación 14-17. Presentamos aquí una guía práctica para generar y analizar mosaicos genéticos 24 marcas a través de las neuronas DA MARCM (Análisis de mosaico con un marcador de células reprimibles) 1,10,25 y Flp-out 22,26,27 técnicas (que se resumen en la Fig. 1.).

Watch this Scientific Journal Video about Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics at JoVE.com

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

Arborización dendrítica de las neuronas sensoriales de la<em&gt; Drosophila</em&gt; Larval sistema nervioso periférico son modelos útiles para dilucidar tanto generales como específicos de la clase de neuronas mecanismos de diferenciación de las neuronas. Se presenta una guía práctica para generar y analizar la arborización dendrítica de neuronas mosaicos genéticos.

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 ...

morphological-analysis-drosophila-larval-peripheral-sensory-neuron

Research

JoVE Journal

Neuroscience

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

15.2K vistas.  RIKEN Brain Science Institute. Arborización dendrítica de las neuronas sensoriales de la Drosophila Larval sistema nervioso periférico son modelos útiles para dilucidar tanto generales como específicos de la clase de neuronas mecanismos de diferenciación de las neuronas. Se presenta una guía práctica para generar y analizar la arborización dendrítica de neuronas mosaicos genéticos.

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

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different neurological diseases. Defects in this essential process can have detrimental effects on neuronal functioning and development. We have developed a dissection protocol that is designed to expose the Drosophila larval segmental nerves to view axonal transport in real time. We have adapted this protocol for live imaging from the one published by Hurd and Saxton (1996) used for immunolocalizatin of larval segmental nerves. Careful dissection and proper buffer conditions are critical for maximizing the lifespan of the dissected larvae. When properly done, dissected larvae have shown robust vesicle transport for 2-3 hours under physiological conditions. We use the UAS-GAL4 method 1 to express GFP-tagged APP or synaptotagmin vesicles within a single axon or many axons in larval segmental nerves by using different neuronal GAL4 drivers. Other fluorescently tagged markers, for example mitochrondria (MitoTracker) or lysosomes (LysoTracker), can be also applied to the larvae before viewing. GFP-vesicle movement and particle movement can be viewed simultaneously using separate wavelengths.

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

This protocol discusses the live dissection of Drosophila larvae for the purpose of imaging the movement of GFP tagged axonal vesicles on microtubule tracks.

Visualización en vivo de las vesículas sinápticas En Drosophila segmentaria axones larvas

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

Investigación

Neurociencias

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

To understand how differences in complex cell shapes are achieved, it is important to accurately follow microtubule organization. The Drosophila larval body wall contains several cell types that are models to study cell and tissue morphogenesis. For example tracheae are used to examine tube morphogenesis1, and the dendritic arborization (DA) sensory neurons of the Drosophila larva have become a primary system for the elucidation of general and neuron-class-specific mechanisms of dendritic differentiation2-5 and degeneration6.
The shape of dendrite branches can vary significantly between neuron classes, and even among different branches of a single neuron7,8. Genetic studies in DA neurons suggest that differential cytoskeletal organization can underlie morphological differences in dendritic branch shape4,9-11. We provide a robust immunological labeling method to assay in vivo microtubule organization in DA sensory neuron dendrite arbor (Figures 1, 2, Movie 1). This protocol illustrates the dissection and immunostaining of first instar larva, a stage when active sensory neuron dendrite outgrowth and branching organization is occurring 12,13.
In addition to staining sensory neurons, this method achieves robust labeling of microtubule organization in muscles (Movies 2, 3), trachea (Figure 3, Movie 3), and other body wall tissues. It is valuable for investigators wishing to analyze microtubule organization in situ in the body wall when investigating mechanisms that control tissue and cell shape.

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

To understand how complex cell shapes, such as neuronal dendrites, are achieved during development, it is important to be able to accurately assay microtubule organization. Here we describe a robust immunohistological labeling method to examine microtubule organization of dendritic arborization neuron sensory dendrites, trachea, muscle, and other Drosophila larva body wall tissues.

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 ...

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

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

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

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

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

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

Voltage-sensitive Dye Recording from Axons, Dendrites and Dendritic Spines of Individual Neurons in Brain Slices

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding how the brain works. The primary function of any nerve cell is to process electrical signals, usually from multiple sources. Electrical properties of neuronal processes are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such a measurement one would, ideally, like to be able to monitor, at multiple sites, subthreshold events as they travel from the sites of origin on neuronal processes and summate at particular locations to influence action potential initiation. This goal has not been achieved in any neuron due to technical limitations of measurements that employ electrodes. To overcome this drawback, it is highly desirable to complement the patch-electrode approach with imaging techniques that permit extensive parallel recordings from all parts of a neuron. Here, we describe such a technique - optical recording of membrane potential transients with organic voltage-sensitive dyes (Vm-imaging) - characterized by sub-millisecond and sub-micrometer resolution. Our method is based on pioneering work on voltage-sensitive molecular probes 2. Many aspects of the initial technology have been continuously improved over several decades 3, 5, 11. Additionally, previous work documented two essential characteristics of Vm-imaging. Firstly, fluorescence signals are linearly proportional to membrane potential over the entire physiological range (-100 mV to +100 mV; 10, 14, 16). Secondly, loading neurons with the voltage-sensitive dye used here (JPW 3028) does not have detectable pharmacological effects. The recorded broadening of the spike during dye loading is completely reversible 4, 7. Additionally, experimental evidence shows that it is possible to obtain a significant number (up to hundreds) of recordings prior to any detectable phototoxic effects 4, 6, 12, 13. At present, we take advantage of the superb brightness and stability of a laser light source at near-optimal wavelength to maximize the sensitivity of the Vm-imaging technique. The current sensitivity permits multiple site optical recordings of Vm transients from all parts of a neuron, including axons and axon collaterals, terminal dendritic branches, and individual dendritic spines. The acquired information on signal interactions can be analyzed quantitatively as well as directly visualized in the form of a movie.

An imaging technique for monitoring of membrane potential changes with sub-micrometer spatial and sub-millisecond temporal resolution is described. The technique, based on laser excitation of voltage-sensitive dyes, allows measurements of signals in axons and axon collaterals, terminal dendritic branches, and individual dendritic spines.

Sensibles al voltaje Dye grabación de axones, dendritas y espinas dendríticas de las neuronas individuales en rodajas de cerebro

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding ...

Functional Analysis of the Larval Feeding Circuit in Drosophila

The serotonergic feeding circuit in Drosophila melanogaster larvae can be used to investigate neuronal substrates of critical importance during the development of the circuit. Using the functional output of the circuit, feeding, changes in the neuronal architecture of the stomatogastric system can be visualized. Feeding behavior can be recorded by observing the rate of retraction of the mouth hooks, which receive innervation from the brain. Locomotor behavior is used as a physiological control for feeding, since larvae use their mouth hooks to traverse across an agar substrate. Changes in feeding behavior can be correlated with the axonal architecture of the neurites innervating the gut. Using immunohistochemistry it is possible to visualize and quantitate these changes. Improper handling of the larvae during behavior paradigms can alter data as they are very sensitive to manipulations. Proper imaging of the neurite architecture innervating the gut is critical for precise quantitation of number and size of varicosities as well as the extent of branch nodes. Analysis of most circuits allow only for visualization of neurite architecture or behavioral effects; however, this model allows one to correlate the functional output of the circuit with the impairments in neuronal architecture.

Functional Analysis of the Larval Feeding Circuit in Drosophila

The feeding circuit in Drosophila melanogaster larvae serves a simple yet powerful model that allows changes in feeding rate to be correlated with alterations in the stomatogastric neural circuitry. This circuit is composed of central serotonergic neurons that send projections to the mouth hooks as well as the foregut.

Análisis funcional del circuito de alimentación de las larvas en Drosophila</i

The serotonergic  feeding circuit in Drosophila  melanogaster larvae can be used to investigate neuronal substrates of  critical importance during 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 ...

Production and Isolation of Axons from Sensory Neurons for Biochemical Analysis Using Porous Filters

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is required to shape functional neuronal circuits. The protocol described here explains how to use cell culture inserts bearing a porous membrane (filter) to obtain large amounts of pure axonal preparations suitable for examination by conventional biochemical or immunocytochemical techniques. The functionality of these filter inserts will be demonstrated with models of developmental pruning and Wallerian degeneration, using explants of embryonic dorsal root ganglion. Axonal integrity and function is compromised in a wide variety of neurodegenerative pathologies. Indeed, it is now clear that axonal dysfunction appears much earlier in the course of the disease than neuronal soma loss in several neurodegenerative diseases, indicating that axonal-specific processes are primarily targeted in these disorders. By obtaining pure axonal samples for analysis by molecular and biochemical techniques, this technique has the potential to shed new light into mechanisms regulating the physiology and pathophysiology of axons. This in turn will have an impact in our understanding of the processes that drive degenerative diseases of the nervous system.

This article describes a neuronal culture system that can be used to obtain large quantities of pure axonal samples for biochemical and immunocytochemical analyses. This technique will allow an improved understanding of normal axonal physiology and signaling pathways leading to neurodegeneration.

Producción y aislamiento de los axones de las neuronas sensoriales para el análisis bioquímico Usando porosos Filtros

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is ...

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in communication with the epidermal cells they innervate and thus their analysis benefits from in situ visualization of both neuronally and epidermally expressed proteins by immunofluorescence. Traditional methods of preparing larval fillets for immunofluorescence experiments leave intact the muscle tissue that covers most of the body wall, presenting several challenges to imaging neuronal and epidermal proteins. Here we describe a method for removing muscle tissue from Drosophila larval fillets. This protocol enables imaging of proteins that are otherwise obscured by muscle tissue, improves signal to noise ratio, and facilitates the use of super-resolution microscopy to study da neuron development.

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Studies of neuronal morphogenesis using Drosophila larval dendritic arborization (da) neurons benefit from in situ visualization of neuronal and epidermal proteins by immunofluorescence. We describe a procedure that improves immunofluorescence analysis of da neurons and surrounding epidermal cells by removing muscle tissue from the larval body wall.

Eliminación de<em&gt; Drosophila</em&gt; Tejido muscular de larvas Filetes para el análisis de inmunofluorescencia de las neuronas sensoriales y las células epidérmicas

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

Uso de los canales lineales de agarosa para estudiar<em&gt; Drosophila</em&gt; Comportamiento de las larvas de arrastre

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

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 ...