Agradecemos a Gary Laevsky útil para los debates sobre la microscopía. Este trabajo fue financiado por el NIH subvenciones R01GM061107 y R01GM067758 a ERG

Nota: El procedimiento para la eliminación de músculo (Figura 1) es una modificación de los métodos descritos anteriormente para la preparación de filetes de larvas. Los pasos que preceden y siguen a la eliminación del músculo se describen brevemente y se remite al lector a los trabajos previos 10, 11 para una descripción más detallada. 1. Dissect Larva en solución salina fría <ol><li> Preparar una dilución de trabajo de solución salina fría HL3.1 15 o Ca 2 + libre frío HL3.1 solución salina 11 (Tabla 1). Coloque la larva en una placa de elastómero de silicona con suficiente solución salina fría para cubrir el fondo del plato. Nota: Véase la discusión con respecto a la elección de la solución salina. </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> solución salina HL3.1 1x (pH 7,2) </td></tr><tr><td> HEPES 5 mM </td></tr><tr><td> NaCl 70 mM </td></tr><tr&gt; <td> KCl 5 mM </td></tr><tr><td> 1,5 mM de CaCl2 (omitir para Ca2 + solución salina exento) </td></tr><tr><td> 4 mM MgCl2 </td></tr><tr><td> 10 mM NaHCO 3 </td></tr><tr><td> trehalosa 5 mM </td></tr><tr><td> sacarosa 115 mM </td></tr><tr><td></td></tr><tr><td> Se esteriliza con filtro y se almacena a 4 ° C </td></tr><tr><td> imita la composición de insectos hemolinfa: Nota </td></tr></tbody></table> Tabla 1. Composición de frío Saline. <ol start="2"><li> Coloque la larva con el lado ventral hacia arriba. La superficie ventral de la larva se puede identificar por las cintas dentical abdominales y el lado dorsal por los tubos traqueales larvas primarias que funcionan de anterior a posterior. Estirar la larva en la dirección antero-posterior y el pin de la cabeza y la cola a unaelastómero de silicona disección plato usando pasadores de insectos. Con unas tijeras de disección finas para cortar a lo largo de la línea media ventral, comenzando en un extremo y progresando hacia el otro. NOTA: Esta orientación conserva el clúster dorsal comúnmente estudiada de las neuronas DA. </li><li> Después de la larva se corta abierta, fijar las cuatro esquinas del plato a la disección como si la apertura de un libro. El uso de fórceps para agarrar y extraer los órganos internos, incluyendo el sistema nervioso central, intestino, y la tráquea. Ajuste pasadores de insectos de manera que el filete es tensa pero no estirada al máximo. NOTA: Los músculos están ancladas a la pared del cuerpo en los límites segmentarias. Aunque los músculos cubren la mayor parte de la pared del cuerpo, que están ausentes en una zona estrecha cerca de la línea media dorsal. </li></ol> La eliminación 2. Músculo <ol><li> Busque la línea media dorsal de la larva, donde el tejido muscular está ausente. Coloque un solo diente fórceps de tal manera que se puede insertar debajo del tejido muscular en la orientación más plana posible. </li><li> A partir dela línea media dorsal, cerca del límite anterior del segmento, deslice cuidadosamente el diente fórceps entre el músculo y la epidermis, teniendo cuidado para minimizar el contacto entre la pinza y la epidermis. </li><li> Tire de la pinza hacia arriba para romper la unión del músculo para la pared del cuerpo en un punto de anclaje. Repita este proceso para el hemi-segmento (s) restante de interés. NOTA: Este protocolo optimiza la preservación de la parte posterior de cada campo de dendritas de las neuronas DA. Para preservar la parte anterior del campo de dendritas, lo mejor es insertar la clavija de fórceps en el extremo posterior de cada segmento. </li><li> Reajustar pasadores de insectos tales que el filete de larvas se estira al máximo en todas las direcciones. </li><li> Fijar el filete mientras que todavía se fija en el plato de disección usando, recién preparada formaldehído frío 4% en PBS durante 25 min. </li><li> Enjuagar 5 veces en PBS. </li><li> El uso de fórceps para extraer con cuidado el tejido muscular de los restantes puntos de anclaje, teniendo cuidado para minimizar contact con la epidermis. </li><li> Desanclar y quitar el filete del plato de la disección a un tubo de microcentrífuga de 1,5 ml. Llevar a cabo todas lavado, bloqueo, y los pasos de tinción de inmunofluorescencia posteriores, como se describió anteriormente 11. </li></ol> 3. Monte el filete de larvas <ol><li> Saca primero la cabeza y la cola con unas tijeras de disección finas con el fin de hacer la muestra lo más plana posible. Montar el filete en un cubreobjetos en medio de montaje antifade con la superficie interior del filete frente al cubreobjetos. </li><li> Colocar un portaobjetos de microscopio en el cubreobjetos y presionar suavemente para dispersar el medio de montaje. Da la vuelta al portaobjetos de microscopio una y sellar el cubreobjetos sobre el portaobjetos usando esmalte de uñas. Las diapositivas se pueden almacenar a -20 ° C durante al menos un mes. </li></ol>

<ol>
	<li>Jan, Y. N., Jan, L. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Branching+out:+mechanisms+of+dendritic+arborization.">Branching out: mechanisms of dendritic arborization.</a> Nat Rev Neurosci. 11 (5), 316-328 (2010).</li><li>Corty, M. M., Matthews, B. J., Grueber, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecules+and+mechanisms+of+dendrite+development+in+Drosophila.">Molecules and mechanisms of dendrite development in Drosophila.</a> Development. 136 (7), 1049-1061 (2009).</li><li>Parrish, J. Z., Emoto, K., Kim, M. D., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+that+regulate+establishment,+maintenance,+and+remodeling+of+dendritic+fields.">Mechanisms that regulate establishment, maintenance, and remodeling of dendritic fields.</a> Annu Rev Neurosci. 30, 399-423 (2007).</li><li>Kim, M. E., Shrestha, B. R., Blazeski, R., Mason, C. A., Grueber, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrins+establish+dendrite-substrate+relationships+that+promote+dendritic+self-avoidance+and+patterning+in+Drosophila+sensory+neurons.">Integrins establish dendrite-substrate relationships that promote dendritic self-avoidance and patterning in Drosophila sensory neurons.</a> Neuron. 73 (1), 79-91 (2012).</li><li>Han, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrins+regulate+repulsion-mediated+dendritic+patterning+of+Drosophila+sensory+neurons+by+restricting+dendrites+in+a+2D+space.">Integrins regulate repulsion-mediated dendritic patterning of Drosophila sensory neurons by restricting dendrites in a 2D space.</a> Neuron. 73 (1), 64-78 (2012).</li><li>Parrish, J. Z., Xu, P., Kim, C. C., Jan, L. Y., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microRNA+bantam+functions+in+epithelial+cells+to+regulate+scaling+growth+of+dendrite+arbors+in+Drosophila+sensory+neurons.">The microRNA bantam functions in epithelial cells to regulate scaling growth of dendrite arbors in Drosophila sensory neurons.</a> Neuron. 63 (6), 788-802 (2009).</li><li>Jiang, N., Soba, P., Parker, E., Kim, C. C., Parrish, J. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microRNA+bantam+regulates+a+developmental+transition+in+epithelial+cells+that+restricts+sensory+dendrite+growth.">The microRNA bantam regulates a developmental transition in epithelial cells that restricts sensory dendrite growth.</a> Development. 141 (13), 2657-2668 (2014).</li><li>Meltzer, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermis-derived+Semaphorin+promotes+dendrite+self-avoidance+by+regulating+dendrite-substrate+adhesion+in+Drosophila+sensory+neurons.">Epidermis-derived Semaphorin promotes dendrite self-avoidance by regulating dendrite-substrate adhesion in Drosophila sensory neurons.</a> Neuron. 89 (4), 741-755 (2016).</li><li>Han, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+cells+are+the+primary+phagocytes+in+the+fragmentation+and+clearance+of+degenerating+dendrites+in+Drosophila.">Epidermal cells are the primary phagocytes in the fragmentation and clearance of degenerating dendrites in Drosophila.</a> Neuron. 81 (3), 544-560 (2014).</li><li>Brent, J. R., Werner, K. M., 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+dissection.">Drosophila larval NMJ dissection.</a> J Vis Exp. (24), (2009).</li><li>Karim, M. R., Moore, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+analysis+of+Drosophila+larval+peripheral+sensory+neuron+dendrites+and+axons+using+genetic+mosaics.">Morphological analysis of Drosophila larval peripheral sensory neuron dendrites and axons using genetic mosaics.</a> J Vis Exp. (57), e3111 (2011).</li><li>Gustafsson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surpassing+the+lateral+resolution+limit+by+a+factor+of+two+using+structured+illumination+microscopy.">Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy.</a> J Microscopy. 198 (2), 82-87 (2000).</li><li>Ashdown, G. W., Cope, A., Wiseman, P. W., Owen, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+flow+quantified+beyond+the+diffraction+limit+by+spatiotemporal+image+correlation+of+structured+illumination+microscopy+data.">Molecular flow quantified beyond the diffraction limit by spatiotemporal image correlation of structured illumination microscopy data.</a> Biophys J. 107 (9), L21-L23 (2014).</li><li>Zhang, W., Yan, Z., Jan, L. Y., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sound+response+mediated+by+the+TRP+channels+NOMPC,+NANCHUNG,+and+INACTIVE+in+chordotonal+organs+of+Drosophila+larvae.">Sound response mediated by the TRP channels NOMPC, NANCHUNG, and INACTIVE in chordotonal organs of Drosophila larvae.</a> PNAS. 10 (33), 13612-13617 (2013).</li><li>Feng, Y., Ueda, A., Wu, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+minimal+hemolymph-like+solution,+HL3.1,+for+physiological+recordings+at+the+neuromuscular+junctions+of+normal+and+mutant+Drosophila+larvae.">A modified minimal hemolymph-like solution, HL3.1, for physiological recordings at the neuromuscular junctions of normal and mutant Drosophila larvae.</a> J Neurogenet. 18 (2), 377-402 (2004).</li><li>Babcock, D. T., Landry, C., Galko, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+signaling+mediates+UV-induced+nociceptive+sensitization+in+Drosophila+larvae.">Cytokine signaling mediates UV-induced nociceptive sensitization in Drosophila larvae.</a> Curr Biol. 19 (10), 799-806 (2009).</li></ol>

Los autores no tienen nada que revelar.

Aquí un protocolo se describe para la extracción manual de tejido muscular de Drosophila filetes de larvas. Este protocolo descrito previamente modifica la técnica de disección de las larvas 10,11. Después de la larva se diseca en una placa de elastómero de silicona, la línea media dorsal se encuentra. Una sola pinzas de punta, en su orientación más plana posible, se inserta cuidadosamente entre el tejido muscular y la epidermis, cerca de la línea media dorsal. El fórceps se tira suavement...

Arborización dendrítica (da), las neuronas de larvas de Drosophila proporcionan un modelo valioso para el estudio del desarrollo neuronal debido a su susceptibilidad a la manipulación genética y la facilidad con la que pueden obtenerse imágenes. Estas neuronas sensoriales han sido instrumentales en la identificación de numerosas vías que controlan la morfogénesis de dendritas 1-3. Cuatro clases de neuronas DA (clase I - IV) inervar la epidermis de las larvas. Estas neuronas se encuentran entre la membrana basal y la epidermis, con sus dendritas formando matrices en gran medida de dos dimensiones 4,5. De las cuatro clases, clase IV da neuronas tienen las glorietas más altamente ramificada y, al igual que las neuronas sensoriales de otros animales, la elaboración de estas pérgolas requiere factores intrínsecos, así como las señales de los tejidos vecinos, particularmente en la epidermis, para su desarrollo 6-9 . Los estudios para determinar las modalidades de dicha neuronal y hecho de extra-neuronalORS controlar beneficio morfogénesis dendrita de la capacidad de detectar la expresión de proteínas in situ por inmunofluorescencia. La cutícula exterior de la larva es impenetrable a los anticuerpos, pero este impedimento es fácilmente superada por la preparación de filetes de larvas a través de métodos de disección bien establecidos 10,11. Sin embargo, el tejido muscular de la pared del cuerpo que se encuentra justo interior a la membrana basal presenta varios retos hacia la visualización de las neuronas DA y las células epidérmicas. En primer lugar, el tejido muscular, lo que la mayoría de las líneas de la pared del cuerpo, oscurece considerablemente señales fluorescentes que emanan de tejido neuronal o epidérmica. Esto reduce sustancialmente la relación señal a ruido en la muestra. En segundo lugar, muchas proteínas relevantes pueden expresarse en el tejido muscular, así como en las neuronas o epidermis. Esta señal de fluorescencia derivada de músculo es probable que aún más la detección oscura de una señal de fluorescencia de la neurona o epidermis. Por último, los avances en las tecnologías de microscopía sinw imágenes permiso de muestras a una resolución de sub-difracción y puede ser especialmente útil en el discernimiento de la localización de las proteínas que se expresan en las neuronas y células de la epidermis que rodea 12,13. Sin embargo, las imágenes a través de super-resolución de microscopía se beneficia de una fuerte relación señal a ruido y la proximidad de la muestra al cubreobjetos. Además de reducir la relación señal a ruido, la pared del cuerpo distancias musculares larvas da a las neuronas de la cubreobjetos, limitando de este modo la resolución de imagen mejorado que se puede conseguir con métodos de microscopía super-resolución. Además de retos para el análisis de inmunofluorescencia, el tejido muscular presenta una barrera para el registro electrofisiológico de las neuronas sensoriales en la pared corporal de larvas. Por tanto, su eliminación se beneficia manipulación neurofisiológica de las neuronas sensoriales 14. Aquí se describe un método para la extracción manual de Drosophila tejido muscular larval. Se demuestra que nuestro protocolo permits de formación de imágenes de inmunofluorescencia de las proteínas que se oscurece de otra manera por el tejido muscular, mejora la relación señal a ruido para la visualización de clase IV neuronas DA, y permite el uso de super-resolución microscopía para discriminar mejor las relaciones espaciales de proteínas y estructuras celulares en Da neuronas y la epidermis.

<table><tbody><tr><td>Dumont #5 tweezers</td><td>Electron Microscopy Sciences</td><td>72701-D</td><td></td></tr><tr><td>Micro Scissors, 8 cm, straight, 5 mm blades, 0.1 mm tips</td><td>World Precision Instruments</td><td>14003</td><td></td></tr><tr><td>Sylgard 184 silicone elastomer kit</td><td>Dow Corning</td><td>3097358-1004</td><td>for dissecting plates</td></tr><tr><td>Austerlitz insect pins, 0.1 mm</td><td>Fine Science Tools</td><td>26002-10</td><td></td></tr><tr><td>Fostec 8375 light source</td><td>Artisan Technology Group</td><td>62792-4</td><td></td></tr><tr><td>Zeiss Stemi 2000</td><td>Carl Zeiss Microscopy</td><td></td><td></td></tr><tr><td>Vectashield antifade mounting medium</td><td>Vector Laboratories</td><td>H-1000</td><td>for confocal microscopy</td></tr><tr><td>Prolong Diamond antifade mountant</td><td>Life Technologies</td><td>P36970</td><td>for structured illumination microscopy</td></tr><tr><td>Micro cover glass, 22 x 22 mm, No. 1.5</td><td>VWR</td><td>48366-227</td><td></td></tr><tr><td>Superfrost Plus microscope slides, 25 x 75 x 1.0 mm</td><td>Fisherbrand</td><td>12-550-15</td><td></td></tr><tr><td>Mouse anti-Coracle antibody</td><td>Developmental Studies Hybridoma Bank</td><td>C615.16</td><td>supernatant, dilute 1:50</td></tr><tr><td>Mouse anti-Discs large antibody</td><td>Developmental Studies Hybridoma Bank</td><td>4F3</td><td>supernatant, dilute 1:50</td></tr><tr><td>Rabbit anti-dsRed antibody</td><td>Clontech</td><td>632496</td><td>dilute 1:1,000</td></tr><tr><td>Goat anti-rabbit antibody, Alexa Fluor 568 conjugated</td><td>ThermoFisher Scientific</td><td>A-11011</td><td>dilute 1:1,000</td></tr><tr><td>Goat anti-mouse antibody, Alexa Fluor 488 conjugated</td><td>ThermoFisher Scientific</td><td>A-11001</td><td>dilute 1:500</td></tr></tbody></table>

removal of drosophila muscle tissue from larval fillets for immunofluorescence analysis of sensory neurons and epidermal cells

Arborización dendrítica (DA), las neuronas de larvas de Drosophila son un modelo popular para la investigación de los mecanismos de morfogénesis neuronal. Da neuronas desarrollan en comunicación con las células epidérmicas que inervan y por tanto su análisis de beneficios de la visualización in situ de las dos proteínas expresadas neuronalmente y epidérmicamente por inmunofluorescencia. Los métodos tradicionales de preparación de filetes de larvas para experimentos de inmunofluorescencia dejan intacto el tejido muscular que cubre la mayor parte de la pared del cuerpo, que presenta varios desafíos para la formación de imágenes proteínas neuronales y epidérmicas. Aquí se describe un método para eliminar el tejido muscular de Drosophila filetes de larvas. Este protocolo permite que las imágenes de las proteínas que se oscurece de otra manera por el tejido muscular, mejora la relación señal a ruido, y facilita el uso de super-resolución microscopía para estudiar da el desarrollo de la neurona.

Se demuestra la utilidad del procedimiento de extracción muscular para mejorar la relación señal a ruido en los experimentos de inmunofluorescencia para co-visualizar las proteínas de unión tabicado Coracle (Cora) y discos de gran (DLG), junto con la clase IV neuronas DA, marcadas con el marcador de membrana CD4 tdTomato. Cora se ha utilizado anteriormente para identificar áreas de muestreo donde da las dendritas de las neuronas están rodeados por cél...

Watch this Scientific Journal Video about Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells at JoVE.com

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

Los estudios de la morfogénesis neuronal utilizando Drosophila arborización dendrítica larval (DA) se benefician de las neuronas en la visualización in situ de las proteínas neuronales y epidérmicas por inmunofluorescencia. Se describe un procedimiento que mejora el análisis de inmunofluorescencia de DA neuronas y las células epidérmicas que rodean mediante la eliminación de tejido muscular de la pared del cuerpo de la larva.

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 ...

removal-drosophila-muscle-tissue-from-larval-fillets-for

Research

JoVE Journal

Neuroscience

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

8.9K vistas.  Princeton University.  Los estudios de la morfogénesis neuronal utilizando Drosophila arborización dendrítica larval (DA) se benefician de las neuronas en la visualización in situ de las proteínas neuronales y epidérmicas por inmunofluorescencia. Se describe un procedimiento que mejora el análisis de inmunofluorescencia de DA neuronas y las células epidérmicas que rodean mediante la eliminación de tejido muscular de la pared del cuerpo de la larva. 

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

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The Drosophila larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila is well known for the ease of powerful genetic manipulations and the larval nervous system has proven particularly useful in studying not only normal function but also perturbations that accompany some neurological disease (Lloyd and Taylor, 2010). Many key synaptic molecules found in Drosophila are also found in mammals and like most CNS excitatory synapses in mammals, the Drosophila NMJ is glutamatergic and demonstrates activity-dependent remodeling (Kohet al. , 2000). Additionally, Drosophila neurons can be individually identified because their innervation patterns are stereotyped and repetitive making it possible to study identified synaptic terminals, such as those between motor neurons and the body-wall muscle fibers that they innervate (Keshishian and Kim, 2004). The existence of evolutionarily conserved synapse components along with the ease of genetic and physical manipulation make the Drosophila model ideal for investigating the mechanisms underlying synaptic function (Budnik, 1996).
The active zones at synaptic terminals are of particular interest because these are the sites of neurotransmitter release. NC82 is a monoclonal antibody that recognizes the Drosophila protein Bruchpilot (Brp), a CAST1/ERC family member that is an important component of the active zone (Waghet al. , 2006). Brp was shown to directly shape the active zone T-bar and is responsible for effectively clustering Ca2+ channels beneath the T-bar density (Fouquetet al. , 2009). Mutants of Brp have reduced Ca2+ channel density, depressed evoked vesicle release, and altered short-term plasticity (Kittelet al. , 2006). Alterations to active zones have been observed in Drosophila disease models. For example, immunofluorescence using the NC82 antibody showed that the active zone density was decreased in models of amyotrophic lateral sclerosis and Pitt-Hopkins syndrome (Ratnaparkhiet al. , 2008; Zweieret al. , 2009). Thus, evaluation of active zones, or other synaptic proteins, in Drosophila larvae models of disease may provide a valuable initial clue to the presence of a synaptic defect.
Preparing whole-mount dissected Drosophila larvae for immunofluorescence analysis of the NMJ requires some skill, but can be accomplished by most scientists with a little practice. Presented is a method that provides for multiple larvae to be dissected and immunostained in the same dissection dish, limiting environmental differences between each genotype and providing sufficient animals for confidence in reproducibility and statistical analysis.

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The neuromuscular junction (NMJ) of Drosophila melanogaster is an important model system for studying normal synaptic function as well as perturbations to synaptic function found in certain neurological diseases. We present a protocol for dissection of the Drosophila larval motor system and immunostaining for active zone proteins within the NMJ.

La disección y la imagen de zonas activas en el Drosophila La unión neuromuscular

The Drosophila  larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila  is well known for ...

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 ...

Dissection of Drosophila melanogaster Flight Muscles for Omics Approaches

Drosophila flight muscle is a powerful model to study diverse processes such as transcriptional regulation, alternative splicing, metabolism, and mechanobiology, which all influence muscle development and myofibrillogenesis. Omics data, such as those generated by mass spectrometry or deep sequencing, can provide important mechanistic insights into these biological processes. For such approaches, it is beneficial to analyze tissue-specific samples to increase both selectivity and specificity of the omics fingerprints. Here we present a protocol for dissection of fluorescent-labeled flight muscle from live pupae to generate highly enriched muscle samples for omics applications. We first describe how to dissect flight muscles at early pupal stages (&#60;48 h after puparium formation [APF]), when the muscles are discernable by green fluorescent protein (GFP) labeling. We then describe how to dissect muscles from late pupae (&#62;48 h APF) or adults, when muscles are distinguishable under a dissecting microscope. The accompanying video protocol will make these technically demanding dissections more widely accessible to the muscle and Drosophila research communities. For RNA applications, we assay the quantity and quality of RNA that can be isolated at different time points and with different approaches. We further show that Bruno1 (Bru1) is necessary for a temporal shift in myosin heavy chain (Mhc) splicing, demonstrating that dissected muscles can be used for mRNA-Seq, mass spectrometry, and reverse transcription polymerase chain reaction (RT-PCR) applications. This dissection protocol will help promote tissue-specific omics analyses and can be generally applied to study multiple biological aspects of myogenesis.

Dissection of Drosophila melanogaster Flight Muscles for Omics Approaches

Drosophila flight muscle is a powerful model to study transcriptional regulation, alternative splicing, metabolism, and mechanobiology. We present a protocol for dissection of fluorescent-labeled flight muscle from live pupae to generate highly enriched samples ideal for proteomics and deep-sequencing. These samples can offer important mechanistic insights into diverse aspects of muscle development.

Disección de los músculos de vuelo de Drosophila melanogaster para los enfoques de Omics

Drosophila flight muscle is a powerful model to study diverse processes such as transcriptional regulation, alternative splicing, metabolism, and ...

Genética

Drosophila Larval NMJ Dissection

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 35 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. In order to access the NMJ for study, it is necessary to carefully dissect each larva. In this article we demonstrate how to properly dissect Drosophila larvae for study of the NMJ by removing all internal organs while leaving the body wall intact. This technique is suitable to prepare larvae for imaging, immunohistochemistry, or electrophysiology.

This protocol demonstrates how to dissect Drosophila larvae in preparation for immunohistochemistry and/or imaging of the neuromuscular junction.

Drosophila larval NMJ Disección

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

Biología

Drosophila Larval NMJ Immunohistochemistry

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 31 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. Immunohistochemistry can be used to clearly image the components of the NMJ. In this article, we demonstrate how to use antibody staining to visualize the Drosophila larval NMJ.

Drosophila Larval NMJ Immunohistochemistry

This protocol demonstrates how to perform immunohistochemistry on dissected Drosophila larva.

Drosophila larval NMJ inmunohistoquímica

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

Las secciones incluidas en parafina y congeladas de Drosophila Los músculos de Adultos

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

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.

Uso de la unión neuromuscular de las larvas de Drosophila y células musculares para visualizar la red de microtúbulos

The microtubule network is an essential component of the nervous system. Mutations in many microtubules regulatory proteins are associated with ...

Ablación de una sola neurona en Drosophila para estudiar la adaptación del sistema nervioso

Laser Cell Ablation in Intact Drosophila Larvae Reveals Synaptic Competition

The protocol describes single-neuron ablation with a 2-photon laser system in the central nervous system (CNS) of intact Drosophila melanogaster larvae. Using this non-invasive method, the developing nervous system can be manipulated in a cell-specific manner. Disrupting the development of individual neurons in a network can be used to study how the nervous system can compensate for the loss of synaptic input. Individual neurons were specifically ablated in the giant fiber system of Drosophila, with a focus on two neurons: the presynaptic giant fiber (GF) and the postsynaptic tergotrochanteral motor neuron (TTMn). The GF synapses with the ipsilateral TTMn, which is crucial to the escape response. Ablating one of the GFs in the 3rd instar brain, just after the GF starts axonal growth, permanently removes the cell during the development of the CNS. The remaining GF reacts to the absent neighbor and forms an ectopic synaptic terminal to the contralateral TTMn. This atypical, bilaterally symmetric terminal innervates both TTMns, as demonstrated by dye coupling, and drives both motor neurons, as demonstrated by electrophysiological assays. In summary, the ablation of a single interneuron demonstrates synaptic competition between a bilateral pair of neurons that can compensate for the loss of one neuron and restore normal responses to the escape circuit.

Autor destacado: Investigación de los mecanismos de ensamblaje de circuitos neuronales y formación de sinapsis en Drosophila

This protocol demonstrates the laser cell ablation of individual neurons in intact Drosophila larvae. The method enables the study of the effect of reducing competition between neurons in the developing nervous system.

La ablación celular con láser en larvas intactas de Drosophila revela una competencia sináptica

The protocol describes single-neuron ablation with a 2-photon laser system in the central nervous system (CNS) of intact Drosophila melanogaster larvae. ...

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Drosophila larval NMJ Disección

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Las secciones incluidas en parafina y congeladas de Drosophila Los músculos de Adultos

Uso de la unión neuromuscular de las larvas de Drosophila y células musculares para visualizar la red de microtúbulos

Uso de la unión neuromuscular de las larvas de Drosophila y células musculares para visualizar la red de microtúbulos

Uso de la unión neuromuscular de las larvas de Drosophila y células musculares para visualizar la red de microtúbulos

La ablación celular con láser en larvas intactas de Drosophila revela una competencia sináptica